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Podocin Inactivation in Mature Kidneys Causes Focal Segmental Glomerulosclerosis and

Ge´raldine Mollet,*† Julien Ratelade,*† Olivia Boyer,*†‡ Andrea Onetti Muda,*§ ʈ Ludivine Morisset,*† Tiphaine Aguirre Lavin,*† David Kitzis,*† Margaret J. Dallman, ʈ Laurence Bugeon, Norbert Hubner,¶ Marie-Claire Gubler,*† Corinne Antignac,*†** and Ernie L. Esquivel*†

*INSERM, U574, Hoˆpital Necker-Enfants Malades, Paris, France; †Faculte´deMe´ decine Rene´ Descartes, Universite´ Paris Descartes, Paris, France; ‡Pediatric Nephrology Department and **Department of Genetics, Hoˆpital Necker- Enfants Malades, Assistance Publique-Hoˆpitaux de Paris, Paris, France; §Department of Pathology, Campus ʈ Biomedico University, Rome, Italy; Department of Biological Sciences, Imperial College London, London, England; and ¶Max-Delbruck Center for Molecular Medicine, Berlin, Germany

ABSTRACT Podocin is a critical component of the glomerular slit diaphragm, and genetic mutations lead to both familial and sporadic forms of steroid-resistant nephrotic syndrome. In mice, constitutive absence of podocin leads to rapidly progressive renal disease characterized by mesangiolysis and/or mesangial sclerosis and nephrotic syndrome. Using established Cre-loxP technology, we inactivated podocin in the adult mouse in a -specific manner. Progressive loss of podocin in the recapitu- lated albuminuria, hypercholesterolemia, hypertension, and renal failure seen in nephrotic syndrome in humans. Lesions of FSGS appeared after 4 wk, with subsequent development of diffuse glomerulosclerosis and tubulointerstitial damage. Interestingly, conditional inactivation of podocin at birth resulted in a gradient of glomerular lesions, including mesangial proliferation, demonstrating a developmental stage dependence of renal histologic patterns of injury. The development of significant albuminuria in this model occurred only after early and focal foot process effacement had progressed to diffuse involvement, with complete absence of podocin immunolabeling at the slit diaphragm. Finally, we identified novel potential mediators and perturbed molecular pathways, including cellular proliferation, in the course of progression of renal disease leading to glomerulosclerosis, using global expression profiling.

J Am Soc Nephrol 20: 2181–2189, 2009. doi: 10.1681/ASN.2009040379

FSGS is a clinicopathologic syndrome character- Recent discoveries have established the genetic ized by podocyte injury and progressive scarring bases of some familial forms of FSGS,4–8 as well as in the renal glomerulus. Epidemiologic studies the role of in enhancing susceptibility to glo- have shown an increasing incidence of FSGS in the United States,1,2 particularly among black in- Received April 8, 2009. Accepted June 10, 2009. dividuals, making it the most common cause of Published online ahead of print. Publication date available at ESRD as a result of primary glomerular diseases www.jasn.org. 2 in both white and black individuals. Given the J.R., O.B., and A.O.M. contributed equally to this work. rising prevalence of chronic kidney disease,3 ef- Correspondence: Dr. Corinne Antignac, INSERM U574, 6e`me forts to understand the pathophysiologic and ge- e´tage, Tour Lavoisier, Hoˆpital Necker-Enfants Malades, 149 rue netic mechanisms leading to FSGS are crucial for de Se`vres, 75015 Paris, France. Phone: ϩ33-1-4449-4552; Fax: developing strategies aimed at prevention and ϩ33-1-4449-0290; E-mail: [email protected] therapy. Copyright ᮊ 2009 by the American Society of Nephrology

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mice of constitutive absence of podocin or ex- pression of a podocin missense mutant and iden- tified genetic and environmental modifiers of the renal disease.17–19 Podocin mutant mice present with albuminuria at birth and develop lesions of mesangiolysis and mesangial sclerosis. Their early death (within the first few weeks of life, depend- ing on the genetic background17–19) precludes ex- tensive study into the mechanisms of glomerular disease. We therefore generated a novel murine model in which podocin was inactivated in the adult mouse kidney in a podocyte-specific man- ner, using established Cre-loxP technology, and here detail the physiologic, ultrastructural, and transcriptional changes leading to FSGS in this model.

RESULTS

Generation of Podocyte-Specific Nphs2 Knockout Mice To study the effects of podocin inactivation in the mature kidney, we generated triallelic Nphs2lox2/ ϩ Ϫ,Cre mice, bearing a floxed Nphs2 2 allele (Figure 1A), a null Nphs2 allele,17 and a podocyte- expressed, tamoxifen-responsive Cre recombi- nase transgene20 (Supplemental Figures S1 and S2). Mice bearing a null allele in the heterozygous state do not demonstrate glomerular disease17 and neither do Nphs2lox2/lox2 mice, even after 8 wk of follow-up (data not shown). Administration of Figure 1. The Nphs2 gene encoding podocin shows conditional inactivation. tamoxifen to phenotypically normal 6-wk-old (A) The Nphs2 gene was targeted by homologous recombination using a Nphs2lox2/Ϫ, Creϩ mice resulted in nuclear trans- construct in which exon 2 was flanked by loxP sites. The Neo selection cassette location of Cre recombinase in approximately was removed by mating with an Flp deleter strain of mice, and excision of exon 70% of and in excision of exon 2. A 2 by a podocyte-expressed Cre recombinase was achieved on administration of progressive decrease in podocin expression was tamoxifen. (B) Real-time PCR revealed downregulation of podocin mRNA as early as 1 wk after Cre activation, using primers and probe that encompass seen as early as 7 d after Cre induction, both at the 2 to 3. Expression levels are shown relative to those in control mice, using mRNA level (shown using real-time PCR; Figure 18S rRNA as a reference gene. (C) Western blotting of total kidney extracts 1B) and at the level (demonstrated by revealed progressive decreases in podocin expression at the protein level over Western blotting [Figure 1C] and by quantifica- time. (D and E) Levels of podocin were quantified by measurement of pixel tion of fluorescence signal intensity of podocin in intensity of immunofluorescence labeling of podocin (in red) in glomeruli delin- glomeruli [Figure 1, D and E]). eated using an anti-nidogen antibody (in green) and normalized to glomerular surface area. *P Ͻ 0.0001. Podocin Loss Leads to Nephrotic Syndrome Inactivation of podocin resulted in death of Ϫ ϩ merular disease.9,10 The NPHS2 gene, which encodes the slit Nphs2lox2/ ,Cre mice at a median time of 11 wk after the start diaphragm protein podocin, not only accounts for 43% of fa- of tamoxifen administration (Figure 2A). Albuminuria was de- milial and 10% of sporadic forms of nephrotic syndrome, but tected on a Coomassie blue–stained protein gel (Figure 2B) also some genetic variants may increase the risk for glomerular after a mean of 10 d (range 8 to 13 d). This progressed to disease.11,12 Podocin acts as a structural scaffold in podocyte massive, nonselective proteinuria by 4 wk (Figure 2B). Tail- foot processes and interacts with slit diaphragm to cuff plethysmography measurements, carried out 4 wk after facilitate cellular signaling events.13–16 podocin inactivation, showed a modest but significant increase Previously, we described the phenotypic consequences in in the BP of null mice (Figure 2C). Finally, plasma levels of

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consistently present only 6 wk after Cre recombi- nase induction. Mice that were killed after 9 wk demonstrated progression of tubular injury, with basement membrane thickening and interstitial fibrosis (Figure 3F).

Developmental Stage Effects on Renal Disease Given the discrepancy between the renal pheno- types resulting from constitutive and conditional inactivation of podocin, we investigated the effects of Nphs2 inactivation at birth, during which nephrogenesis is not completed in mice. After ad- ministration of tamoxifen to nursing mothers for 3 d after birth, resulting in tamoxifen delivery via Ϫ milk,21,22 albuminuria developed in Nphs2lox2/ , Creϩ pups after 2 wk (data not shown). When mas- sive proteinuria was present at 4 wk by Coomassie blue staining, mice were killed. The renal histology showed a gradient of lesions. Superficial outer cor- tical glomeruli, which were not present at the time Figure 2. Conditional Nphs2 knockout mice have various phenotypes. (A) of birth, were normal and podocin expression was Ϫ Kaplan-Meier survival curve of control and Nphs2lox2/ mice upon Cre activation intact (Figure 4, A and B). In midcortical and the by tamoxifen shows median survival of 11 wk upon podocin loss. (B) Mice majority of juxtamedullary glomeruli, in which Cre develop detectable albuminuria at 2 wk by Coomassie staining of 12% SDS- recombinase was active and podocin was dimin- PAGE. This progresses to massive nonspecific proteinuria by 4 wk. (C) Tail-cuff ished (Figure 4A), lesions of mesangial proliferation Ϫ plethysmography of Nphs2lox2/ mice 4 wk after Cre activation shows mild were observed (Figure 4B). In addition, in the most lox2/Ϫ hypertension. Experiments were performed on 12 control and 12 Nphs2 severely affected juxtamedullary glomeruli, podocin Ͻ mice. §P 0.05 after two-tailed t test. (D) Elevations in serum levels of choles- was absent (Figure 4A) and lesions of FSGS were terol 4 wk after podocin loss precede increases in serum urea and creatinine. present (Figure 4B). Control values at all time points were unchanged and were pooled. Data are means Ϯ SEM, with at least five mice in each group. #P Ͻ 0.001 by one-way Ultrastructural Studies ANOVA. We performed serial ultrastructural studies in cholesterol were significantly elevated by 4 wk, whereas plasma adult Nphs2lox2/Ϫ,Creϩ mice, after Cre induction, to under- urea and creatinine levels began to rise only at 6 wk (Figure stand the role of foot process effacement (FPE) in the develop- 2D). ment of proteinuria. Whereas foot processes in control mice were evenly spaced and separated by a slit diaphragm (Figure Ϫ ϩ Mice Develop FSGS and Tubular Damage 5A), foot processes in Nphs2lox2/ ,Cre mice were focally ef- Despite downregulation of podocin expression, the renal his- faced at 1 and 2 wk after Cre induction (Figure 5, B and C). tology of Nphs2lox2/Ϫ,Creϩ mice at the light microscopic level 1 Morphometric measurements revealed a near doubling of the wk (data not shown) after induction of Cre recombinase activ- mean foot process width as early as 1 wk (Figure 5E). The ity was similar to those of control (Nphs2lox2/Ϫ,CreϪ and degree of FPE progressed to diffuse effacement 4 wk (Figure 5D) Nphs2lox2/ϩ,Creϩ) mice (Figure 3A). After 2 wk, occasional after tamoxifen administration. Progression of FPE corresponded podocyte hypertrophy was noted, with minimal mesangial ma- to the worsening of albumin leak, eventually to massive, nonse- trix expansion in some glomeruli (Figure 3B). Four weeks after lective proteinuria. Contrary to the findings of others implicating Cre induction, FSGS was observed in many glomeruli, with podocyte detachment and subsequent glomerular basement varying degrees of severity (Figure 3C). Glomerular pseudo- membrane (GBM) denudation as a mechanism of glomerular crescents were observed in at least 30% of glomeruli beginning sclerosis,23 ultrastructural studies did not reveal areas of denuded at 4 wk (Figure 3D). GBM in Nphs2lox2/Ϫ,Creϩ mice at any of the time points investi- Glomerulosclerosis continued to worsen by 6 wk, with in- gated. creasing segmental involvement (Figure 3E). At or near the Using immunogold electron microscopic labeling, we local- time of death, global sclerosis was evident in the majority of ized podocin at the slit diaphragm in controls (Figure 6A) and glomeruli (Figure 3F). Tubulointerstitial injury characterized in areas where foot processes were maintained in Nphs2lox2/Ϫ, by diffuse tubular dilation, tubular atrophy and necrosis, and Creϩ mice at 1 and 2 wk (Figure 6B). No labeling was seen in the presence of proteinaceous casts (Figure 3, E and F) was parietal epithelial, endothelial, or mesangial cells. In some ar-

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S2) and canonical pathway perturbations (Supple- mental Table S3). Functional annotation, using the Ingenuity Pathway Analysis Program, showed early and significant perturbations of cell-cycle regulation and cellular proliferation pathways. Proliferation of resident cells in the glomerulus during the develop- ment of glomerulosclerosis has been inconsistently demonstrated in humans and animal models. In Nphs2lox2/Ϫ,Creϩ mice, immunostaining for Ki-67 identified a significant increase in the percentage of proliferating resident glomerular cells (Figure 7A), involving podocytes and endothelial and mesangial cells (Figure 7B). Immunostaining for CD133 and CD24, markers of progenitor cells, were negative, suggesting that proliferative cells were intrinsic to the glomerulus (data not shown).

DISCUSSION

Animal models of podocyte injury, using toxic agents, immunologic damage, renal ablation, gene targeting, and transgenesis or via expression of HIV component proteins, have offered tre- mendous insights into the pathophysiologic mechanisms leading to glomerulosclerosis.24 We chose to develop murine models of podocyte in- jury on the basis of the targeted inactivation of the podocin gene, mutations in which account for both familial and sporadic cases of steroid-resis- tant nephrotic syndrome.5,11,25 In addition, dys- Figure 3. Renal lesions progress upon Nphs2 inactivation in the mature kidney. regulation of podocin expression was described in (A) Control mice show normal glomerular and tubular morphologies. (B) At 2 wk, an experimental model of membranous nephrop- there were no significant glomerular changes besides mild glomerular enlarge- athy,26 in FSGS,27 and in other forms of acquired ment and occasional podocyte hypertrophy. (C) There is segmental accumula- 28,29 tion of collagen (arrow) in many glomeruli 4 wk after Cre induction. (D) In at least proteinuric diseases, therein highlighting its 30% of glomeruli starting at 4 wk, pseudocrescents were observed. (E) There is pivotal role in the pathogenesis of glomerular dis- progression of lesions of FSGS 6 wk later, accompanied by tubular dilation and ease development. protein casts. (F) At 9 wk, diffuse global sclerosis was present, along with marked The early demise of two previous mouse mod- tubular dilation, atrophy, and interstitial fibrosis. Bars ϭ 50 ␮m. Slides were els of podocin inactivation and the presence of stained with periodic acid-Schiff stain. Magnification: ϫ400 in A, B, C, and E; histologic lesions of mesangiolysis and mesangial ϫ1000 in D and F. sclerosis, which are not characteristic of human disease because of podocin mutations, impelled eas of focal effacement at 1 and 2 wk, gold particles were dis- us to generate a model of podocin inactivation in the mature placed within the cytoplasm of effaced podocytes, sometimes kidney. Using an established Cre deleter line,20 expression and clustering along the plasma membrane facing the urinary space nuclear translocation of Cre recombinase in approximately (Figure 6C). Only after 4 wk was podocin completely absent in 70% of podocytes resulted in a 50% downregulation of podo- diffusely effaced foot processes (Figure 6D). cin expression as early as 1 wk after induction. By 4 wk, podo- cin was nearly absent in all glomeruli, suggesting that podocin Global Profiling of Mutant Glomeruli loss was secondarily achieved through a vicious cycle of dam- To identify molecular pathways perturbed upon podocin loss, aged podocytes’ damaging neighboring podocytes.30,31 This we performed global gene expression profiling of glomeruli may explain immunogold labeling studies showing displaced isolated from mutant and control mice at weeks 1, 2, and 4. We podocin in some focally effaced foot processes, reflecting validated microarray results using quantitative real-time PCR podocytes in which Cre recombinase was not activated. Alter- (Supplemental Figure S3; Supplemental Table S1) and revealed a natively, these particles may represent residual podocin in the complex array of transcriptional changes (Supplemental Table process of cytosolic degradation. Indeed, failure of podocin

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Figure 4. Inactivation at birth reveals devel- opmental stage dependence of phenotypes. (A) At 4 wk of life, podocin expression (in red) demonstrated a gradient from the outer cor- tical (OC) to midcortical (MC) and to jux- tamedullary (JM) glomeruli, marked with ni- dogen (in green). Because OC glomeruli are not formed at birth, podocin expression is intact, whereas MC glomeruli, which are im- mature at birth, have decreased podocin ex- pression. At birth, JM glomeruli are mature and show near absence of podocin. (B) His- tologic analysis showed normal OC glomer- uli, glomerular hypertrophy and lesions of mesangial proliferation in MC glomeruli and lesions of FSGS in severely affected JM glo- meruli. Slides were stained with periodic acid- Schiff. Magnification, ϫ400. targeting to sites of action on the plasma membrane has been shown both in vitro and in vivo to result in loss of func- tion.18,32,33 Podocyte injury in our model leads to progressive renal disease, ultimately recapitulating features of nephrotic syn- drome in humans, such as hyperlipidemia, hypertension, and renal insufficiency. Heterogeneity between mutant mice in the evolution of renal disease may partly be due to individual differences resulting from their underlying mixed genetic background. Less phenotypic variability has been seen in ongoing studies on congenic mice (C.A., un- published observations). In contrast to our previously described podocin knockout model in which foot processes are effaced at birth,17 podocyte FPE evolved from focal to diffuse involvement with inactiva- tion in the mature kidney; however, despite the presence of focally effaced foot processes at 1 wk, we found no significant albuminuria until the second week. The absence of albumin- uria at 1 wk may reflect the limits of detection of our assay or, alternatively, may represent a time when the capacity of com- pensatory proximal tubular reabsorption of albumin34 has not yet been saturated. Studies to investigate the tubular mechanisms of albumin handling in these mice are ongoing. Our findings contrast with models in which proteinuria has been described in the absence of FPE35 but are in agreement with two previous studies conducted in human samples36 and in the puromycin aminoglycoside nephrosis model37 showing absence of proteinuria despite extensive FPE. Finally, the ap- Figure 5. FPE develops upon podocin loss. (A) Ultrastructural pearance of massive, nonselective albuminuria coincided in studies in a control mouse revealed elaboration of podocytes (P) our study with diffuse FPE, but we cannot exclude concomi- into regularly spaced foot processes (FP), on the opposite side of tant changes in GBM composition, although no gross abnor- the GBM as the fenestrated endothelial cells (En) lining the cap- illary lumen (L). (B and C) At 1 (B) and 2 wk (C) after Cre induction, malities in the GBM were noted by electron microscopy. FPE was focal. (D) After 4 wk, foot processes were globally ef- Our results demonstrate that the evolution of renal pheno- faced. (E) Morphometric studies revealed progressive increase in types of mice in which podocin has been inactivated in the foot process width over time. Calculations are based on 15 cap- mature kidney is different from those of the previous Nphs2 illary loops each in three mice at each time point and compared models in which functional podocin was absent during kidney with five control mice. Magnification, ϫ15,000. *P Ͻ 0.0001. development. Rather than displaying lesions of mesangiolysis

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ing from congenital to late-onset disease has been described in NPHS2 cases, but renal bi- opsy data have been limited. Contrary to mice, in which nephrogenesis continues until the sec- ond week of life, nephrogenesis in humans is complete at birth. One could speculate that cer- tain podocin mutations, however, may exert deleterious effects that affect developmental pathways in the renal glomerulus. A deeper un- derstanding of the role of podocin in intraglo- merular cross-talk is necessary to clarify whether a developmental stage dependence of renal disease evolution in nephrotic syndrome is of clinical relevance in humans. The transcriptional changes occurring during the course of glomerular disease development are Figure 6. Podocin is immunolocalization with progressive renal disease. (A) poorly understood. We used microarrays to re- Immunogold labeling of podocin shows discrete localization to the slit dia- veal the perturbations in gene expression in iso- phragms bridging foot processes (FP) in control mice. (B) At 1 wk, podocin lated glomeruli at various time points after podo- remained localized mostly to the slit diaphragms. (C) At 2 wk, podocin was cin inactivation and found a molecular signature localized mostly to cytosolic areas of podocytes (P), especially in areas of focal unique from other, previously published models FP effacement. (D) Podocin expression was essentially absent in areas of dif- of FSGS,43–45 thus, confirming the heterogeneity fusely effaced FP. No podocin expression was observed in glomerular endothe- of FSGS depending on the mechanism by which ϫ lial cells (En) lining the capillary lumen (L). Magnifications: 25,000 in A through podocyte injury is incurred. ϫ C; 15,000 in D. Intriguingly, we found that genes involved and mesangial sclerosis seen in Nphs2 knockout or the in cell-cycle regulation were the earliest genes to be tran- Ϫ ϩ Nphs2R140Q/R140Q mutant mice,17,18 Nphs2lox2/ ,Cre mice de- scriptionally activated in our model. Several other mouse veloped lesions of FSGS in the setting of nephrotic-range pro- models of glomerular disease failed to reveal a prominent teinuria, recapitulating the most common lesion found in re- role of proliferation of resident cells of the glomerulus, be- nal biopsies of patients bearing NPHS2 mutations.5,38 When sides parietal epithelial cells.46–48 Conversely, we found inactivation was carried out at birth, during which nephrons proliferation not only of podocytes, as seen in models of are continuing to form and mature, the spectrum of lesions HIV-associated nephropathy,49 but also of mesangial and included mesangial proliferation in the intermediate zone of endothelial cells. This may represent dedifferentiation of the renal cortex. These data suggest that podocin loss disrupts terminally differentiated cells in the glomerulus in response podocyte–mesangial cell–endothelial cell cross-talk in the glo- to podocyte injury in our model. It will be interesting to merulus differently in mature and in developing glomeruli. determine whether cellular proliferation is a compensatory The importance of cross-talk in glomerular assembly and mat- or a deleterious event in glomerulosclerosis. uration, mediated by the vascular endothelial growth factor Conditional inactivation of podocin in the adult murine pathway, for instance, has been highlighted by recent stud- kidney represents a novel model system of nephrotic syndrome ies.39,40 The developmental stage dependence of cystic renal as a result of FSGS, thereby recapitulating human disease re- disease evolution has similarly been elucidated in a Pkd1 sulting from mutations in the podocin gene and providing a knockout mouse model.21 vehicle for better understanding key pathophysiologic mecha- In humans, the age of onset of renal disease as a result of nisms of glomerular disease development. Furthermore, it re- podocin mutations is broad, and although minimal change vealed important phenotypic differences from previous consti- and FSGS are the predominant histologic lesions, mesangial tutive knockout models of podocin, therein highlighting the lesions have likewise been reported.38 It is noteworthy that importance of conditional targeting of other genes implicated the predominant renal biopsy findings in patients who bear in nephrotic syndrome. mutations in the NPHS1 gene, which encodes , and largely present with nephrotic syndrome at birth are lesions of mesangial hypercellularity with varying degrees of mes- CONCISE METHODS angial sclerosis.41 Recently, we identified a cohort of pa- tients who bear compound heterozygous NPHS1 mutations Generation of Mice and present with later onset nephrotic syndrome (mean 3 The Nphs2 mutant mouse line was established at the Mouse Clinical yr), in whom renal biopsy, by contrast, revealed minimal- Institute–Institut Clinique de la Souris (http://www.mci.u-strasbg.fr; change nephropathy and FSGS.42 A similar spectrum rang- Illkirch, France). The mouse genome was modified to allow for exci-

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Phenotypic Characterization Survival was assessed by following a cohort of mice until death. There- after, five control and five null mice were killed at weeks 1, 2, 4, and 6 after tamoxifen administration. Blood was obtained via cardiac punc- ture, centrifuged at 5000 rpm for 5 min to obtain plasma, and blood urea nitrogen, creatinine, and cholesterol were measured using an Olympus AU-400 multiparametric analyzer. Time of onset of albu- minuria was determined by daily collection of spontaneously voided urine after initiation of tamoxifen induction, and 2 ␮l of urine was examined on a 12% SDS-PAGE gel stained with Coomassie blue. One kidney was extracted and snap-frozen in liquid nitrogen for protein and RNA analysis. When podocin was inactivated at birth, pups were genotyped at day 5 and albuminuria was tested thereafter. Litters were killed at 4 wk and characterized. Four weeks after initiation of tamoxifen administration, mice weighing 20 to 25 g were habituated to tail-cuff plethysmography over a 2-d period. Thereafter, repeated BP measurements (at least 10 per session per mouse) were obtained over 3 d, during which the arterial pulse was detected by a piezoelectric detector. Signal acquisition and processing were performed by PowerLab 4sp instruments using Chart 4.1.1 (ADInstruments, Spechbach, Germany). Kidneys fixed in alco- holic Bouin solution were paraffin-embedded, and 3-␮-thick sections were stained with periodic acid-Schiff and Masson-Trichrome.

Protein and RNA Analysis See supplemental data for detailed protocols on Western blotting, immunofluorescence, real-time PCR, and microarray analysis.

Figure 7. Cell proliferation in resident glomerular cells with Transmission and Immunogold Electron Microscopy progressive glomerulosclerosis is shown. (A) Quantification of Methods for tissue preparation for electron microscopy may be found Ki-67–positive cells in 10 glomeruli each in two control and three in supplemental data. Electron micrographs were obtained using a null mice at each time point showed an increase in proliferating Jeol JEM-100CX II transmission electron microscope. Eight to 10 cells as early as week 1 and progressing until week 4, with a photographs, covering at least 15 open random capillary loops in subsequent decrease at 6 wk. $P Ͻ 0.01; *P Ͻ 0.0001. (B) Immu- several glomeruli per mouse were taken; negatives were digitized, and lox2/Ϫ ϩ nofluorescence labeling in a representative Nphs2 ,Cre images with a final magnification of ϫ15,000 were obtained. With the mouse after 4 wk demonstrated proliferation of all resident cells in use of ImageJ software (National Institutes of Health; http://rsbweb.nih. the glomerulus, including podocytes (labeled with anti-nephrin gov/ij/features.html), the length of the peripheral GBM was measured; Ab, red), mesangial cells (labeled with anti-desmin Ab, red), and the number of slit pores overlying this GBM length was then manually endothelial cells (labeled with anti-CD31 Ab, red). Proliferating counted. The average foot process width, expressed in microns, was cal- cells were labeled with anti–Ki-67 Ab (green) and nuclei with Topro 3 (blue). Magnification, ϫ400. culated by dividing the total GBM length measured in the single glomer- ulus by the total number of slits counted. The value obtained was multi- ␲ sion of exon 2 of the Nphs2 gene upon tamoxifen administration. See plied by /4, a correction factor for the random orientation in which the supplemental data for further details regarding construct generation foot processes were sectioned.50 and mating scheme. Except for studies on neonatal mice, experiments were performed on 6-wk-old male and female triallellic Nphs2lox2/Ϫ, Statistical Analysis ϩ ϩ ϩ ϩ Ϯ Cre mice and their littermate controls: Nphs2lox2/ ,Cre , Nphs2lox2/ , All data are presented as means SEM and compared using two- CreϪ, or Nphs2lox2/Ϫ,CreϪ. No gender effects were seen. Mice were of tailed t test or Mann-Whitney test for independent samples, using Ͻ mixed genetic background. Cre recombinase was induced by intra- GraphPad Prism. P 0.05 was considered statistically significant. peritoneal administration of tamoxifen (33 mg/kg per d for 5 d; Sigma, Steinheim, Germany). In nursing mothers, 5 mg of tamoxifen was administered intraperitoneally during the first 3 d after delivery. ACKNOWLEDGMENTS Mice were maintained in a pathogen-free environment, and experi- ments were conducted in accordance with French government poli- This work was supported by grants from the EuReGene Network, an cies (Services Ve´te´rinaires de la Sante´et de la Production Animale, integrated project (5085) of the 6th Framework of the European Com- Ministe`re de l’Agriculture). mission (C.A.), GIS-Institut des Maladies Rares (C.A.), Agence Nationale

J Am Soc Nephrol 20: 2181–2189, 2009 Conditional Podocin Loss and FSGS 2187 BASIC RESEARCH www.jasn.org de la Recherche (C.A. and E.L.E.), and Genzyme Renal Innovations Pro- Daskalakis N, Kwan SY, Ebersviller S, Burchette JL, Pericak-Vance MA, gram (C.A. and E.L.E.). E.L.E. received a National Institutes of Health/ Howell DN, Vance JM, Rosenberg PB: A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science National Institute of Diabetes and Digestive and Kidney Diseases Ruth 308: 1801–1804, 2005 Kirchstein National Research Service Award (DK065409). In addition, 9. Kim JM, Wu H, Green G, Winkler CA, Kopp JB, Miner JH, Unanue ER, grants from the Ministe`re de l’Enseignement Supe´rieur et de la Re- Shaw AS: CD2-associated protein haploinsufficiency is linked to glo- cherche and the Fondation de la Recherche Me´dicale provided PhD merular disease susceptibility. Science 300: 1298–1300, 2003 thesis and master’s thesis support to J.R. and O.B., respectively. The 10. Kopp JB, Smith MW, Nelson GW, Johnson RC, Freedman BI, Bowden DW, Oleksyk T, McKenzie LM, Kajiyama H, Ahuja TS, Berns JS, Briggs mutant mouse line was established at the Mouse Clinical Institute W, Cho ME, Dart RA, Kimmel PL, Korbet SM, Michel DM, Mokrzycki (Institut Clinique de la Souris, Illkirch, France) in the Targeted MH, Schelling JR, Simon E, Trachtman H, Vlahov D, Winkler CA: MYH9 Mutagenesis and Transgenesis Department. is a major-effect risk gene for focal segmental glomerulosclerosis. Nat We thank Viviane Beau at INSERM U574 and Nicholas Sorhaindo Genet 40: 1175–1184, 2008 and Martine Muffat-Joly at Hoˆpital Bichat (IFR2-Laboratoire de Bio- 11. Machuca E, Hummel A, Nevo F, Dantal J, Martinez F, Al-Sabban E, Baudouin V, Abel L, Grunfeld JP, Antignac C: Clinical and epidemio- chimie, Paris, France) for mouse genotyping and biochemical, and BP logical assessment of steroid-resistant nephrotic syndrome associated measurements; Me´riem Garfa (Plateau d’imagerie cellulaire, IRNEM, with the NPHS2 R229Q variant. Kidney Int 75: 727–735, 2009 Paris, France) for confocal microscopy and Metamorph analysis; and 12. McKenzie LM, Hendrickson SL, Briggs WA, Dart RA, Korbet SM, Mokr- members of the Animal Facility at Hoˆpital Necker/IRNEM, Paris, for zycki MH, Kimmel PL, Ahuja TS, Berns JS, Simon EE, Smith MC, Tracht- the care and maintenance of the mice used for this study. man H, Michel DM, Schelling JR, Cho M, Zhou YC, Binns-Roemer E, Kirk GD, Kopp JB, Winkler CA: NPHS2 variation in sporadic focal segmental glomerulosclerosis. J Am Soc Nephrol 18: 2987–2995, 2007 13. 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2188 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 2181–2189, 2009 www.jasn.org BASIC RESEARCH

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Kuusniemi AM, Merenmies J, Lahdenkari AT, Holmberg C, Salmela K, Machida J, Sakamoto T, Endo F, Hattori S: In situ evaluation of Karikoski R, Rapola J, Jalanko H: Glomerular sclerosis in kidneys with podocin in normal and glomerular diseases. Kidney Int 64: 2092– congenital nephrotic syndrome (NPHS1). Kidney Int 70: 1423–1431, 2006 2099, 2003 42. Philippe A, Nevo F, Esquivel EL, Reklaityte D, Gribouval O, Tete MJ, 28. Schmid H, Cohen CD, Henger A, Schlondorff D, Kretzler M: Gene Loirat C, Dantal J, Fischbach M, Pouteil-Noble C, Decramer S, Hoehne expression analysis in renal biopsies. Nephrol Dial Transplant 19: M, Benzing T, Charbit M, Niaudet P, Antignac C: Nephrin mutations 1347–1351, 2004 can cause childhood-onset steroid-resistant nephrotic syndrome. 29. Koop K, Eikmans M, Baelde HJ, Kawachi H, De Heer E, Paul LC, Bruijn J Am Soc Nephrol 19: 1871–1878, 2008 JA: Expression of podocyte-associated molecules in acquired human 43. 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Shi S, Yu L, Chiu C, Sun Y, Chen J, Khitrov G, Merkenschlager M, ages podocytes: Autonomous vicious cycle that drives local spread of Holzman LB, Zhang W, Mundel P, Bottinger EP: Podocyte-selective glomerular sclerosis. Curr Opin Nephrol Hypertens 14: 205–210, 2005 deletion of dicer induces proteinuria and glomerulosclerosis. JAm 32. Huber TB, Simons M, Hartleben B, Sernetz L, Schmidts M, Gundlach E, Soc Nephrol 19: 2159–2169, 2008 Saleem MA, Walz G, Benzing T: Molecular basis of the functional 46. Asano T, Niimura F, Pastan I, Fogo AB, Ichikawa I, Matsusaka T: podocin-nephrin complex: Mutations in the NPHS2 gene disrupt Permanent genetic tagging of podocytes: Fate of injured podocytes in nephrin targeting to lipid raft microdomains. Hum Mol Genet 12: a mouse model of glomerular sclerosis. J Am Soc Nephrol 16: 2257– 3397–3405, 2003 2262, 2005 33. Roselli S, Moutkine I, Gribouval O, Benmerah A, Antignac C: Plasma 47. Harvey SJ, Jarad G, Cunningham J, Goldberg S, Schermer B, Harfe membrane targeting of podocin through the classical exocytic path- BD, McManus MT, Benzing T, Miner JH: Podocyte-specific deletion of way: Effect of NPHS2 mutations. Traffic 5: 37–44, 2004 dicer alters cytoskeletal dynamics and causes glomerular disease. 34. Christensen EI, Birn H, Rippe B, Maunsbach AB: Controversies in J Am Soc Nephrol 19: 2150–2158, 2008 nephrology: Renal albumin handling, facts, and artifacts! Kidney Int 48. Zuo Y, Matsusaka T, Zhong J, Ma J, Ma LJ, Hanna Z, Jolicoeur P, Fogo 72: 1192–1194, 2007 AB, Ichikawa I: HIV-1 genes vpr and nef synergistically damage podocytes, 35. Kalluri R: Proteinuria with and without renal glomerular podocyte leading to glomerulosclerosis. J Am Soc Nephrol 17: 2832–2843, 2006 effacement. J Am Soc Nephrol 17: 2383–2389, 2006 49. Barisoni L, Kriz W, Mundel P, D’Agati V: The dysregulated podocyte 36. Lahdenkari AT, Lounatmaa K, Patrakka J, Holmberg C, Wartiovaara J, phenotype: A novel concept in the pathogenesis of collapsing idio- Kestila M, Koskimies O, Jalanko H: Podocytes are firmly attached to pathic focal segmental glomerulosclerosis and HIV-associated ne- glomerular basement membrane in kidneys with heavy proteinuria. phropathy. J Am Soc Nephrol 10: 51–61, 1999 J Am Soc Nephrol 15: 2611–2618, 2004 50. Gundersen HJ, Seefeldt T, Osterby R: Glomerular epithelial foot pro- 37. Ryan GB, Karnovsky MJ: An ultrastructural study of the mechanisms of cesses in normal man and rats: Distribution of true width and its intra- proteinuria in aminonucleoside nephrosis. Kidney Int 8: 219–232, 1975 and inter-individual variation. Cell Tissue Res 205: 147–155, 1980 38. Ruf RG, Lichtenberger A, Karle SM, Haas JP, Anacleto FE, Schultheiss M, Zalewski I, Imm A, Ruf EM, Mucha B, Bagga A, Neuhaus T, Fuchshuber A, Bakkaloglu A, Hildebrandt F: Patients with mutations in NPHS2 (podocin) do not respond to standard steroid treatment of See related editorial, “Surprising Results following Conditional Podocyte Inac- tivation,” on pages 2086–2088. nephrotic syndrome. J Am Soc Nephrol 15: 722–732, 2004 39. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Supplemental information for this article is available online at http://www. Kikkawa Y, Miner JH, Quaggin SE: Glomerular-specific alterations of jasn.org/.

J Am Soc Nephrol 20: 2181–2189, 2009 Conditional Podocin Loss and FSGS 2189

CONDITIONAL PODOCIN INACTIVATION SUPPLEMENTARY DATA

SUPPLEMENTARY FIGURES AND TABLES: Supplementary Figure S1. Generation of floxed Nphs2 exon 2 allele and confirmation

of gene targeting. A. A 7.7-kb targeting vector consisting of a loxP site in intron 1-2 and a loxP-Frt-Neo-Frt cassette in intron 2-3 was generated by PCR and inserted into an MCI proprietary vector. The linearized vector was electroporated into ES cells. B. Screening of 400 ES cell clones by Southern blot hybridization, using a Neo probe and a 3' probe, revealed two correctly targeted clones 121 and 130.

A

Target vector 1 2 NEO

5’ end 3’ end 3’ probe Wild-type allele 1 2 3 9.2 kb Xmn I Xmn I

5’ end neo probe Floxed allele 1 2 NEO 3 5 kb Xmn I Xmn I 7.8 kb Nde I Nde I

B Neo probe, NdeI 3’ probe, XmnI WT 121 130 121 130 MW MW

- 1 -

Supplementary Figure S2. Generation of Nphs2lox2/-,Cre+ mice and excision of exon 2

upon Cre recombinase induction. A. Triallelic Nphs2lox2/-,Cre+ mice were obtained by mating phenotypically normal Nphs2lox2/lox2 mice with Nphs2+/-,Cre+ mice. Mendelian

inheritance of these alleles was observed. B. Genotypes were verified by multiplex PCR

of tail genomic DNA. C. Cre recombinase activity was induced upon tamoxifen

administration, leading to excision of the floxed exon 2 of the Nphs2 gene. D. Cre

recombinase activity in the kidney was verified by PCR using a set of forward and

reverse primers designed around exon 2 and demonstrating a 692-bp product (before Cre)

and a 316-bp product (after Cre) using genomic DNA extracted from the renal cortex.

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A B

C D

F R

FR

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Supplementary Figure S3. Significant correlation (r = 0.827, p<0.0001) between the fold-change in expression levels obtained from the microarray experiment and quantitative real-time PCR of 15 genes at weeks 1, 2, and 4 (n = 45).

10 8 6 4 2

-10 -8 -6 -4 -2 -2 2 4 6 8 10 -4 -6 qRT-PCR fold-change -8 -10

Microarray fold-change

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Supplementary Table S1. Primers and probe sets used for the verification of microarray data using quantitative real-time PCR.

Applied Biosystems Gene Gene product Reference 18S 18S ribosomal RNA Hs99999901_s1

Adm Adrenomedullin Mm00437438_g1

Dock1 Dedicator of cytokinesis 1 Mm01269874_m1

Frk Fyn-related kinase Mm00456656_m1

Gas2l3 Growth arrest-specific 2 like 3 Mm02600495_m1

Mitogen-activated protein kinase-activated protein Mapkapk3 Mm00628676_m1 kinase 3

Nphs1 Nephrin Mm00497828_m1

Nphs2 Podocin Mm00499928_m1

Pbk PDZ binding kinase Mm00517793_m1

Ptpro Protein tyrosine , receptor type, O Mm00478480_m1

Smad3 SMAD homolog 3 Mm00489641_m1

Smurf1 SMAD specific E3 ubiquitin protein 1 Mm00547102_m1

Tgfb1 Transforming growth factor-beta 1 Mm03024053_m1

Tgfb3 Transforming growth factor-beta 3 Mm01307959_m1

Tgfbr1 Transforming growth factor-beta receptor, type 1 Mm03024015_m1

Tgfbr2 Transforming growth factor-beta receptor, type 2 Mm03024091_m1

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Supplementary Table S2. Global gene expression profiling, using microarrays, at 1, 2 and 4 weeks after podocin inactivation, in isolated glomeruli of Nphs2lox2/-,Cre+ mice, as compared to control mice. Using thresholds of a t-test p value < 0.001 and at least ± 1.5 fold-change in expression, we found significant perturbations in 42, 62 and 738 annotated and non-annotated genes at weeks 1, 2 and 4, respectively.

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WEEK 1

Probesets Symbol Title Fold-change p 1421244_at Esr1 1 (alpha) 93,81 1,97E-05 1452242_at Cep55 centrosomal protein 55 7,17 1,08E-04 1426817_at Mki67 antigen identified by monoclonal antibody Ki 67 6,35 4,77E-04 1454694_a_at Top2a topoisomerase (DNA) II alpha 6,11 6,40E-04 1434815_a_at Mapkapk3 mitogen-activated protein kinase-activated protein kinase 3 5,75 4,49E-06 1448226_at Rrm2 ribonucleotide reductase M2 5,15 1,94E-04 1450920_at Ccnb2 cyclin B2 4,87 3,06E-04 1448627_s_at Pbk PDZ binding kinase 4,85 2,34E-04 1428852_at Dock3 dedicator of cyto-kinesis 3 4,76 8,54E-04 1424278_a_at Birc5 baculoviral IAP repeat-containing 5 4,59 5,49E-04 1416802_a_at Cdca5 cell division cycle associated 5 4,40 7,20E-05 1423775_s_at Prc1 protein regulator of cytokinesis 1 4,08 1,86E-04 1437716_x_at Kif22 kinesin family member 22 3,85 6,94E-04 1436847_s_at Cdca8 cell division cycle associated 8 3,80 7,45E-04 1439377_x_at Cdc20 cell division cycle 20 homolog 3,26 6,12E-04 1453416_at Gas2l3 growth arrest-specific 2 like 3 3,08 3,05E-04 1419153_at 2810417H13Rik RIKEN cDNA 2810417H13 gene 2,63 6,15E-04 1455990_at Kif23 kinesin family member 23 2,40 1,71E-05 1449171_at Ttk Ttk protein kinase 2,37 5,67E-04 1437370_at Sgol2 shugoshin-like 2 2,35 7,32E-04 1422430_at Fignl1 fidgetin-like 1 2,30 4,31E-04 1436707_x_at Ncaph non-SMC condensin I complex, subunit H 2,27 8,90E-04 1418281_at Rad51 RAD51 homolog 2,25 7,52E-04 1422944_a_at Diap3 diaphanous homolog 3 2,13 5,34E-04 1452040_a_at Cdca3 cell division cycle associated 3 2,05 1,32E-04 1426858_at Inhbb inhibin beta-B 2,00 2,27E-04 1416698_a_at Cks1b CDC28 protein kinase 1b 2,00 7,31E-04 1417445_at Kntc2 kinetochore associated 2 1,94 4,61E-04 1429295_s_at Trip13 interactor 13 1,88 1,23E-04 1431087_at Spbc24 spindle pole body component 24 homolog 1,86 7,90E-04 1438434_at Arhgap11a Rho GTPase activating protein 11A 1,85 8,77E-04 1437580_s_at Nek2 NIMA (never in mitosis gene a)-related expressed kinase 2 1,78 5,34E-04 1416431_at Tubb6 tubulin, beta 6 1,76 3,83E-04 1452305_s_at Cenpn-201 protein N Gene 1,75 5,13E-04 1432417_a_at Tspan2 tetraspanin 2 1,70 1,32E-04 1435773_at 4930547N16Rik RIKEN cDNA 4930547N16 gene 1,55 6,34E-04 1424766_at Ercc6l excision repair cross-complementing rodent repair deficiency, complementation group 6-like 1,55 8,57E-04 1458560_at Aspm asp (abnormal spindle)-like, microcephaly associated 1,53 3,20E-04 1418369_at Prim1 DNA primase, p49 subunit 1,52 3,14E-04 1448878_at Mxd3 Max dimerization protein 3 1,52 7,49E-04 1419838_s_at Plk4 polo-like kinase 4 1,51 2,79E-04

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WEEK 2

Probesets Symbol Title Fold-change p UPREGULATED 1421244_at Esr1 estrogen receptor 1 (alpha) 90,40 1,46E-06 1438009_at Hist1h2ao histone cluster 1, H2ao gene 7,93 8,16E-04 1416077_at Adm adrenomedullin 3,69 4,99E-04 1434815_a_at Mapkapk3 mitogen-activated protein kinase-activated protein kinase 3 3,50 1,04E-04 1429637_at 2210419I08Rik RIKEN cDNA 2210419I08 gene 2,63 4,62E-04 1416805_at 1110032E23Rik RIKEN cDNA 1110032E23 gene 2,45 3,42E-04 1422128_at Rpl14 ribosomal protein L14 2,44 4,78E-05 1428852_at Dock3 dedicator of cyto-kinesis 3 2,42 1,05E-04 1460601_at Myrip myosin VIIA and Rab interacting protein 2,37 2,82E-04 1448698_at Ccnd1 cyclin D1 1,98 1,03E-04 1418780_at Cyp39a1 cytochrome P450, family 39, subfamily a, polypeptide 1 1,96 6,95E-04 1459860_x_at Trim2 tripartite motif protein 2 1,93 8,49E-06 1454613_at Dpysl3 dihydropyrimidinase-like 3 1,84 9,93E-04 1433509_s_at Reep1 receptor accessory protein 1 1,80 1,89E-05 1431777_a_at Hmgn3 high mobility group nucleosomal binding domain 3 1,78 9,24E-04 1428713_s_at Gins2 GINS complex subunit 2 1,73 1,01E-03 1416205_at Glb1 galactosidase, beta 1 1,69 6,16E-04 1450455_s_at Akr1c12 aldo-keto reductase family 1, member C12 1,65 4,40E-04 1447787_x_at Gja7 gap junction membrane channel protein alpha 7 1,63 5,03E-04 1417719_at Sap30 sin3 associated polypeptide 1,58 1,99E-04 1439887_at Rnf152 ring finger protein 152 1,56 4,98E-04 1426569_a_at Frk fyn-related kinase 1,56 9,10E-04 1426652_at Mcm3 minichromosome maintenance deficient 3 1,55 5,76E-04 1449577_x_at Tpm2 tropomyosin 2, beta 1,54 5,30E-04 1439514_at Cks1b CDC28 protein kinase 1b 1,52 4,13E-04 1423488_at Mmd monocyte to macrophage differentiation-associated 1,52 4,54E-04 1436448_a_at Ptgs1 prostaglandin-endoperoxide synthase 1 1,51 1,62E-04 DOWNREGULATED 1440854_at 2810403A07Rik -1,50 2,71E-04 1436871_at Sfrs7 splicing factor, arginine/serine-rich 7 -1,51 3,30E-04 1455092_at Zfp207 protein 207 -1,52 9,75E-04 1456659_at Sytl3 synaptotagmin-like 3 -1,52 8,05E-04 1419499_at Gpam glycerol-3-phosphate acyltransferase, mitochondrial -1,54 9,19E-04 1430152_at Eps15 epidermal growth factor receptor pathway substrate 15 -1,55 3,04E-04 1457771_at Ptpro Protein tyrosine phosphatase, receptor type, O -1,56 3,69E-04 1457302_at Slc20a2 Solute carrier family 20, member 2 -1,58 8,06E-04 1443088_at Sdccag10 serologically defined colon cancer antigen 10 -1,60 4,44E-04 1437181_at Peli2 pellino 2 -1,62 7,98E-04 1456956_at Ikzf2 IKAROS family zinc finger 2 -1,62 2,11E-04 1459381_at Dach1 Dachshund 1 -1,64 5,73E-04 1439769_at Zfp644 Zinc finger protein 644 -1,65 9,85E-04 1415822_at Scd2 stearoyl-Coenzyme A desaturase 2 -1,65 3,36E-04 1440621_at Kitl Kit ligand -1,68 3,70E-04 1452815_at P2ry10 purinergic receptor P2Y, G-protein coupled 10 -1,69 8,50E-04 1438838_at B230206F22Rik RIKEN cDNA B230206F22 gene -1,82 3,29E-04 1443184_at Cdc14a CDC14 cell division cycle 14 homolog A -1,94 6,38E-04 1451702_at Cmtm7 CKLF-like MARVEL transmembrane domain containing 7 -2,01 1,13E-04 1437605_at Nphs2 nephrosis 2 homolog, podocin -2,09 8,76E-04 1429144_at Prei4 preimplantation protein 4 -2,13 2,98E-05 1432352_at Colec12 collectin sub-family member 12 -2,26 7,41E-04

- 8 -

1438295_at Glcci1 Glucocorticoid induced transcript 1 -2,28 9,93E-04 1429951_at Ssbp2 single-stranded DNA binding protein 2 -3,63 2,17E-04

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WEEK 4

Probesets Symbol Title Fold-change p UPREGULATED 1421244_at Esr1 estrogen receptor 1 (alpha) 58,98 5,87E-06 1426547_at Gc group specific component 12,06 2,46E-04 1431379_a_at Slc13a1 solute carrier family 13 (sodium/sulphate symporters), member 1 9,41 1,49E-04 1448541_at Kns2 kinesin 2 8,29 1,59E-04 1421239_at Il6st interleukin 6 signal transducer 8,00 1,94E-04 1431385_a_at Mbtps1 membrane-bound peptidase, site 1 7,67 3,43E-04 1434366_x_at C1qb complement component 1, q subcomponent, beta polypeptide 7,49 3,50E-04 1425810_a_at Csrp1 cysteine and glycine-rich protein 1 6,76 1,28E-04 1449067_at Slc2a2 solute carrier family 2 (facilitated glucose transporter), member 2 6,60 2,56E-04 1417752_at Coro1c coronin, actin binding protein 1C 6,57 1,22E-04 1449416_at Fzd4 frizzled homolog 4 6,35 3,76E-05 1452638_s_at Dnm1l 1-like 6,30 1,81E-04 1450473_at Pcdh12 protocadherin 12 6,21 3,53E-04 1460661_at Edg3 endothelial differentiation, sphingolipid G-protein-coupled receptor 3 6,21 1,63E-04 1418614_at Kcnj1 potassium inwardly-rectifying channel, subfamily J, member 1 6,18 7,66E-05 1452242_at Cep55 centrosomal protein 55 6,02 3,56E-04 1431030_a_at Rnf14 ring finger protein 14 5,92 4,68E-04 1420924_at Timp2 tissue inhibitor of 2 5,88 3,01E-04 1419157_at Sox4 SRY-box containing gene 4 5,72 5,69E-04 1439109_at Ccdc68 coiled-coil domain containing 68 5,55 3,66E-04 1449551_at Myo1c myosin IC 5,42 1,47E-04 1419378_a_at Fxyd2 FXYD domain-containing ion transport regulator 2 5,35 6,62E-04 1431261_at Clic5 chloride intracellular channel 5 5,26 7,05E-04 1421866_at Nr3c1 subfamily 3, group C, member 1 5,18 6,01E-04 1450379_at Msn moesin 5,18 9,32E-04 1416190_a_at Sec61a1 Sec61 alpha 1 subunit 5,14 1,42E-04 1422606_at C1qtnf3 C1q and tumor necrosis factor related protein 3 5,02 6,67E-05 1424278_a_at Birc5 baculoviral IAP repeat-containing 5 5,01 6,89E-04 1451927_a_at Mapk14 mitogen activated protein kinase 14 4,97 5,86E-04 1433492_at Epb4.1l2 erythrocyte protein band 4.1-like 2 4,88 1,68E-04 1420948_s_at Atrx alpha thalassemia/mental retardation syndrome X-linked homolog 4,85 2,81E-04 1447513_at Kcnd3 potassium voltage-gated channel, Shal-related family, member 3 4,84 2,04E-04 1447252_s_at Mep1a meprin 1 alpha 4,65 9,99E-04 1448964_at S100g S100 calcium binding protein G 4,64 4,91E-04 1419099_x_at Stom 4,56 3,30E-04 1421991_a_at Igfbp4 insulin-like growth factor binding protein 4 4,56 4,35E-04 1422856_at Slc12a3 solute carrier family 12, member 3 4,55 9,72E-04 1449549_at Efnb2 ephrin B2 4,53 3,98E-04 1438661_a_at Arf2 ADP-ribosylation factor 2 4,53 9,40E-05 1448207_at Lasp1 LIM and SH3 protein 1 4,53 4,14E-04 1432344_a_at Aplp2 amyloid beta (A4) precursor-like protein 2 4,50 1,67E-04 1422341_s_at Lypla3 lysophospholipase 3 4,46 2,55E-04 1415893_at Sgpl1 sphingosine phosphate 1 4,41 7,09E-05 1417033_at Ube2g2 ubiquitin-conjugating E2G 2 4,39 6,09E-05 1419573_a_at Lgals1 lectin, galactose binding, soluble 1 4,38 5,00E-04 1416601_a_at Dscr1 Down syndrome critical region homolog 4,37 8,58E-04 1436847_s_at Cdca8 cell division cycle associated 8 4,36 3,77E-04 1425711_a_at Akt1 thymoma viral proto-oncogene 1 4,33 8,72E-04 1417396_at Podxl podocalyxin-like 4,28 2,51E-04 1421832_at Twsg1 twisted gastrulation homolog 1 4,23 9,95E-04

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1419679_at Lats2 large tumor suppressor 2 4,20 3,63E-04 1432646_a_at AC153583.10 Uncharacterized protein 4,19 2,01E-04 1416572_at Mmp14 matrix metallopeptidase 14 (membrane-inserted) 4,19 9,54E-05 1425444_a_at Tgfbr2 transforming growth factor, beta receptor II 4,17 2,04E-04 1454106_a_at Cxxc1 CXXC finger 1 (PHD domain) 4,12 2,52E-04 1421890_at St3gal2 ST3 beta-galactoside alpha-2,3-sialyltransferase 2 4,10 9,97E-06 1453960_a_at Capzb capping protein (actin filament) muscle Z-line, beta 4,05 6,56E-05 1418453_a_at Atp1b1 ATPase, Na+/K+ transporting, beta 1 polypeptide 4,01 7,46E-04 1421026_at Gna12 guanine nucleotide binding protein, alpha 12 4,00 1,29E-04 1437932_a_at Cldn1 claudin 1 3,99 1,01E-03 1422512_a_at Ogfr opioid growth factor receptor 3,99 5,06E-09 1422959_s_at Zfp313 zinc finger protein 313 3,95 4,40E-05 1426085_a_at Pxn paxillin 3,91 3,00E-05 1421198_at Itgav integrin alpha V 3,90 2,46E-04 1422017_s_at 4833439L19Rik RIKEN cDNA 4833439L19 gene 3,90 6,06E-04 1460191_at Ykt6 YKT6 homolog 3,88 1,65E-05 1417025_at H2-Eb1 histocompatibility 2, class II antigen E beta 3,84 5,78E-04 1450920_at Ccnb2 cyclin B2 3,83 5,90E-04 1451843_a_at Ggta1 glycoprotein galactosyltransferase alpha 1, 3 3,81 6,54E-05 1452534_a_at Hmgb2 high mobility group box 2 3,79 1,21E-04 1420975_at Baz1b bromodomain adjacent to zinc finger domain, 1B 3,77 1,71E-04 1422862_at Pdlim5 PDZ and LIM domain protein 5 3,76 3,20E-04 1439151_at Msrb3 methionine sulfoxide reductase B3 3,76 6,40E-04 1421415_s_at Gcnt2 glucosaminyl (N-acetyl) 2, I-branching enzyme 3,75 1,08E-04 1423049_a_at Tpm1 tropomyosin 1, alpha 3,74 1,85E-04 1438860_a_at Slc44a2 solute carrier family 44, member 2 3,72 8,06E-05 1421313_s_at Cttn cortactin 3,71 3,32E-05 1415949_at Cpe carboxypeptidase E 3,69 3,42E-04 1455627_at Col8a1 procollagen, type VIII, alpha 1 3,64 1,97E-04 1424332_at Rab40c Rab40c, member RAS oncogene family 3,61 3,55E-06 1428850_x_at Cd99 CD99 antigen 3,60 1,57E-04 1425855_a_at Crk v-crk sarcoma virus CT10 oncogene homolog 3,59 6,99E-05 1426888_at Ehmt2 euchromatic histone lysine N-methyltransferase 2 3,53 9,65E-04 1439051_a_at Mark4 MAP/microtubule affinity-regulating kinase 4 3,53 1,51E-04 1430834_at Gprin3 GPRIN family member 3 3,51 6,89E-05 1450154_at Folh1 folate 3,51 2,03E-04 1417457_at Cks2 CDC28 protein kinase regulatory subunit 2 3,47 5,08E-04 1450846_at Bzw1 basic and W2 domains 1 3,47 4,82E-04 1448348_at Gpiap1 GPI-anchored membrane protein 1 3,36 4,30E-04 1438406_at Scarf2 scavenger receptor class F, member 2 3,32 7,05E-05 1437513_a_at Serinc1 serine incorporator 1 3,32 8,48E-05 1421321_a_at Net1 neuroepithelial cell transforming gene 1 3,30 1,76E-04 1425654_a_at Lima1 LIM domain and actin binding 1 3,25 6,29E-04 1448949_at Car4 carbonic anhydrase 4 3,23 6,18E-04 1437591_a_at Wdr1 WD repeat domain 1 3,21 3,40E-05 1419416_a_at Rarg , gamma 3,21 3,68E-04 1432436_a_at Ak3 adenylate kinase 3 3,19 4,27E-04 1441968_at Tspan9 tetraspanin 9 3,18 3,39E-05 1423505_at Tagln transgelin 3,18 3,45E-05 1416704_at Mapk14 mitogen activated protein kinase 14 3,17 1,13E-04 1420610_at Prkacb protein kinase, cAMP dependent, catalytic, beta 3,15 7,64E-04 1426191_a_at Bcl2l1 Bcl2-like 1 3,14 4,59E-04 1433515_s_at Etnk1 ethanolamine kinase 1 3,14 3,36E-05 1421215_a_at Slmap sarcolemma associated protein 3,12 2,76E-04 1430172_a_at Cyp4f16 cytochrome P450, family 4, subfamily f, polypeptide 16 3,06 5,81E-04

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1422853_at Shc1 src homology 2 domain-containing transforming protein C1 3,05 6,71E-05 1431196_at Atp2c1 ATPase, Ca++-sequestering 3,03 1,59E-04 1432466_a_at Apoe apolipoprotein E 3,03 7,56E-04 1422313_a_at Igfbp5 insulin-like growth factor binding protein 5 3,02 3,74E-04 1447222_at Hspa12a heat shock protein 12A 3,02 5,35E-04 1426538_a_at Trp53 transformation related protein 53 3,01 4,24E-04 1422974_at Nt5e 5' , ecto 3,00 8,36E-04 1424303_at Depdc7 DEP domain containing 7 2,97 6,65E-04 1417229_at Capn1 calpain 1 2,96 8,71E-05 1454184_a_at Ikbkb inhibitor of kappaB kinase beta 2,96 1,13E-05 1417613_at Ier5 immediate early response 5 2,95 4,57E-04 1425170_a_at Adam15 a and metallopeptidase domain 15 (metargidin) 2,95 7,52E-04 1430634_a_at Pfkp phosphofructokinase, platelet 2,93 5,21E-05 1422249_s_at Zfa; Zfx zinc finger protein, autosomal; zinc finger protein X-linked 2,92 4,76E-04 1420651_at Ate1 arginine-tRNA-protein transferase 1 2,92 4,43E-05 1426849_at Sec24b SEC24 related gene family, member B 2,91 8,65E-05 1425368_a_at Numb numb gene homolog 2,91 3,61E-05 1437772_s_at Fuca1 fucosidase, alpha-L- 1, tissue 2,90 6,89E-04 1431464_a_at Pmm2 phosphomannomutase 2 2,89 8,45E-04 1424307_at Arhgap1 Rho GTPase activating protein 1 2,87 1,70E-04 1431061_s_at Peli1 pellino 1 2,86 7,91E-04 1415989_at Vcam1 vascular cell adhesion molecule 1 2,86 7,10E-04 1453054_at Scamp1 secretory carrier membrane protein 1 2,85 6,24E-04 1432262_at 4930504E06Rik RIKEN cDNA 4930504E06 gene 2,83 3,53E-04 1448458_at Top2b topoisomerase (DNA) II beta 2,83 4,44E-04 1450124_a_at Atp2a3 ATPase, Ca++ transporting, ubiquitous 2,82 2,73E-04 1430332_a_at Gusb glucuronidase, beta 2,80 7,50E-05 1417588_at Galnt3 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 2,80 4,37E-05 1425621_at Trim35 tripartite motif-containing 35 2,80 1,00E-03 1424382_at Rcn3 reticulocalbin 3, EF-hand calcium binding domain 2,80 3,50E-05 1429246_a_at Anxa6 annexin A6 2,79 2,39E-05 1450378_at Tapbp TAP binding protein 2,79 3,00E-04 1451072_a_at Rnf4 ring finger protein 4 2,78 7,98E-04 1427967_at Srgap2 SLIT-ROBO Rho GTPase 2,78 8,52E-04 1439214_a_at Api5 apoptosis inhibitor 5 2,78 3,81E-04 1415957_a_at Nnp1 novel nuclear protein 1 2,77 1,07E-04 1423308_at Tgoln1 trans-golgi network protein 2,76 1,75E-04 1436508_at 2410014A08Rik RIKEN cDNA 2410014A08 gene 2,74 6,75E-04 1423590_at Napsa napsin A aspartic peptidase 2,71 8,29E-04 1420646_at Nus1 nuclear undecaprenyl pyrophosphate synthase 1 homolog 2,70 3,14E-04 1424791_a_at Bcam basal cell adhesion molecule 2,70 8,52E-04 1421095_a_at Trpc1 transient receptor potential cation channel, subfamily C, member 1 2,69 3,59E-04 1429029_at Sgms2 sphingomyelin synthase 2 2,68 1,85E-04 1427844_a_at Cebpb CCAAT/enhancer binding protein (C/EBP), beta 2,67 2,16E-04 1430692_a_at Sel1l sel-1 suppressor of lin-12-like 2,64 1,68E-05 1451358_a_at Racgap1 Rac GTPase-activating protein 1 2,63 3,11E-04 1424158_at Ehd2 similar to EH-domain containing 3 2,62 1,86E-05 1427414_at Prkar2a protein kinase, cAMP dependent regulatory, type II alpha 2,61 1,92E-04 1449405_at Tns1 tensin 1 2,60 4,34E-04 1455061_a_at Acaa2 acetyl-Coenzyme A acyltransferase 2 2,60 3,09E-04 1449207_a_at Kif20a kinesin family member 20A 2,59 9,49E-04 1450183_a_at Sh2b3 SH2B adaptor protein 3 2,57 3,12E-04 1431274_a_at Hspa9 heat shock protein 9 2,56 2,74E-04 1421860_at Clstn1 calsyntenin 1 2,55 7,88E-05 1448844_at Cyb5b cytochrome b5 type B 2,55 1,22E-04

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1416698_a_at Cks1b CDC28 protein kinase 1b 2,54 7,31E-04 1420954_a_at Add1 adducin 1 (alpha) 2,53 3,26E-05 1423392_at Clic4 chloride intracellular channel 4 (mitochondrial) 2,52 1,11E-04 1436482_a_at Sdc3 syndecan 3 2,52 2,00E-05 1423048_a_at Tollip toll interacting protein 2,51 9,32E-04 1431197_at Arl6ip2 ADP-ribosylation factor-like 6 interacting protein 2 2,51 3,40E-04 1421448_at Garnl1 GTPase activating RANGAP domain-like 1 2,51 7,20E-04 1416740_at Col5a1 procollagen, type V, alpha 1 2,51 7,15E-05 1427490_at Abcb7 ATP-binding cassette, sub-family B (MDR/TAP), member 7 2,50 4,67E-04 1448732_at Ctsb cathepsin B 2,50 2,71E-04 1456616_a_at Bsg basigin 2,50 1,44E-04 1419372_at Gosr2 golgi SNAP receptor complex member 2 2,50 2,67E-04 1434357_a_at Kpnb1 karyopherin (importin) beta 1 2,50 4,94E-04 1426348_at Col4a1 procollagen, type IV, alpha 1 2,49 9,45E-04 1460049_s_at 1500015O10Rik RIKEN cDNA 1500015O10 gene 2,49 2,06E-04 1453623_a_at Rad23a RAD23a homolog (S. cerevisiae) 2,48 2,93E-04 1434492_at A130022J15Rik RIKEN cDNA A130022J15 gene 2,48 6,16E-04 1422845_at Canx calnexin 2,48 4,38E-04 1449107_at Nudt4 nudix (nucleoside diphosphate linked moiety X)-type motif 4 2,47 5,42E-04 1425796_a_at Fgfr3 fibroblast growth factor receptor 3 2,45 8,00E-05 1450747_at Keap1 kelch-like ECH-associated protein 1 2,45 4,47E-04 1431645_a_at Gdi2 guanosine diphosphate (GDP) dissociation inhibitor 2 2,44 9,54E-04 1439827_at Adamts12 a disintegrin-like and metallopeptidase with motifs 12 2,44 7,62E-04 1427510_at Gnai1 guanine nucleotide binding protein, alpha inhibiting 1 2,44 9,11E-04 1424128_x_at Aurkb aurora kinase B 2,43 2,12E-04 1453736_s_at B230219D22Rik RIKEN cDNA B230219D22 gene 2,42 1,35E-04 1416454_s_at Acta2 actin, alpha 2, smooth muscle, aorta 2,41 2,86E-04 1450472_s_at Smad3 MAD homolog 3 2,41 2,62E-04 1453307_a_at Anapc5 anaphase-promoting complex subunit 5 2,41 1,56E-04 1416691_at Gtpbp2 GTP binding protein 2 2,41 1,07E-04 1422039_at Tnfrsf22 tumor necrosis factor receptor superfamily, member 22 2,40 3,50E-06 1424131_at Col6a3 procollagen, type VI, alpha 3 2,40 4,64E-04 1451109_a_at Nedd4 neural precursor cell expressed, developmentally down-regulated gene 4 2,39 5,38E-04 1426319_at Pdgfd platelet-derived growth factor, D polypeptide 2,38 9,36E-04 1452496_at Atp11c Atpase, class VI, type 11C 2,38 3,03E-04 1421954_at Crkl v-crk sarcoma virus CT10 oncogene homolog (avian)-like 2,37 7,77E-04 1422969_s_at Ihpk1 inositol hexaphosphate kinase 1 2,37 6,59E-05 1427567_a_at Tpm3 tropomyosin 3, gamma 2,37 1,85E-04 1419191_at Hipk3 homeodomain interacting protein kinase 3 2,37 4,94E-04 1416130_at Prnp prion protein 2,36 7,65E-04 1460279_a_at Gtf2 general transcription factor II I 2,36 1,72E-04 1421848_at Slc22a5 solute carrier family 22 (organic cation transporter), member 5 2,36 8,75E-04 1421491_a_at Tmem49 transmembrane protein 49 2,35 7,84E-04 1416986_a_at Sirpa signal-regulatory protein alpha 2,35 9,62E-04 1456255_at Ecm29 Proteasome-associated protein ECM29 homolog 2,34 7,00E-04 1423588_at Arpc4 actin related protein 2/3 complex, subunit 4 2,34 8,83E-04 1436858_at Mbnl2 muscleblind-like 2 2,32 3,72E-04 1426399_at Vwa1 von Willebrand factor A domain containing 1 2,32 5,81E-04 1432272_a_at Ercc4 excision repair cross-complementing rodent repair deficiency, complementation group 4 2,32 4,12E-04 1448883_at Lgmn legumain 2,32 4,35E-04 1422763_at Gipc1 GIPC PDZ domain containing family, member 1 2,31 7,97E-04 1421063_s_at Snrpn small nuclear ribonucleoprotein N 2,31 4,21E-04 1452187_at Rbm5 RNA binding motif protein 5 2,31 2,68E-04 1450413_at Pdgfb platelet derived growth factor, B polypeptide 2,30 7,21E-04 1420684_at Acox3 acyl-Coenzyme A oxidase 3, pristanoyl 2,30 3,74E-04

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1431052_at Arhgap12 Rho GTPase activating protein 12 2,30 2,63E-04 1432269_a_at Sh3kbp1 SH3-domain kinase binding protein 1 2,30 8,30E-05 1450089_a_at Srprb signal recognition particle receptor, B subunit 2,29 1,56E-04 1448375_at Tm9sf3 transmembrane 9 superfamily member 3 2,28 8,73E-04 1422063_a_at Pex5 peroxisome biogenesis factor 5 2,28 3,94E-05 1449325_at Fads2 fatty acid desaturase 2 2,27 1,69E-04 1453757_at Herc5 hect domain and RLD 5 2,27 8,74E-04 1448127_at Rrm1 ribonucleotide reductase M1 2,27 5,61E-05 1450512_at Ntn4 netrin 4 2,26 9,91E-04 1450029_s_at Itga9 integrin alpha 9 2,26 6,51E-05 1419049_at Pcnx pecanex homolog 2,26 1,11E-05 1448332_at Pex19 peroxisome biogenesis factor 19 2,24 4,27E-04 1452893_s_at 2310040A07Rik RIKEN cDNA 1110065P19 gene; RIKEN cDNA 2310040A07 gene 2,24 2,61E-04 1435071_at Zfyve1 zinc finger, FYVE domain containing 1 2,23 3,67E-04 1426063_a_at Gem GTP binding protein (gene overexpressed in skeletal muscle) 2,23 8,75E-04 1423746_at Txndc5 thioredoxin domain containing 5 2,23 9,45E-04 1448605_at Rhoc ras homolog gene family, member C 2,23 4,85E-06 1427276_at Smc4 structural maintenance of 4 2,22 2,72E-04 1420867_at Tmed2 transmembrane emp24 domain trafficking protein 2 2,22 7,91E-04 1420812_at Hdac7a histone deacetylase 7A 2,22 1,92E-05 1437637_at Phtf2 putative homeodomain transcription factor 2 2,22 1,02E-04 1453605_s_at Ccdc91 coiled-coil domain containing 91 2,21 3,44E-04 1421940_at Stag1 stromal antigen 1 2,21 3,77E-04 1416818_at Parva parvin, alpha 2,20 4,26E-04 1418527_a_at Fusip1 FUS interacting protein (serine-arginine rich) 1 2,20 2,41E-04 1431102_at Cep350 centrosomal protein 350 2,19 4,00E-04 1425679_a_at Mapk8ip1 mitogen activated protein kinase 8 interacting protein 1 2,19 5,07E-05 1429564_at Pcgf5 polycomb group ring finger 5 2,19 2,58E-04 1450104_at Adam10 a disintegrin and metallopeptidase domain 10 2,19 7,25E-04 1423661_s_at Ctdsp2 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 2 2,18 1,11E-04 1460376_a_at Cox15 COX15 homolog, assembly protein 2,18 9,88E-04 1426004_a_at Tgm2 transglutaminase 2, C polypeptide 2,17 6,84E-05 1438196_at Gpd1l glycerol-3-phosphate dehydrogenase 1-like 2,17 3,68E-04 1453751_at Dhx38 DEAH (Asp-Glu-Ala-His) box polypeptide 38 2,17 4,91E-04 1420894_at Tgfbr1 transforming growth factor, beta receptor I 2,15 1,70E-04 1424511_at Aurka aurora kinase A 2,15 2,11E-04 1426024_a_at Dbn1 drebrin 1 2,14 8,41E-04 1434370_s_at Faf1 Fas-associated factor 1 2,14 6,10E-04 1460233_at Guca2b guanylate cyclase activator 2b (retina) 2,14 2,07E-05 1450767_at Nedd9 neural precursor cell expressed, developmentally down-regulated gene 9 2,12 6,34E-05 1453995_a_at Htf9c HpaII tiny fragments 9c 2,12 3,15E-04 1421936_at Dapp1 dual adaptor for phosphotyrosine and 3-phosphoinositides 1 2,12 7,73E-04 1421842_a_at Scamp4 secretory carrier membrane protein 4 2,11 4,30E-05 1424997_at Sfrs8 splicing factor, arginine/serine-rich 8 2,11 2,32E-04 1424513_at Atp6v0a2 ATPase, H+ transporting, lysosomal V0 subunit A2 2,11 2,41E-04 1421869_at Trim44 tripartite motif-containing 44 2,11 6,52E-04 1451743_at D19Wsu162e Outcome predictor in acute leukemia 1 homolog 2,10 4,53E-04 1419022_a_at Eno1 enolase 1, alpha non-neuron 2,10 4,16E-05 1425102_a_at Ace2 angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 2,09 9,62E-04 1433479_at Ubr7 ubiquitin protein ligase E3 component n-recognin 7 2,09 4,74E-05 1418452_at Gng2 guanine nucleotide binding protein (G protein), gamma 2 subunit 2,09 1,64E-04 1434767_at C79407 MKIAA1903 protein fragment 2,09 6,68E-04 1438361_at 2310035C23Rik RIKEN cDNA 2310035C23 gene 2,08 6,68E-04 1443991_at Dock1 dedicator of cytokinesis 1 2,08 1,52E-04 1432411_a_at Fbxw2 F-box and WD-40 domain protein 2 2,08 7,78E-04

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1425326_at Acly ATP citrate lyase 2,07 2,93E-04 1451353_at Tm6sf1 transmembrane 6 superfamily member 1 2,07 5,94E-05 1451782_a_at Slc29a1 solute carrier family 29 (nucleoside transporters), member 1 2,07 5,33E-04 1415976_a_at Carhsp1 calcium regulated heat stable protein 1 2,06 2,41E-04 1426764_at Oaz2 ornithine decarboxylase antizyme 2 2,06 6,98E-04 1451792_a_at Pja1 praja1, RING-H2 motif containing 2,06 5,47E-04 1422793_at Pafah1b2 platelet-activating factor acetylhydrolase, isoform 1b, alpha2 subunit 2,06 4,42E-04 1454721_at 1110018G07Rik RIKEN cDNA 1110018G07 gene 2,05 7,97E-05 1430289_a_at Wdr77 WD repeat domain 77 2,04 1,96E-04 1422879_at Sypl synaptophysin-like protein 2,04 8,24E-04 1419738_a_at Tpm2 tropomyosin 2, beta 2,03 1,23E-04 1417271_a_at Eng endoglin 2,03 3,76E-04 1423720_a_at Sar1a SAR1 gene homolog A (S. cerevisiae) 2,02 2,69E-04 1425840_a_at Sema3f semaphorin 3f 2,02 3,00E-04 1426923_at Hrb HIV-1 Rev binding protein 2,01 1,58E-04 1422491_a_at Bnip2 BCL2/adenovirus E1B interacting protein 1, NIP2 2,01 6,99E-04 1452457_a_at Prkab1 protein kinase, AMP-activated, beta 1 non-catalytic subunit 2,01 1,67E-05 1451064_a_at Psat1 phosphoserine aminotransferase 1 2,01 1,97E-05 1427447_a_at Triobp TRIO and F-actin binding protein 2,01 9,03E-05 1453946_a_at Sdccag8 serologically defined colon cancer antigen 8 2,01 1,55E-04 1431220_at 2810416G20Rik RIKEN cDNA 2810416G20 gene 2,00 2,47E-05 1425273_s_at Emp2 epithelial membrane protein 2 2,00 8,85E-04 1422535_at Ccne2 cyclin E2 2,00 6,04E-04 1422577_at Cs citrate synthase 2,00 9,53E-05 1421879_at Mtmr1 myotubularin related protein 1 2,00 6,55E-04 1416978_at Fcgrt Fc receptor, IgG, alpha chain transporter 1,99 2,93E-04 1417018_at Efemp2 EGF-containing fibulin-like extracellular matrix protein 2 1,99 4,38E-04 1423596_at Nek6 NIMA (never in mitosis gene a)-related expressed kinase 6 1,99 4,46E-04 1428209_at Bex4 brain expressed gene 4 1,99 1,47E-04 1453849_s_at Hnrpab heterogeneous nuclear ribonucleoprotein A/B 1,99 2,49E-04 1415773_at Ncl nucleolin 1,99 1,36E-04 1419385_a_at Ubqln1 ubiquilin 1 1,98 9,39E-04 1416437_a_at Mapk8ip3 mitogen-activated protein kinase 8 interacting protein 3 1,98 9,26E-04 1423603_at Zfpm1 zinc finger protein, multitype 1 1,97 7,42E-04 1448990_a_at Myo1b myosin IB 1,97 2,10E-04 1424443_at Hdgfrp3 hepatoma-derived growth factor, related protein 3 1,97 3,97E-06 1417401_at Rai14 retinoic acid induced 14 1,96 1,83E-04 1419451_at Fzr1 fizzy/cell division cycle 20 related 1 1,96 3,54E-05 1424453_at Pcyt1a phosphate cytidylyltransferase 1, choline, alpha isoform 1,96 6,47E-04 1431646_a_at Stx6 syntaxin 6 1,95 7,53E-05 1431285_at Mgrn1 mahogunin, ring finger 1 1,95 2,42E-05 1416805_at 1110032E23Rik RIKEN cDNA 1110032E23 gene 1,95 6,14E-04 1416388_at Pip5k2c phosphatidylinositol-4-phosphate 5-kinase, type II, gamma 1,95 7,26E-05 1419279_at Pip5k2a phosphatidylinositol-4-phosphate 5-kinase, type II, alpha 1,94 1,50E-04 1442116_at Gpr176 G protein-coupled receptor 176 1,94 1,69E-04 1451795_at Tom1l2 target of myb1-like 2 1,94 6,49E-05 1417240_at Zyx zyxin 1,94 5,42E-04 1425227_a_at Atp6v0a1 ATPase, H+ transporting, lysosomal V0 subunit A1 1,93 6,39E-04 1429549_at Col27a1 procollagen, type XXVII, alpha 1 1,93 2,36E-04 1420637_at Prps2 phosphoribosyl pyrophosphate synthetase 2 1,93 2,34E-04 1428396_at Smurf1 SMAD specific E3 ubiquitin protein ligase 1 1,93 6,41E-04 1427711_a_at Ceacam1 CEA-related cell adhesion molecule 1 1,92 2,83E-04 1450676_at Tceb3 transcription elongation factor B (SIII), polypeptide 3 1,92 7,66E-04 1451981_at Gtrgeo22 gene trap ROSA b-geo 22 1,92 4,00E-05 1421146_at Rapgef1 Rap guanine nucleotide exchange factor (GEF) 1 1,91 5,18E-04

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1423734_at Rac1 RAS-related C3 botulinum substrate 1 1,91 2,19E-04 1421871_at Sh3bgrl SH3-binding domain glutamic acid-rich protein like 1,91 9,87E-04 1417716_at Got2 glutamate oxaloacetate transaminase 2, mitochondrial 1,91 4,08E-05 1417350_at Pldn pallidin 1,90 7,49E-04 1427533_at Als2cl ALS2 C-terminal like 1,90 1,64E-04 1417308_at Pkm2 pyruvate kinase, muscle 1,90 5,99E-04 1416525_at Spop speckle-type POZ protein 1,90 6,12E-04 1428016_a_at Rasip1 Ras interacting protein 1 1,89 9,05E-04 1430811_a_at Nuf2 NUF2, NDC80 kinetochore complex component, homolog 1,89 5,47E-04 1452684_at Akt1s1 AKT1 substrate 1 (proline-rich) 1,89 5,18E-04 1449219_at Fads3 fatty acid desaturase 3 1,88 9,75E-04 1421708_a_at Stat6 signal transducer and activator of transcription 6 1,88 3,05E-05 1453367_a_at Abhd12 abhydrolase domain containing 12 1,88 8,40E-04 1417031_at Tmem206 transmembrane protein 206 1,88 2,69E-04 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta 1420828_s_at Ywhaq polypeptide 1,88 5,74E-04 1431760_a_at Sdccag3 serologically defined colon cancer antigen 3 1,88 8,34E-04 1442176_at Arid5b AT rich interactive domain 5B (Mrf1 like) 1,87 4,78E-04 1418961_at Necap2 NECAP endocytosis associated 2 1,87 7,73E-04 1429187_at Tmed7 transmembrane emp24 protein transport domain containing 7 1,87 7,19E-04 1448851_a_at Dnajc5 DnaJ (Hsp40) homolog, subfamily C, member 5 1,87 7,29E-05 1460208_at Fbn1 fibrillin 1 1,86 3,45E-04 1428132_at Cdc42se1 CDC42 small effector 1 1,86 2,08E-04 1422972_s_at Gcn5l2 GCN5 general control of amino acid synthesis-like 2 1,86 9,08E-04 solute carrier family 35 (UDP-N-acetylglucosamine (UDP-GlcNAc) transporter), member 1424580_at Slc35a3 A3 1,85 2,70E-04 1426499_at Sh3glb2 SH3-domain GRB2-like endophilin B2 1,85 6,59E-04 1418632_at Ube2h ubiquitin-conjugating enzyme E2H 1,85 1,11E-05 1456412_a_at Pps inositol polyphosphate 5-phosphatase K 1,85 8,19E-05 1450161_at Ikbkg inhibitor of kappaB kinase gamma 1,84 6,02E-04 1425676_a_at Elovl1 elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 1 1,84 3,14E-04 1422661_at Lgals8 lectin, galactose binding, soluble 8 1,84 4,03E-05 1417341_a_at Ppp1r2 1, regulatory (inhibitor) subunit 2 1,84 2,36E-04 1428150_at Coro7 coronin 7 1,84 4,20E-04 1449481_at Slc25a13 calcium-binding mitochondrial carrier protein Aralar2 1,84 4,74E-06 1435861_at Ppfia1 liprin-alpha-1 1,83 9,56E-04 1452801_at Pigk phosphatidylinositol glycan anchor biosynthesis, class K 1,83 5,44E-04 1430385_a_at Glb1l galactosidase, beta 1-like 1,83 8,76E-04 1418581_a_at Limk2 LIM motif-containing protein kinase 2 1,83 6,72E-04 1451056_at Psmd7 proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 1,82 8,21E-04 1418972_at Bcl10 B-cell leukemia/lymphoma 10 1,81 1,05E-04 1416213_x_at Surf4 surfeit gene 4 1,81 2,46E-05 1453712_a_at Map2k5 mitogen activated protein kinase kinase 5 1,81 4,77E-04 1450396_at Stag2 stromal antigen 2 1,81 8,27E-04 1423010_at Sacs sacsin 1,80 9,91E-04 1434181_at Plekhc1 pleckstrin homology domain containing, family C (with FERM domain) member 1 1,80 7,16E-04 1454142_a_at Pwp1 PWP1 homolog (S. cerevisiae) 1,80 7,81E-04 1460513_a_at Ednra endothelin receptor type A 1,80 9,48E-04 1437938_x_at Dnm2 dynamin 2 1,80 1,10E-04 1429878_a_at Nupl1 nucleoporin like 1 1,79 1,17E-04 1429765_at 1500005K14Rik RIKEN cDNA 1500005K14 gene 1,79 1,38E-04 1423543_at Swap70 SWA-70 protein 1,79 8,52E-05 1452424_at Gpr23 G protein-coupled receptor 23 1,78 8,05E-04 1416182_at Apba3 amyloid beta (A4) precursor protein-binding, family A, member 3 1,78 2,06E-05 1450166_at Ids iduronate 2- 1,78 4,58E-04

- 16 -

1421242_at Rnf144 ring finger protein 144 1,78 8,14E-04 1460476_s_at 1200015N20Rik RIKEN cDNA 1200015N20 gene 1,77 8,97E-04 1416304_at Litaf LPS-induced TN factor 1,77 1,00E-03 1452742_at Trak1 trafficking protein, kinesin binding 1 1,77 2,35E-04 1451841_a_at Ncor2 nuclear receptor co-repressor 2 1,76 7,40E-04 1432281_a_at Itgb6 integrin beta 6 1,76 2,77E-04 1448662_at Fzd6 frizzled homolog 6 (Drosophila) 1,76 9,22E-04 1423053_at Arf4 ADP-ribosylation factor 4 1,76 4,42E-04 1423586_at Axl AXL receptor tyrosine kinase 1,76 9,27E-04 1415795_at Spin spindlin 1,75 1,55E-04 1430869_a_at Habp4 hyaluronic acid binding protein 4 1,75 2,84E-04 1426514_at 4631426J05Rik RIKEN cDNA 4631426J05 gene 1,75 3,19E-04 1451897_a_at Nbr1 neighbor of Brca1 gene 1 1,74 6,65E-04 1450384_at Bace1 beta-site APP cleaving enzyme 1 1,74 1,87E-04 1454037_a_at Flt1 FMS-like tyrosine kinase 1 1,74 4,62E-04 1450981_at Cnn2 calponin 2 1,73 1,09E-04 1423446_at Dapk3 death-associated kinase 3 1,73 1,34E-04 1415956_a_at Pctk1 PCTAIRE-motif protein kinase 1 1,73 1,54E-04 1418364_a_at Ftl1 ferritin light chain 1 1,73 7,39E-04 1452162_at Wdr48 WD repeat domain 48 1,73 3,35E-04 1431208_a_at Slc9a3r2 solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regu... 1,72 3,22E-04 1417868_a_at Ctsz 1,72 6,42E-04 1448346_at Cfl1 cofilin 1, non-muscle 1,71 7,17E-05 1426442_at Gpm6a glycoprotein m6a 1,71 7,30E-04 1418692_at Rab8a RAB8A, member RAS oncogene family 1,71 8,38E-04 1418290_a_at Ezh1 enhancer of zeste homolog 1 (Drosophila) 1,71 5,83E-04 1435587_at Pcid2 PCI domain containing 2 1,70 7,52E-04 1456651_a_at Tpr translocated promoter region 1,70 3,32E-04 1426677_at Flna filamin, alpha 1,70 1,01E-03 1431345_a_at Taf1b TATA box binding protein (Tbp)-associated factor, RNA polymerase I, B 1,70 6,74E-04 1426540_at Endod1 domain containing 1 1,70 6,07E-04 1425139_at Sesn2 sestrin 2 1,70 2,58E-04 1435075_at Tmem106b transmembrane protein 106B 1,70 3,29E-04 1416808_at Nid1 nidogen 1 1,70 6,48E-04 1420869_at Mllt10 myeloid/lymphoid or mixed lineage-leukemia translocation to 10 homo... 1,69 6,97E-04 1431926_a_at 1300010M03Rik RIKEN cDNA 1300010M03 gene 1,69 4,12E-04 1450408_at Clcn7 chloride channel 7 1,68 1,22E-04 1452428_a_at B2m beta-2 microglobulin 1,67 4,96E-04 1434905_at Ndufa4l2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 1,67 2,90E-04 1417421_at S100a1 S100 calcium binding protein A1 1,67 3,32E-04 1437611_x_at Kif2c kinesin family member 2C 1,66 1,73E-06 1416132_at Efr3a EFR3 homolog A 1,66 2,80E-04 1448339_at Tmem30a transmembrane protein 30A 1,66 4,75E-04 1451011_at Zfp358 zinc finger protein 358 1,66 7,58E-04 1453051_at Zkscan1 zinc finger with KRAB and SCAN domains 1 1,66 2,04E-04 1423524_at Mastl microtubule associated serine/threonine kinase-like 1,66 2,36E-05 1451805_at Phip pleckstrin homology domain interacting protein 1,66 2,83E-04 1424882_a_at Nt5dc2 5'-nucleotidase domain containing 2 1,65 9,00E-04 1421600_a_at Trim26 tripartite motif protein 26 1,65 4,00E-04 1422747_at Chek2 CHK2 checkpoint homolog (S. pombe) 1,65 8,92E-04 1420920_a_at Arf1 ADP-ribosylation factor 1 1,64 8,81E-04 1452734_at Rnaset2 T2 1,64 3,11E-04 1450399_at Psen1 presenilin 1 1,64 5,80E-04 1450841_at Stt3a STT3, subunit of the oligosaccharyltransferase complex, homolog A 1,63 2,57E-05 1427391_a_at Col12a1 procollagen, type XII, alpha 1 1,63 7,92E-05

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1433499_at 2010005J08Rik RIKEN cDNA 2010005J08 gene 1,63 4,54E-04 1450354_a_at Ptdss2 phosphatidylserine synthase 2 1,63 6,16E-04 1435276_a_at Dgcr2 DiGeorge syndrome critical region gene 2 1,63 4,44E-04 1425551_at Hip1r huntingtin interacting protein 1 related 1,63 2,30E-04 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 1441389_at Adamts5 5 (-2) 1,62 9,97E-04 1428739_at 2310040A07Rik RIKEN cDNA 2310040A07 gene 1,62 1,00E-03 1448603_at Srpk2 serine/arginine-rich protein specific kinase 2 1,61 2,58E-04 1417509_at Rnf19 ring finger protein (C3HC4 type) 19 1,61 4,58E-04 1430552_a_at Sbf1 SET binding factor 1 1,61 4,14E-04 1429038_at Dda1 DET1 and DDB1 associated 1 1,61 7,73E-04 1428527_at Snx7 sorting nexin 7 1,61 5,50E-04 1416746_at H2afx H2A histone family, member X 1,61 2,02E-04 1454754_a_at Aamp angio-associated migratory protein 1,60 1,22E-05 1449966_s_at Cab39l calcium binding protein 39-like 1,60 6,96E-04 1418292_at Asna1 arsA (bacterial) arsenite transporter, ATP-binding, homolog 1 1,60 7,72E-04 1420997_a_at Gpi1 glucose phosphate 1 1,60 2,22E-05 1426737_at Gspt1 G1 to S phase transition 1 1,60 8,28E-04 1422771_at Smad6 MAD homolog 6 1,59 7,31E-04 1453226_at Kif18b kinesin family member 18B 1,59 1,02E-04 1450852_s_at F2r coagulation factor II (thrombin) receptor 1,59 3,07E-04 1417330_at Slc23a2 solute carrier family 23 (nucleobase transporters), member 2 1,58 1,47E-04 1448350_at Asl argininosuccinate lyase 1,58 4,57E-05 1458636_at Rnf219 ring finger protein 219 1,58 3,36E-05 1455044_at Tmem44 transmembrane protein 44 1,58 3,81E-04 1433460_at Ttc7b tetratricopeptide repeat domain 7B 1,58 2,77E-04 1421121_at Akap10 A kinase (PRKA) anchor protein 10 1,58 2,73E-04 1417773_at Nans N-acetylneuraminic acid synthase (sialic acid synthase) 1,57 1,19E-04 1424435_a_at Gart phosphoribosylglycinamide formyltransferase 1,57 9,64E-04 1452881_at Gins2 GINS complex subunit 2 1,57 7,80E-04 1448159_at Rab7 RAB7, member RAS oncogene family 1,57 2,37E-04 1438220_at Foxj3 forkhead box J3 1,57 6,75E-04 1423077_at Snx9 sorting nexin 9 1,56 3,91E-04 1460288_a_at Ppp4c protein phosphatase 4, catalytic subunit 1,56 2,53E-04 1428049_a_at Nudt16l1 nudix (nucleoside diphosphate linked moiety X)-type motif 16-like 1 1,56 5,57E-04 1415918_a_at Tpi1 triosephosphate isomerase 1 1,56 3,12E-04 1424129_at Mfsd1 major facilitator superfamily domain containing 1 1,56 7,03E-04 1439713_at Itga1 integrin alpha 1 1,56 7,50E-05 1451908_a_at Sec14l1 SEC14-like 1 1,55 9,37E-04 1431299_a_at 2310014H01Rik RIKEN cDNA 2310014H01 gene 1,55 1,87E-04 1423382_a_at Hnrpf heterogeneous nuclear ribonucleoprotein F 1,55 4,12E-04 1448624_at Cd2bp2 CD2 antigen (cytoplasmic tail) binding protein 2 1,55 5,73E-04 1415860_at Kpna2 karyopherin (importin) alpha 2 1,55 8,67E-04 1415670_at Copg coatomer protein complex, subunit gamma 1,55 2,48E-04 1422399_a_at Rab23 RAB23, member RAS oncogene family 1,55 5,44E-04 1454021_a_at Exosc10 exosome component 10 1,55 1,23E-04 1417200_at Tmem183a transmembrane protein 183A 1,55 8,12E-04 1419508_at Ripk1 receptor (TNFRSF)-interacting serine-threonine kinase 1 1,55 8,20E-04 1449293_a_at Skp2 S-phase kinase-associated protein 2 (p45) 1,54 9,36E-04 1452920_a_at Ppil2 peptidylprolyl isomerase (cyclophilin)-like 2 1,54 2,00E-05 1426529_a_at Tagln2 transgelin 2 1,54 6,10E-04 1448751_at Ap3m2 adaptor-related protein complex 3, mu 2 subunit 1,54 3,92E-04 1450632_at Rhoa ras homolog gene family, member A 1,54 5,44E-04 1431062_a_at Exoc4 exocyst complex component 4 1,54 1,21E-04 1420653_at Tgfb1 transforming growth factor, beta 1 1,54 7,06E-04

- 18 -

1416819_at Cdc37 cell division cycle 37 homolog 1,54 2,37E-04 1431707_a_at Pscd3 pleckstrin homology, Sec7 and coiled-coil domains 3 1,54 3,06E-04 1416557_a_at Eftud2 elongation factor Tu GTP binding domain containing 2 1,54 7,52E-05 1452057_at Actr1b ARP1 actin-related protein 1 homolog B 1,53 2,57E-05 1435800_a_at Csda cold shock domain protein A 1,52 8,22E-04 1453865_a_at Otud5 OTU domain containing 5 1,52 8,82E-04 1426379_at Eif4b eukaryotic translation initiation factor 4B 1,52 6,34E-04 1422552_at Rprm reprimo, TP53 dependent G2 arrest mediator candidate 1,52 4,05E-04 1418712_at Cdc42ep5 CDC42 effector protein (Rho GTPase binding) 5 1,52 1,76E-04 1419329_at Sorbs3 sorbin and SH3 domain containing 3 1,51 9,46E-04 1434086_at Gpr107 G protein-coupled receptor 107 1,50 8,84E-04 1423814_at Ddx41 DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 1,50 5,54E-04 1428750_at Cdc42ep2 CDC42 effector protein (Rho GTPase binding) 2 1,50 4,07E-04 1416830_at 0610031J06Rik RIKEN cDNA 0610031J06 gene 1,50 4,44E-04 1460510_a_at Coq10b coenzyme Q10 homolog B 1,50 9,07E-04 1416837_at Bax Bcl2-associated X protein 1,50 1,10E-04 1423488_at Mmd monocyte to macrophage differentiation-associated 1,50 2,80E-05 DOWNREGULATED 1418029_at Faim Fas apoptotic inhibitory molecule -1,50 1,97E-04 1429002_at Snw1 SNW domain containing 1 -1,50 1,57E-04 1421904_at Tgs1 trimethylguanosine synthase homolog -1,50 1,65E-04 1418271_at Bhlhb5 basic helix-loop-helix domain containing, class B5 -1,50 9,47E-04 1417935_at Mkrn2 makorin, ring finger protein, 2 -1,51 5,36E-05 1426604_at Rnasel ribonuclease L (2', 5'-oligoisoadenylate synthetase-dependent) -1,51 2,16E-04 1429394_at AL365322.1 Uncharacterized protein -1,51 6,69E-04 1449664_s_at Rnf20 ring finger protein 20 -1,51 8,42E-04 1434768_at Tpp1 tripeptidyl peptidase I -1,51 7,88E-05 1420510_at Lox lysyl oxidase -1,51 3,11E-04 1434749_at BC067068 Uncharacterized protein -1,51 9,79E-06 1435818_at Khlh15 kelch-like 15 -1,52 8,45E-04 1449772_at Tbc1d14 TBC1 domain family, member 14 -1,52 6,97E-04 1436122_at Zfp667 zinc finger protein 667 -1,52 3,45E-04 1419981_at Riok3 RIO kinase 3 (yeast) -1,52 6,49E-04 1434039_at Appbp2 amyloid beta precursor protein (cytoplasmic tail) binding protein 2 -1,52 8,74E-05 1440250_at Col4a4 procollagen, type IV, alpha 4 -1,52 2,90E-04 1455671_at Commd8 COMM domain containing 8 -1,52 3,54E-04 1433856_at Hisppd1 histidine domain containing 1 -1,52 2,77E-04 1429233_at C87414 Uncharacterized protein -1,53 2,82E-04 1452310_at Tada2l transcriptional adaptor 2 (ADA2 homolog, yeast)-like -1,53 4,15E-04 1440231_at Mtap9 microtubule-associated protein 9 -1,53 3,90E-04 1426952_at Arhgap18 Rho GTPase activating protein 18 -1,53 8,57E-04 1452880_at Znhit3 zinc finger, HIT type 3 -1,53 1,49E-04 1429155_at 4933411K20Rik RIKEN cDNA 4933411K20 gene -1,53 6,56E-04 1434478_at Heca headcase homolog (Drosophila) -1,53 8,15E-04 1418996_a_at Lyrm5 LYR motif containing 5 -1,54 1,68E-04 1455403_at Manea mannosidase, endo-alpha -1,54 6,16E-04 1455611_at Pias1 protein inhibitor of activated STAT 1 -1,54 8,70E-04 1435632_at Nufip2 nuclear fragile X mental retardation protein interacting protein 2 -1,54 2,33E-04 1416852_a_at Ncdn neurochondrin -1,54 6,28E-04 1425216_at Ffar2 free fatty acid receptor 2 -1,54 7,68E-04 1434717_at Cul3 cullin 3 -1,55 1,27E-04 1415725_at Rrn3 RRN3 RNA polymerase I transcription factor homolog (yeast) -1,55 3,51E-04 1423753_at Bambi BMP and activin membrane-bound inhibitor, homolog (Xenopus laevis) -1,55 5,19E-04 1424516_at B230354K17Rik RIKEN cDNA B230354K17 gene -1,55 8,25E-04 1448722_s_at Ppcs phosphopantothenoylcysteine synthetase -1,55 2,76E-04

- 19 -

1440989_at Mrpl35 mitochondrial ribosomal protein 35 -1,55 1,76E-06 1452445_at Slc41a2 solute carrier family 41, member 2 -1,55 1,10E-04 1424243_at Rwdd4a RWD domain containing 4A -1,55 9,37E-04 1418660_at Clock circadian locomoter output cycles kaput -1,55 2,05E-04 1428926_at 1110003O08Rik RIKEN cDNA 1110003O08 gene -1,55 9,85E-04 1434161_at Esrrb estrogen related receptor, beta -1,56 5,63E-04 1442003_at Diap2 diaphanous homolog 2 -1,56 9,55E-04 1418040_at Tmem186 transmembrane protein 186 -1,56 7,13E-04 1454631_at Gtf2a1 General transcription factor II A, 1 -1,57 5,45E-04 1426692_at Ccdc97 coiled-coil domain containing 97 -1,57 6,80E-04 1419267_at Nfyb nuclear transcription factor-Y beta -1,57 5,20E-04 1419359_at Hexim1 hexamethylene bis-acetamide inducible 1 -1,57 8,98E-04 1441646_at Vta1 Vps20-associated homolog 1 -1,57 7,02E-04 1451968_at Xrcc5 X-ray repair complementing defective repair in Chinese hamster cells 5 -1,58 5,79E-04 1443779_s_at Lcor ligand dependent nuclear receptor corepressor -1,58 4,04E-04 1437424_at Syde2 synapse defective 1, Rho GTPase, homolog 2 -1,58 2,10E-04 1424074_at Btf3l4 basic transcription factor 3-like 4 -1,58 8,37E-04 1442006_at Vps8 vacuolar protein sorting 8 homolog -1,58 9,41E-05 1453061_at Elac1 elaC homolog 1 -1,58 6,53E-04 1452199_at 2700094F01Rik RIKEN cDNA 2700094F01 gene -1,58 4,83E-04 1434764_at Akap11 A kinase (PRKA) anchor protein 11 -1,59 5,21E-04 1416486_at Scye1 small inducible cytokine subfamily E, member 1 -1,59 6,96E-06 1451558_at Fbxw7 F-box and WD-40 domain protein 7, archipelago homolog -1,59 4,02E-04 1435299_at Pik3r1 phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 (p85 alpha) -1,59 1,27E-04 1438216_at Rreb1 ras responsive element binding protein 1 -1,59 2,45E-04 1450411_at 2900009I07Rik RIKEN cDNA 2900009I07 gene -1,60 7,59E-04 1419951_at Lman1 Lectin, mannose-binding, 1 -1,60 1,69E-04 1423302_a_at Paxip1 PAX interacting (with transcription-activation domain) protein 1 -1,60 2,78E-04 1454808_at Efha1 EF hand domain family A1 -1,60 7,40E-04 1455496_at Pfas phosphoribosylformylglycinamidine synthase (FGAR amidotransferase) -1,60 6,62E-04 1456403_at Pag1 phosphoprotein associated with glycosphingolipid microdomains 1 -1,60 5,90E-04 1450268_at Fign fidgetin -1,61 9,21E-04 1427934_at Lyrm2 LYR motif containing 2 -1,61 1,23E-05 1431012_a_at Peci peroxisomal delta3, delta2-enoyl-Coenzyme A isomerase -1,61 9,98E-04 1438187_at Slc25a29 solute carrier family 25 (mitochondrial carrier, palmitoylcarnitine... -1,62 3,04E-04 1444425_at Zfp53 Zinc finger protein 53 -1,62 7,13E-04 1436026_at Zfp703 zinc finger protein 703 -1,62 8,51E-04 1420132_s_at Pttg1ip Pituitary tumor-transforming 1 interacting protein -1,63 4,08E-04 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated 1429205_at Mllt3 to, 3 -1,63 4,20E-04 1429455_at Gapvd1 GTPase activating protein and VPS9 domains 1 -1,64 2,11E-05 1433842_at Lrrfip1 leucine rich repeat (in FLII) interacting protein 1 -1,64 9,17E-04 1454945_at Lrriq2 leucine-rich repeats and IQ motif containing 2 -1,64 3,20E-05 1415758_at Fryl furry homolog-like -1,64 7,38E-05 1434629_at Zbtb26 zinc finger and BTB domain containing 26 -1,64 1,05E-04 1433795_at Tgfbr3 transforming growth factor, beta receptor III -1,65 2,76E-04 1416796_at Nck2 non-catalytic region of tyrosine kinase adaptor protein 2 -1,65 7,36E-04 1418524_at Pcm1 1 -1,65 1,16E-04 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, 1455246_at Smarcc1 member 1 -1,65 1,42E-04 1416007_at Satb1 special AT-rich sequence binding protein 1 -1,65 8,04E-04 1451356_at Anp32e acidic (leucine-rich) nuclear phosphoprotein 32 family, member E -1,65 5,51E-04 1439433_a_at Slc35a2 solute carrier family 35 (UDP-galactose transporter), member 2 -1,65 6,18E-04 1447864_s_at Pogk pogo transposable element with KRAB domain -1,66 1,44E-04 1435991_at Nr3c2 nuclear receptor subfamily 3, group C, member 2 -1,66 7,09E-04

- 20 -

1424013_at Etf1 eukaryotic translation termination factor 1 -1,67 5,38E-04 1438961_s_at Blmh bleomycin hydrolase -1,67 4,12E-04 1423845_at Csdc2 cold shock domain containing C2, RNA binding -1,68 3,53E-04 1454648_s_at D10Wsu102e Uncharacterized protein -1,68 3,68E-04 1455508_at A530082C11Rik RIKEN cDNA A530082C11 gene -1,69 5,82E-06 1434471_at BC003331 Protein odr-4 homolog (mODR-4) -1,69 3,19E-04 1451360_at Ergic2 ERGIC and golgi 2 -1,70 4,41E-04 1424034_at Rora RAR-related orphan receptor alpha -1,71 8,10E-04 1456126_at Malt1 mucosa associated lymphoid tissue lymphoma translocation gene 1 -1,71 4,73E-04 1419946_s_at Rab2 RAB2, member RAS oncogene family -1,71 1,07E-04 1425512_at Map2k7 mitogen-activated protein kinase kinase 7 -1,72 2,21E-04 1438026_at Zfp560 zinc finger protein 560 -1,72 9,32E-04 1418471_at Pgf placental growth factor -1,72 7,28E-04 1442989_at Nars2 Asparaginyl-tRNA synthetase 2 (mitochondrial)(putative) -1,72 8,08E-04 1435713_at Mettl2 methyltransferase like 2 -1,72 6,61E-04 1435867_at Agk-201 acylglycerol kinase -1,72 1,95E-04 1430137_at Map3k13 mitogen-activated protein kinase kinase kinase 13 -1,73 6,85E-04 1422697_s_at Jarid2 jumonji, AT rich interactive domain 2 -1,73 3,75E-04 1441318_at Jarid1a jumonji, AT rich interactive domain 1A (Rbp2 like) -1,73 9,82E-04 1426961_at Phf20 PHD finger protein 20 -1,74 1,07E-05 1419967_at Seh1l SEH1-like (S. cerevisiae -1,74 8,38E-04 1438078_at Dgke diacylglycerol kinase, epsilon -1,75 4,16E-04 1435253_at Rab11b RAB11B, member RAS oncogene family -1,75 6,37E-04 1425543_s_at Plekha5 pleckstrin homology domain containing, family A member 5 -1,75 1,68E-04 1455493_at Syne1 synaptic nuclear envelope 1 -1,75 5,29E-04 1455217_at Lrig2 leucine-rich repeats and immunoglobulin-like domains 2 -1,75 1,37E-05 1457674_at Vps13a vacuolar protein sorting 13A (yeast) -1,75 2,39E-04 1452988_at 2610306M01Rik RIKEN cDNA 2610306M01 gene -1,76 1,09E-05 1449893_a_at Lrig1 leucine-rich repeats and immunoglobulin-like domains 1 -1,76 5,61E-04 1431428_a_at Nosip nitric oxide synthase interacting protein -1,76 1,71E-04 1417247_at AI597479 expressed sequence AI597479 -1,76 3,00E-04 1455468_at Zfp536 zinc finger protein 536 -1,76 3,49E-05 1433477_at Abr active BCR-related gene -1,78 6,57E-04 1416926_at Trp53inp1 transformation related protein 53 inducible nuclear protein 1 -1,78 8,09E-04 1435537_at Ptprd protein tyrosine phosphatase, receptor type, D -1,78 6,00E-04 1459997_s_at Tmem17 transmembrane protein 17 -1,78 7,78E-05 1435052_at 4930455F23Rik RIKEN cDNA 4930455F23 gene -1,79 6,63E-04 1424371_at Psmf1 proteasome (prosome, macropain) inhibitor subunit 1 -1,79 1,73E-04 1422896_at Vamp4 vesicle-associated membrane protein 4 -1,79 4,18E-04 1428675_at 1110049F12Rik RIKEN cDNA 1110049F12 gene -1,79 4,31E-05 1421223_a_at Anxa4 annexin A4 -1,80 1,13E-04 1443880_at Zbtb39 zinc finger and BTB domain containing 39 -1,81 7,26E-04 1417904_at Dclre1a DNA cross-link repair 1A, PSO2 homolog -1,81 7,61E-04 1429242_at 1110054O05Rik RIKEN cDNA 1110054O05 gene -1,81 8,22E-04 1440381_at 2410085M17Rik RIKEN cDNA 2410085M17 gene -1,81 8,15E-04 1449813_at Zfp30 zinc finger protein 30 -1,82 9,45E-04 1437363_at Homer1 homer homolog 1 -1,82 1,64E-04 1455396_at Atp8b1 ATPase, class I, type 8B, member 1 -1,82 9,44E-04 1433905_at Akap7 A kinase (PRKA) anchor protein 7 -1,82 1,49E-04 1422870_at Hoxc4 homeo box C4 -1,83 3,46E-04 1435170_at Tsr2 TSR2, 20S rRNA accumulation, homolog -1,83 9,40E-04 1434675_at 1700065O13Rik RIKEN cDNA 1700065O13 gene -1,84 2,77E-04 1429430_at Pcmtd1 protein-L-isoaspartate O-methyltransferase domain-containing protein 1 -1,84 3,61E-04 1435664_at Zfp397 zinc finger protein 397 -1,84 8,52E-04 1435752_s_at Abcc9 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 -1,85 3,87E-04

- 21 -

1449160_at Npr1 natriuretic peptide receptor 1 -1,85 1,87E-04 1420907_at Cd2ap CD2-associated protein -1,86 2,82E-04 1425052_at Isoc1 Isochorismatase domain containing 1 -1,87 1,87E-04 1415712_at Zranb1 zinc finger, RAN-binding domain containing 1 -1,87 2,00E-04 1448960_at Cxxc5 CXXC finger 5 -1,89 4,08E-04 1436570_at AC131942.1 Transcribed locus -1,90 2,01E-04 1434806_at Mtx3 metaxin 3 -1,92 4,68E-04 1446622_at Ntrk3 neurotrophic tyrosine kinase, receptor, type 3 -1,92 3,89E-04 1455582_at C1qtnf1 C1q and tumor necrosis factor related protein 1 -1,92 8,28E-04 1417281_a_at Mmp23 matrix metallopeptidase 23 -1,93 4,14E-04 1428299_at Dyrk1a dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a -1,93 2,48E-04 1422706_at Tmepai prostate transmembrane protein, androgen induced 1 -1,95 9,16E-04 1449056_at Agk acylglycerol kinase -1,96 4,45E-05 1424030_at Grhl1 grainyhead-like 1 -1,98 7,08E-04 1417801_a_at Ppfibp2 liprin-beta 2 -1,99 9,96E-05 1452907_at Galc galactosylceramidase -2,00 8,78E-04 1432033_at Ntng2 netrin G2 -2,02 6,91E-04 1442525_at Ccdc47 coiled-coil domain containing 47 -2,02 3,17E-04 1428026_at Tshz2 teashirt zinc finger family member 2 -2,03 5,05E-04 1433965_at Atp8a1 ATPase, aminophospholipid transporter (APLT), class I, type 8A, member 1 -2,04 4,58E-04 1450309_at Astn2 astrotactin 2 -2,05 8,09E-04 1420043_s_at Thoc1 THO complex 1 -2,06 2,33E-04 1429096_at Kank1 KN motif and ankyrin repeat domains 1 -2,06 4,17E-05 1455643_s_at Tsr1 TSR1, 20S rRNA accumulation, homolog -2,07 1,18E-04 1420928_at St6gal1 beta galactoside alpha 2,6 sialyltransferase 1 -2,07 2,05E-04 1455535_at Sox5 SRY-box containing gene 5 -2,08 9,96E-04 1442315_at Foxd2 forkhead box D2 -2,10 5,91E-04 1434639_at Kbtbd9 kelch repeat and BTB (POZ) domain containing 9 -2,11 1,00E-03 1439786_at Gab2 growth factor receptor bound protein 2-associated protein 2 -2,11 6,55E-04 1419309_at Pdpn podoplanin -2,12 9,04E-06 1429216_at Paqr3 progestin and adipoQ receptor family member III -2,12 1,94E-04 1429183_at Pkp2 plakophilin 2 -2,12 1,35E-04 1436365_at Zbtb7c zinc finger and BTB domain containing 7C -2,13 4,47E-04 1449975_a_at Park2 parkin -2,17 1,24E-04 1426578_s_at Snapap SNAP-associated protein -2,18 1,35E-04 1424356_a_at Metrnl meteorin, glial cell differentiation regulator-like -2,19 8,61E-04 1416032_at Tmem109 transmembrane protein 109 -2,21 3,47E-07 1427386_at Arhgef16 Rho guanine nucleotide exchange factor (GEF) 16 -2,21 5,14E-05 1446493_at Cdc14a CDC14 cell division cycle 14 homolog A (S. cerevisiae) -2,21 9,22E-04 1448150_at Nup62 nucleoporin 62 -2,21 6,32E-04 1442226_at Sema3e semaphorin 3e -2,22 4,10E-04 1423175_s_at Pard6b par-6 (partitioning defective 6) homolog beta -2,23 9,53E-04 1438423_at Ssbp2 single-stranded DNA binding protein 2 -2,24 4,33E-04 1460000_at Shisa3 shisa homolog 3 -2,30 8,21E-04 1433907_at Pknox2 Pbx/knotted 1 2 -2,34 1,99E-04 1423499_at Sncaip synuclein, alpha interacting protein (synphilin) -2,35 7,44E-04 1450782_at Wnt4 wingless-related MMTV integration site 4 -2,37 2,65E-04 1457716_at Otud7b OTU domain containing 7B -2,38 1,63E-04 1433761_at Pde4dip 4D interacting protein (myomegalin) -2,41 8,87E-04 1455566_s_at 2810022L02Rik RIKEN cDNA 2810022L02 gene -2,44 9,61E-05 1458324_x_at Aldh1l1 aldehyde dehydrogenase 1 family, member L1 -2,46 6,50E-04 1418533_s_at Fzd2 frizzled homolog 2 -2,47 3,43E-04 1436319_at Sulf1 sulfatase 1 -2,52 2,01E-04 1457424_at Eya1 eyes absent 1 homolog -2,53 9,58E-04 1419054_a_at Ptpn21 protein tyrosine phosphatase, non-receptor type 21 -2,54 7,48E-04

- 22 -

1441687_at AL645468.11 Uncharacterized protein -2,65 8,67E-04 1423174_a_at Pard6b par-6 (partitioning defective 6) homolog beta -2,66 1,45E-04 1434267_at Nek1 NIMA (never in mitosis gene a)-related expressed kinase 1 -2,69 2,92E-04 1436070_at Glo1 glyoxalase 1 -2,70 6,15E-04 1426865_a_at Ncam1 neural cell adhesion molecule 1 -2,72 4,44E-04 1457139_at Auts2 autism susceptibility candidate 2 -2,81 6,71E-04 1435126_at Dusp15 Dual specificity phosphatase-like 15 -2,85 1,61E-04 1452020_a_at Siva1 SIVA1, apoptosis-inducing factor -2,87 1,67E-04 1457698_at 0610012H03Rik RIKEN cDNA 0610012H03 -2,96 9,17E-04 1436115_at Gm266 gene model 266, (NCBI) -2,98 8,45E-04 1420944_at Zfp185 zinc finger protein 185 -3,00 9,41E-04 1438279_at Dpp4 Dipeptidylpeptidase 4 -3,10 4,95E-04 1457110_at Pank1 pantothenate kinase 1 -3,14 7,26E-04 1437312_at Bmpr1b bone morphogenetic protein receptor, type 1B -3,25 6,17E-05 1444228_s_at Herc2 hect domain and RLD 2 -3,27 3,64E-04 1452779_at 3110006E14Rik RIKEN cDNA 3110006E14 gene -3,50 6,29E-04 1451702_at Cmtm7 CKLF-like MARVEL transmembrane domain containing 7 -3,62 6,94E-04

Supplementary Microarray Results:

At week 1, the 42 genes up-regulated were functionally annotated by the

Ingenuity Pathway Analysis Program as being involved in cell cycle (20 genes), cellular

assembly and organization (15), DNA replication, recombination and repair (17), cellular

movement (5) and cellular compromise (5). At week 2, among the 62 perturbed genes, 6

were involved in cell cycle regulation and proliferation, including Ccnd1, Cdc14a, Adm,

Gnai1, Ssbp2, and Frk. In addition, the cytoskeletal remodeling gene1 Dock3 was up-

regulated, whereas Ptpro or Glepp1, involved in foot process remodeling,2 was

downregulated. Finally, analysis of the 738 perturbed genes at 4 weeks with the

Ingenuity Pathways Analysis Program revealed statistically significant enrichment for

several canonical signaling pathways, including ephrin receptor, integrin and SAPK/JNK

signaling (Supplementary Table S3). In addition, both cell cycle and apoptosis

pathways were significantly perturbed at 4 weeks.

Our microarray results differed greatly with previously published data, but

confirmed some prior results. Chugh and colleagues3 investigated transcriptional

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changes in an extensive list of genes in the puromycin aminoglycoside nephrosis model, and our data corroborates their finding showing downregulation of Ptpro, encloding

GLEPP1, early in the course of proteinuria. In addition, we found up-regulation in several genes belonging to the TGF-β family at 4 weeks, as in a study involving laser microdissected human glomeruli with FSGS.4 Not surprisingly, only 2/68 genes up- regulated during the course of glomerulosclerosis in a Dicer knockout model5 were similarly perturbed in our study,

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Supplementary Table S3. Significantly perturbed canonical pathways in isolated glomeruli 4 weeks after conditional inactivation of podocin, using Ingenuity Pathway

Analysis

Canonical Pathways P value

Ephrin Receptor Signaling 4.27 x 10-8

Integrin Signaling 1.82 x 10-6

Amyloid Processing 4.27 x 10-5

Protein Ubiquitination Pathway 1.35 x 10-4

SAPK/JNK Signaling 1.41 x 10-4

Huntington's Disease Signaling 2.09 x 10-4

Axonal Guidance Signaling 3.47 x 10-4

Apoptosis Signaling 1.10 x 10-3

Hypoxia Signaling 1.26 x 10-3

Actin Cytoskeleton Signaling 1.86 x 10-3

Leukocyte Extravasation Signaling 1.91 x 10-3

Cell Cycle: G2/M DNA Damage Checkpoint Regulation 2.40 x 10-3

B Cell Receptor Signaling 3.31 x 10-3

IGF-1 Signaling 3.98 x 10-3

VEGF Signaling 4.37 x 10-3

Cell Cycle: G1/S Checkpoint Regulation 7.24 x 10-3

PI3K/AKT Signaling 8.71 x 10-3

Antigen Presentation Pathway 3.55 x 10-2

Parkinson's Signaling 3.80 x 10-2

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IL-4 Signaling 3.80 x 10-2

ERK/MAPK Signaling 3.89 x 10-2

IL-6 Signaling 4.27 x 10-2

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SUPPLEMENTARY METHODS

Generation of Mice. A 7.7-kb targeting construct consisting of three fragments

generated by PCR and subcloned in an MCI proprietary vector (Figure S1A). The construct spans an intronic region 5’ to exon 1 up to the intronic region 3’ of exon 2, and was modified by the insertion of a loxP site in intron 1-2 and of a loxP-Frt-Neo-Frt

cassette in intron 2-3, the latter serving as the positive selection marker. After

linearization and electroporation in 129S2/SvPAS mouse embryonic stem cells, screening

was performed by long-range PCR using external primers and Southern blot

hybridization using an external probe (Figure S1B). One screen-positive clone was

expanded, karyotyped and injected into C57BL/6 blastocysts. Eight males with >90%

chimerism were mated with female Flp recombinase deleter mice to obtain germline

transmission and simultaneously remove the Neo cassette. Heterozygous Nphs2lox2/+,Flp+ mice were subsequently mated against wild-type counterparts to generate Flp- heterozygous mice.

Homozygous Nphs2lox2/lox2 mice had normal renal histology and serum and urine

biochemical parameters at 2 months of age and RT-PCR confirmed that no aberrant splicing occurs (data not shown). These mice were mated with mice bearing one null

+/- + Nphs2 allele (Nphs2 ) and the βMCM86 transgene (Cre ), in which tamoxifen

administration leads to podocyte-specific activation of Cre recombinase6

(Supplementary Figure S2).

Genotyping. At each mating, genotyping PCRs were performed to identify the offsprings

bearing the desired alleles. Multiplex PCRs were performed to identify the Nphs2 wild-

type and null alleles, using the following protocol: denaturation at 94°C x 5 min

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denaturation, followed by 35 cycles of denaturation at 94°C x 1 min, annealing at 55°C x

1 min, elongation at 72°C x 1 min, and ending with elongation at 72°C x 7 min.

Similarly, multiplex PCRs were performed for the floxed Nphs2 and Cre alleles, using

the following protocol: denaturation at 94°C x 5 min, followed by 40 cycles of

denaturation at 94°C x 30 sec, elongation at 62°C x 45 sec, elongation at 72°C x 30 sec,

and final elongation step at 72°C x 7 min. PCR products were separated on a 2% agarose

gel. The primer pairs used for genotyping are detailed below:

Allele Forward Reverse

Nphs2 wild (+) 5’-GCTGTCCTAGATGGGAAGT-3’ 5’-TGCATACCTGGTGCCTATA-3’

Nphs2 null (-) 5’-GACAGCGTCTCCGACCTGAT-3’ 5’-CGACCGGCTGCAGAACAG-3’

Nphs2 lox2 5’-CCAGCATCCCATTAGATAGATGAGG-3’ 5’-GCATCCAAATGATCAGAGTTCCCAGG-3’

Cre 5’-CGCAGAACCTGAAGATGTTCGCGA-3’ 5’-GGATCATCAGCTACACCAGAGACG-3’

Transmission Electron Microscopy. Mice were anesthetized using Avertine, followed

by intra-cardiac perfusion of 1X PBS and 2.5% glutaraldehyde (diluted in 1X PBS).

Kidneys were removed and 1 mm3 cortical fragments were incubated in fresh 2.5%

glutaraldehyde for 2 hr at room temperature. After several rinses in 1X PBS, samples

were post-fixed in 1% buffered osmium tetroxide for 1 hr, followed by 1X PBS rinse.

Samples were dehydrated in graded ethanols and transferred to propylene oxide prior to

embedding in epoxy resin (Epon 812, Fluka, Saint Louis, MO). Ultra-thin (60-80 nm)

sections from selected areas with glomeruli were collected on nickel or copper grids,

stained with uranyl acetate and lead citrate and observed under a Jeol JEM-100CX II

transmission electron microscope.

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Immunogold Electron Microscopy. Ultrastructural immunolocalization of podocin was

achieved by the post-embedding method. Following anesthesia using Avertine, mouse

kidneys were rapidly dissected, cut in 1 mm-thick sections and immersed in freshly

prepared 4% paraformaldehyde in 0.1M PBS, 4% sucrose solution. After 2 hrs of

fixation, tissues were minced into smaller fragments, thoroughly washed in PBS and low-

temperature embedded in Lowicryl K4M (EMS, Fort Washington, PA) using a freeze-

substitution device (Leica-Reichert, Vienna, Austria). Polymerization was allowed to

take placed at -30°C using UV light at 360 nm. Ultra-thin sections were cut and mounted

on 600-mesh nickel grids. Tissue sections were initially incubated for 10 minutes on a

drop of 0.1M PBS containing 1% BSA, and then with rabbit anti-C-terminal podocin

antibody7 diluted 1:50 in blocking solution (2% BSA in PBS) overnight at 4°C. Grids were then rinsed in PBS-1% BSA buffer and incubated on a drop of secondary antibody,

10 nm gold-conjugated goat-anti-rabbit (EMS, Fort Washington, PA) diluted 1:50 in blocking solution for 45 min at room temperature. After several PBS washes, samples were post-fixed in 2% glutaraldehyde for 15 min, rinsed in water and dried. Sections were counterstained with uranyl acetate and examined with a Jeol electron microscope.

Sections incubated only with secondary antibody served as controls.

Western Blot. Kidneys were excised, proteins were extracted, separated on an SDS-

PAGE and transferred onto nitrocellulose membranes, as described previously.8 After

blocking with TBS-0.2% Tween-5% milk, membranes were incubated overnight at 4°C

with affinity-purified rabbit anti-C-terminal podocin antibody7 diluted 1:1000.

Membranes were washed and incubated with horseradish peroxidase-conjugated donkey

anti-rabbit antibody (diluted 1:1000, GE Healthcare, Buckinghamshire, UK) for one hour.

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Immunoblotting against GAPDH (diluted 1:5000, Chemicon, Temecula, CA) was

performed as a loading control.

Immunofluorescence. Kidneys frozen in OCT were cut in 10-µ sections, fixed in acetone and simultaneously incubated with rabbit anti-podocin (diluted 1:500) and rat anti-nidogen (diluted 1:100, Chemicon, Temecula, CA) antibodies. For proliferation assays, sections were incubated with rabbit anti-Ki-67 (diluted 1:100, Thermo Fisher

Scientific, Fremont, CA) and either mouse anti-desmin (diluted 1:50, Immunotech,

Marseille, France), rat anti-CD31 (diluted 1:50, BD Pharmingen, Franklin Lakes, NJ) or guinea pig anti-nephrin (diluted 1:50, Progen, Heidelberg, Germany) antibodies, which labelled mesangial cells, endothelial cells and podocytes, respectively. After rinsing, slides were incubated with appropriate secondary Alexa Fluor-conjugated antibodies

(Invitrogen, Eugene, OR). Nuclei were labelled using Topro-3 (diluted 1:100,

Invitrogen). Slides were examined using a Zeiss confocal microscope (Carl Zeiss, Jena,

Germany). Podocin was quantified with Metamorph software (Molecular Devices,

Toronto, Canada) using 40X as standard objective on 5 controls and 4 null mice at each time point. For each mouse, the amount of podocin was quantified in 30 glomeruli by dividing fluorescence intensity with the area of the glomerular tuft. Proliferation was quantified in 10 glomeruli each in 2 control and 3 null mice at each time point. In each glomerulus, the number of Ki67-positive nuclei was divided by the total number of

Topro3-positive nuclei.

Isolation of Glomeruli. Mice were sacrificed 1, 2 and 4 weeks after tamoxifen administration. The procedure for isolation of glomeruli was based on a published method with modifications.9 Briefly, mice were perfused with 8 x 107 tosyl-inactivated

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magnetic Dynabeads (Invitrogen, Eugene, OR) in 40 mL of HBSS solution (Invitrogen).

The kidneys of each mouse were dissected, diced into 1 mm3 pieces and digested in

HBSS containing 1 mg/mL A (Sigma, St. Louis, MO) and 100 U/mL DNAse

I (Invitrogen, Eugene, OR) at 37°C for 30 min with agitation. Tissues were then pressed

through a 100-µ cell strainer, and extracts of > 90% purified glomeruli were subsequently

obtained after several rounds of concentration using a magnet.

Quantitative Real-Time PCR. Total glomerular RNA was prepared from Nphs2lox2/-,Cre+ and Nphs2lox2/-,Cre- (controls) as described above. Reverse transcription was performed

using random primers and Superscript II reverse transcriptase, according to

manufacturer’s protocol (Invitrogen). Gene expression was quantified in triplicate using

real-time PCR on an Applied Biosystems 7700 system, commercially available and

optimized TaqMan Assays-on-Demand primer and probe sets (Applied Biosystems,

Foster City, CA) listed in Supplementary Table S1 and the Absolute QPCR ROX Mix

(Thermo Scientific, Surrey, UK). Target gene expression levels, normalized to 18S rRNA,

were calculated using the 2-∆∆CT method10, and are reported as fold-change over control

mice at the same time point. The primers and probe used for podocin measurement

includes exon 2 of the Nphs2 gene.

Microarray. Total glomerular RNA from four Nphs2lox2/-,Cre+ and four Nphs2lox2/-,Cre – mice at 1, 2 and 4 weeks after tamoxifen administration were extracted using the RNeasy

Micro Kit (Qiagen, Hilden, Germany). RNA integrity and quantity were verified using the Agilent 2100 Bioanalyzer (Waldbronn, Germany) system and RNA Nano Chips.

After reverse transcription and labeling, RNAs were hybridized onto Affymetrix

Genechip Mouse Genome 430 2.0 Arrays following the manufacturer's protocol. Arrays

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were subsequently washed and stained in a Fluidics Station 400 and scanned with a

GeneChip Scanner 3000 according to manufacturer protocol. After passing standard

quality controls, a set of RMA normalized expression values were obtained. Gene

expression levels were compared between control and null mice at each time point, using

unpaired Student t-test, assuming unequal variance. Differentially expressed genes

between control and null mice were functionally annotated using Ingenuity Pathway

Analysis tools (www.ingenuity.com, Redwood City, CA).

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2. Wharram BL, Goyal M, Gillespie PJ, Wiggins JE, Kershaw DB, Holzman LB, Dysko RC, Saunders TL, Samuelson LC & Wiggins RC: Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate. J Clin Invest, 106: 1281-1290, 2000

3. Clement LC, Liu G, Perez-Torres I, Kanwar YS, Avila-Casado C & Chugh SS: Early changes in gene expression that influence the course of primary glomerular disease. Kidney Int, 72: 337-347, 2007

4. Bennett MR, Czech KA, Arend LJ, Witte DP, Devarajan P & Potter SS: Laser capture microdissection-microarray analysis of focal segmental glomerulosclerosis glomeruli. Nephron Exp Nephrol, 107: e30-40, 2007

5. Shi S, Yu L, Chiu C, Sun Y, Chen J, Khitrov G, Merkenschlager M, Holzman LB, Zhang W, Mundel P & Bottinger EP: Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis. J Am Soc Nephrol, 19: 2159-2169, 2008

6. Bugeon L, Danou A, Carpentier D, Langridge P, Syed N & Dallman MJ: Inducible gene silencing in podocytes: a new tool for studying glomerular function. J Am Soc Nephrol, 14: 786-791, 2003

7. Roselli S, Gribouval O, Boute N, Sich M, Benessy F, Attie T, Gubler MC & Antignac C: Podocin localizes in the kidney to the slit diaphragm area. Am J Pathol, 160: 131-139, 2002

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8. Philippe A, Weber S, Esquivel EL, Houbron C, Hamard G, Ratelade J, Kriz W, Schaefer F, Gubler MC & Antignac C: A missense mutation in podocin leads to early and severe renal disease in mice. Kidney Int, 2008

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