Papadaki et al., 2009 Supplementary Supplemental Data Index x Supplemental Figures 1-6 x Supplemental Tables 1a, 1b, 2 Papadaki et al., 2009 Supplementary Supplemental Figure 1. Thymocyte restricted inactivation of the Elavl1 locus. + fl (A) Diagrammatic representation of the wild-type (Elavl1P P), floxed (Elavl1P P) and Cre- - recombined (Elavl1P P) Elavl1/HuR loci on mouse chromosome 8; Noted are the loxP sequences (triangles) flanking the selection marker (neo) used in gene targeting and the ATG containing exon 2 (white box); (H) denotes restriction sites for loci mapping. (B) Detection of native (+), targeted (fl) and Cre-recombinant (-) loci in thymocyte DNA extracts from control and test mice following HindIII digestion and Southern blotting. (C) Western blot of total thymic protein extracts probed with ĮHuR Ab + fl/fl indicating the loss of HuR protein in LckCreP PElavl1P P thymi. Į-actin is shown for quantitation. (D) Flow cytometric detection of intracellular mHuR protein in + fl/+ LckCreP PElavl1P P thymocytes (open histogram), and its respective loss in + fl/fl LckCreP PElavl1P P thymocytes (shaded histogram). The dotted histogram depicts the + isotype-matched background staining. (E) Flow cytometric detection of HuRP P or - + + + fl/+ HuRP P cells in gated splenic CD4P Por CD8P P T-cells from 8 week old LckCreP PElavl1P + fl/fl - Pand LckCreP PElavl1P P mice respectively. (F) Enumeration of HuRP P cells in + fl/fl LckCreP PElavl1P P thymocyte subsets and splenic T-cells; Data are percentages (+SEM) derived from the flow cytometric detection of HuR- cells in CD4/CD8/DP and DN gated populations (n=12-15) at 8-10 weeks of age. Papadaki et al., 2009 Supplementary - Supplemental Figure 2. The increased expansion of HuRP PDN relates to - increases in immature thymocytes and the exclusive presence of HuRP PT- lymphocytes in mutant mice. (A) Detection of DN-descendant immature hi lo + lo + fl/+ CD24P PCD5P PSP cells in CD8P PTCRȕP Pgated LckCreP PElavl1P P(open + fl/fl histogram) and LckCreP PElavl1P Pthymi (filled histogram) and their respective + lo counts (+SD) as CD8P PTCRȕP P immature cells; (*)p,0.05. (B)Contour plots - + showing the representation of HuRP P and HuRP P cells expressing surface TCRȕ + fl/+ + fl/fl in gated LckCreP PElavl1P Pand LckCrePPElavl1P PDP and SP thymocytes as well as splenic T cells. Papadaki et al., 2009 Supplementary Supplemental Figure 3. Effects of HuR deletion on the presence of TCR- + + + + restricted thymocytes (A) Detection of HuR in F5P P LckCreP PElavl1fl/P PandPPF5P P + fl/fl LckCreP PElavl1P Pthymocytes expressing the F5-TCR (Vȕ11). (B) Detection of F5 + + transgenic cells as Vȕ11-gated CD4/8 thymocytes or splenic CD8P P T cells in F5P P + fl/fl LckCreP PElavl1P Ptransgenic mice, relative to the corresponding control subsets. + + + fl/fl (C) F5P P T-cell counts in thymi and spleens of F5P P LckCreP PElavl1P Pmice relative controls. Data (+SD) derived from n=7 mice/group at 8 weeks of age; (*)underlined numbers are p values of statistical difference. (D) Detection of HuR in female + + fl/fl tgHYP P LckCreP PElavl1P Ptransgenic thymocytes or splenocytes, expressing the HY- + + fl/+ TCR (T3.70) relative to the corresponding tgHYP P LckCreP PElavl1P Pcontrols. (E) + + Detection of HY-TCRP P transgenic cells as T3.70-gated DN, DP and CD8P PSP + + + fl/fl thymocytes as well as CD8P P splenocytes in female tgHYP P LckCreP PElavl1P Pmice + + relative to the corresponding controls.(F) HY-TCRP P T-cell counts in female tgHYP P + fl/fl LckCreP PElavl1P Pthymi and spleens relative to control values. Data (+SD) from n=7 mice/group at 10 weeks of age; (*) underlined numbers correspond to p values. Papadaki et al., 2009 Supplementary Supplemental Figure 4. Changes in the presence of chemokine - responsive HuRP Pthymocyte subsets are not due to defects in the expression of chemokine receptors. (A) qRT-PCR detection of S1PR1R (EDG- 1), Klf2, CXCR4 and CCR7 mRNAs in thymocyte extracts derived from + fl/fl LckCreP PElavl1P Pmice. Black bar graphs denote expression values (+SD) in + fl/fl LckCreP PElavl1P Pcultures as fold change relative to those from + fl/+ LckCreP PElavl1P Pcontrols (White Bar-Ref). (B) Intracellular flow cytometry histograms showing the expression of S1PR1R (EDG-1), Klf2, CXCR4 or CCR7 + + + fl/+ proteins in gated CD4P P and CD8P P SP thymocytes of LckCreP PElavl1P P(open + fl/fl histogram) and LckCreP PElavl1P P(filled histogram) thymocytes. Dashed histograms refer to isotype match stainings. (C) Representative histograms + fl/+ showing that intracellular HuR levels in SP subsets from LckCreP PElavl1P Pthymi are not altered between resting (open histograms) and chemokine treated (filled histograms) cells. Papadaki et al., 2009 Supplementary 8 - Supplemental Figure 5. Differences in HuRP P thymocyte deletion following antigenic or corticosteroid stimulation and detection of death-related + fl/+ mRNAs. (A) Flow cytometric detection of thymocyte loss in LckCreP PElavl1P + fl/fl Pand LckCreP PElavl1P Pmice following the in vivo injection of ĮCD3 or saline. (B) + + fl/+ + Detection of F5-TCR expressing thymocytes in F5P P LckCreP PElavl1P Pand F5P P + fl/fl LckCreP PElavl1P Pmice injected with variable doses of the NP68 peptide. (C) Response of HuR-deficient thymocytes to dexamethasone induced apoptosis. Thymocytes were cultured in vitro and apoptosis was induced with 100nM dexamethasone. Cells were collected at 0, 4, 16, 24 hours post stimulation. + fl/fl LckCreP PElavl1P Pthymocytes (filled histogram) appear to be as susceptible as control thymocytes (solid line) to dexamethasone induced apoptosis as shown by Annexin V/Propidium iodide staining and flow cytometric analysis. The line + + graphs show both the percentages of dead and apoptotic CD4P PCD8P P cells. Results shown are representative of two independent experiments with n=3 mice/group. (D) qRT-PCR detection of pro-apoptotic mRNAs in + fl/fl LckCreP PElavl1P Pthymocytes. Data (+SD) are fold changes relative to control values (White bar-Ref) and (*) p<0.05. Papadaki et al., 2009 Supplementary Supplemental Figure 6. Expression of known HuR-targets in activated HuR-deficient thymocytes. Microarray analysis of thymocyte mRNAs previously reported to interact with HuR. Shown are normalized relative + +/+ fl/fl expression values from control LckCreP P Elavl1P P (Ɣ) and Elavl1P P (ż) as well + fl/fl as test LckCreP P Elavl1P P (Ɣ) thymocyte cultures stimulated with PMA/ionomycin for 0,4 and 12 hrs. Papadaki et al., 2009-Supplementary @ Supplementary Table 1a. List of Downregulated Genes in HuR-deficient thymocytes before and after mitogenic stimulationP O Hrs En Gene Name GeneIDP Description Score 1* FC** Score 2* FC** Bbc3 170770 Bcl-2 Binding Component 3 -3,601 1,575 -5,283 1,808 Cacna1e 12290 Voltage-Dependent R-Type Calcium Channel Alpha-1e Subunit -2,734 1,883 -6,186 2,148 Camk2n1 66259 Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor -4,542 1,419 -7,791 1,891 Col15a1 12819 Procollagen, Type XV -4,023 1,915 -2,627 2,916 Cyp2j6 74519 Cytochrome P450 2j5 -5,336 1,386 -5,023 1,499 D130020L05Rik 68734 Riken Cdna D130020l05 Gene -3,408 1,409 -2,784 1,206 D15Wsu75e 28075 D15wsu75e Protein -3,235 1,405 -4,176 1,439 Doc2g 60425 Double C2, Gamma -3,183 7,126 -5,934 4,116 Flt4 14257 Vascular Endothelial Growth Factor Receptor 3 Precursor -4,063 1,450 -4,644 1,604 Gpr161 240888 G Protein-Coupled Receptor 161 -5,153 1,755 -2,957 1,796 Gzmm 50701 Complement Factor D Precursor -2,626 1,391 -3,977 1,227 Lpin1 14245 Lipin 1 (Fatty Liver Dystrophy Protein) -3,779 1,346 -3,655 1,374 Lta 16992 Lymphotoxin-Alpha Precursor (Lt-Alpha) -3,646 2,087 -3,095 1,776 Med25 75613 Mediator Of RNA Polymerase II Transcription, Subunit 25 Homolog -2,934 1,147 -2,884 1,152 Oxa1L 69089 Oxidase Assembly 1-Like -3,799 1,420 -4,003 1,379 Phlpp 98432 Ph Domain And Leucine Rich Repeat Protein Prosphatase -3,042 1,368 -2,689 1,628 Ppfibp2 19024 Protein Tyrosine Phosphatase, F Interacting Protein, Binding Protein 2 -5,098 1,625 -3,010 1,346 Q8BZ64-2 654470 Tect1_Mouse Isoform 2 Of Q8bz64 -2,626 1,290 -3,129 1,365 Rtel1 269400 Regulator Of Telomere Elongation Helicase 1 -4,273 1,343 -3,080 1,299 Shf 435684 Src Homology 2 Domain Containing F -4,957 1,427 -10,059 1,449 Slc13a1 55961 Solute Carrier Family 13, Member 1 -3,401 1,913 -3,309 2,114 Ssrp1 20833 Structure-Specific Recognition Protein 1 -2,842 1,287 -3,167 1,182 Stx5a 56389 Syntaxin 5a -3,021 1,350 -4,915 1,359 Tfpi 21788 Tissue Factor Pathway Inhibitor Precursor -9,815 2,178 -10,552 1,757 Tnfrsf25 85030 Tumor Necrosis Factor Receptor Superfamily, Member 25 -3,054 2,133 -3,592 1,886 Unc13a 382018 Unc-13 Homolog A (C. Elegans) -3,057 1,769 -2,710 1,354 4 Hrs Gene Name Gene ID Description Score 1 FC Score 2 FC 1500011H22Rik 68948 Riken Cdna 1500011h22 Gene -7,065 1,403 -2,650 1,292 1700003H04Rik 384775 RIKEN Cdna 1700003H04 GENE -3,359 2,470 -3,232 2,607 4930404H21Rik 73808 RIKEN Cdna 4930404H21 GENE -3,515 1,541 -3,854 1,569 4930579E17Rik 75847 Riken Cdna 4930579e17 Gene -8,278 1,965 -5,110 2,011 4933431D05Rik 381714 RIKEN Cdna 4933431D05 GENE -3,344 1,787 -5,589 2,328 5730557B15Rik 67434 RIKEN Cdna 5730557B15 GENE -3,613 2,324 -3,151 1,992 9430057O19Rik 231093 RIKEN Cdna 9430057O19 GENE -3,344 1,787 -6,657 2,173 Papadaki et al., 2009-Supplementary A830093I24Rik 207921 RIKEN Cdna A830093I24 GENE -3,313 1,319 -11,589 1,839 Abca4 11304 Atp-Binding Cassette, Sub-Family A, Member 4 -5,152 1,651 -2,981 1,289 Atf7ip2 75329 Activating Transcription Factor 7 Interacting Protein 2 -9,664 1,292 -3,818 1,175