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. - 2 - A B C D F R FR - 3 - 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 - 4 - 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 phosphatase, receptor type, O Mm00478480_m1 Smad3 SMAD homolog 3 Mm00489641_m1 Smurf1 SMAD specific E3 ubiquitin protein ligase 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 - 5 - 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. - 6 - WEEK 1 Probesets Symbol Title Fold-change p 1421244_at Esr1 estrogen receptor 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 thyroid hormone receptor 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 centromere 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 - 7 - 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 zinc finger 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 - 9 - 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