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Supp Figures & Tables.Pdf SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. H226 cells express ΔNp63α and functional p53. (A) Western blot of total cell extracts from H226 cells as well as myc tagged p63 isoforms using a pan-p63 antibody and an antibody that specifically recognizes the α-isoforms of p63. (B) FACS analysis of cell cycle profile following staining with propidium iodide. H226 cells were treated with 10uM Nutlin-3, a non-genotoxic activator of p53, for 48 hours. (C) Sulforhodamine B (SRB) assay of cell proliferation following 5 days of knockdown of ΔNp63α and/or p53. (D) Cell cycle profile following 5 days of knockdown of ΔNp63α and/or p53. (E) Apoptotic index of cells following 5 days of knockdown of ΔNp63α. Treatment with 375 µM of the antimetabolite 5-fluorouracil (5FU) for 48 hours serves as a positive control. Cells were stained with annexin V and propidium iodide and analyzed by flow cytometry. Histograms represent the percentage of cells positive for annexin V staining. Supplemental Figure 2. ΔNp63α does not repress 14-3-3σ, GADD45A, NOXA or PUMA in H226 cells. Validation of microarray results by quantitative RT-PCR analysis of additional Class I genes following 12 hours of 10 µM Nutlin-3 treatment, 48 hours of ΔNp63α knockdown, or combination treatment. Supplemental Figure 3. Gene-specific repression by ΔNp63α in diverse cell types. Q-RT-PCR analysis of selected Class I, II, and III target genes following 48 hours of ΔNp63α knockdown in (A) HaCaT cells (immortalized keratinocytes), (B) SCC-13 cells (oral HNSCC), (C) Cal-27 (tongue HNSCC). Supplemental Figure 4. Gene expression classes are conserved following administration of additional p53 activating stimuli. (A) Western blot and (B) Q-RT- PCR analysis of selected Class I, II, and III target genes following 12 hours of p53 activation by the antimetabolite 5FU in H226 cells. Supplemental Figure 5. A schematic representation of p53 and ΔNp63α target gene loci. A linear scale model of selected Class I, II, and III gene loci indicating the locations of the p53/p63 response elements (RE) (REs indicated by asterisk, if more than one RE is present, the RE used for chromatin immunoprecipitation analysis is indicated in red), transcription start sites and exon/intron structure. Coding exons are indicated in black, UTRs in white. Sequence information for the p53/63 RE binding site(s) are indicated to the right of the schematic for each locus. Supplemental Figure 6. Presentation of Figure 2 according to % input for all IPs. ChIP assays of the ZHX2 locus were performed using cell extracts from H226 cells. (A) Extracts from cells before (black lines) and 48 h after (blue lines) ΔNp63α knockdown were utilized. (B) Extracts from control cells (black lines) and DMAP1 constitutive knockdown cells (green lines) were utilized. Antibodies specific for p63, Ser5- phosphorylated RNAPII, DMAP1, RUVBL2 and H2A.Z were used. ChIP-enriched DNA was quantified by real-time PCR. The position of each PCR amplicon is indicated on a linear scale model of the locus. The position of the p53/p63 response element is indicated by a red asterisk. The gray band represents the transcribed region of the locus. Supplemental Figure 7. p53 and ΔNp63α binding profiles are conserved following administration of additional p53 activating stimuli. ChIP assays were performed with whole cell extracts from control cells (white bars) or following 12 h of 375mM 5FU treatment (black bars). Antibodies specific for p53 and p63 were used. ChIP-enriched DNA was quantified by real-time PCR for the p53/p63 response elements of each indicated gene. See Supp. Figure 5 for gene maps and amplicon locations. Supplemental Figure 8. Confirmation of SRCAP complex knockdown effect on Class I and III gene expression. (A) Composition of SRCAP, Tip60, and related complexes. Data combined from reviews by (Jin et al. 2005; Huen et al. 2010; Billon and Côté 2012). (B) Quantitative RT-PCR analysis of gene expression in H226 cells stably expressing a second shRNA against SRCAP subunits SRCAP, ACTR6, ACTL6A, RUVBL1, RUVBL2, DMAP1, VPS72 and YEATS4 and non-SRCAP subunits KAT5, EP400, INO80 and SMARCA2. Supplemental Figure 9. Validation of SRCAP/Tip60 complex member knockdowns. Quantitative RT-PCR validation of SRCAP, ACTR6, ACTL6A, DMAP1, RUVBL1, RUVBL2, VPS72, YEATS4, KAT5, EP400, INO80 and SMARCA2 constitutive knockdown. Supplemental Figure 10. Oncomine analysis of ZHX2 expression. Screenshots from Oncomine (www.oncomine.org) indicating decreased ZHX2 expression in numerous patient (A) squamous cell carcinoma and (B) other carcinoma samples (dark blue bars) as compared to normal lung tissue (light blue bars). Multiple datasets (Hou PLoS One 2010, Estilo BMC Cancer 2009 and Finak Nat Med 2008) were utilized. Supplemental Figure 11. Presentation of Figure 4 according to % input for all IPs. ChIP assays of the ZHX2 locus were performed using cell extracts from H226 cells. (A) Extracts from cells before (black lines) and 48 h after (blue lines) ΔNp63α knockdown were utilized. (B) Extracts from control cells (black lines) and DMAP1 constitutive knockdown cells (green lines) were utilized. Antibodies specific for p63, Ser5- phosphorylated RNAPII, DMAP1, RUVBL2 and H2A.Z were used. ChIP-enriched DNA was quantified by real-time PCR. The position of each PCR amplicon is indicated on a linear scale model of the locus. The position of the p53/p63 response element is indicated by a red asterisk. The gray band represents the transcribed region of the locus. Supplemental Figure 12. Loss of SRCAP and histone variant H2A.Z occupancy upon ΔNp63α knockdown at additional Class III genes. ChIP assays of DMAP1 and RUVBL2 binding to the enhancer regions of the Class III genes NTN4, SAMD9L and IGFBP3 and ChIP assays of H2A.Z binding to the promoter regions of those genes. ChIP was performed with whole cell extracts from control cells (white bars) or following 48 hours of ΔNp63α knockdown (black bars). ChIP-enriched DNA was quantified by real-time PCR. Supplemental Figure 13. Validation of H2A.Z knockdown. (A) Western blot and (B) quantitative RT-PCR validating H2A.Z knockdown following 5 days of treatment with 2ug/mL doxycycline. Supplemental Figure 14. Rescue of ΔNp63α knockdown phenotype by SAMD9L and IGFBP3 knockdown. Time course of cell proliferation using a second shRNA against SAMD9L or IGFBP3. 1x106 cells were seeded at day 0. shp63 cells were pretreated with doxycycline for 5 days prior to seeding to induce p63 knockdown. Supplemental Figure 15. Oncomine analysis of SAMD9L expression. Screenshots from Oncomine (www.oncomine.org) indicating decreased SAMD9L expression in numerous patient (A) lung SCC and (B) other lung cancer samples (dark blue bars) as compared to normal lung tissue (light blue bars). Multiple datasets (Hou PLoS One 2010 and Garber PNAS 2001) utilized. SUPPLEMENTAL TABLES Supplemental Table 1: Microarray results following Nutlin-3 treatment and ΔNp63α knockdown, sorted by fold change. Supplemental Table 2: Microarray results following Nutlin-3 treatment and ΔNp63α knockdown, sorted by p value. Supplemental Table 3: shRNA vector and sequence information. Supplemental Table 4: Antibody information. Supplemental Table 5: Oligonucleotide sequences for quantitative RT-PCR analysis of gene expression and ChIP Q-PCR analysis. Gallant-Behm198069_Supp.Fig1 A B 100 H226 extractmyc-ΔNp63myc-Δαmyc-TAp63Np63γmyc-TAp63α H226γ extractmyc-ΔNp63myc-Δαmyc-TAp63Np63γmyc-TAp63α γ G2/M 170 75 130 S 110 50 G1 ΔNp63α % cells 70 25 Debris 55 40 WB: Pan p63 Ab α-specific p63 Ab Nutlin-3 + C 0.5 D 100 E 80 ) 510 G2/M 75 60 S 0.25 50 40 G1 % cells 25 20 SRB Assay (A Debris Annexin-V positive (%) H226 shp53 shp53 + + shp63 OFF ON OFF shp63 OFF shp63 OFF ON OFF ON 5FU + shp63 ON Gallant-Behm198069_Supp.Fig2 14-3-3σ GADD45A 3 2 2 1 1 NOXA PUMA 4 4 3 3 2 2 Relative mRNA expression Relative mRNA 1 1 shp63 + None Nutlin-3 shp63 Nutlin-3 Gallant-Behm198069_Supp.Fig3 HaCaT 3 shp63 shp63OFF ON ΔNp63α 2 Nucleolin 1 0 SCC-13 14 shp63 shp63OFF ON 10 ΔNp63α 4 Nucleolin 3 2 1 0 Relative mRNA expression Relative mRNA Cal-27 3 shp63 shp63OFF ON ΔNp63α 2 Nucleolin 1 0 4 N p21 BAX T GJB4 ZHX2 N MDM2 PLAC8 IGFBP3 FAM49A SAMD9L Class I: Class II: Class III: p53 induced p53 induced, ΔNp63α repressed ΔNp63α repressed shp63 OFF shp63 ON Gallant-Behm198069_Supp.Fig4 A B Class I p21 BAX MDM2 5 3 8 4 6 3 2 shp63: + + 4 2 1 5FU: + + 1 2 ΔNp63α Class II GJB4 FAM49A PLAC8 p53 2 2 2 p21 1 1 1 Nucleolin Relative mRNA expression Relative mRNA Class III ZHX2 NTN4 SAMD9L 2 2 2 1 1 1 Control 5FU Gallant-Behm198069_Supp.Fig5 p21 AGACTGGGCA/TGTCTGGGCA = 2000bp (CDKN1A) * * GAAGAAGACT/GGGCATGTCT p53R2 TGACATGCCC/AGGCATGTCT (RRM2B) * MDM2 AGTTAAGTCC/TGACTTGTCT ** GGTCAAGTTC/AGACACGTTC PUMA CTGCAAGTCC/TGACTTGTCC (BBC3) * DR5 GGGCATGTCC/GGGCAAGACG (TNFRSF10B) * GADD45A GAACATGTCT/AAGCATGCTG * PCNA GAACAAGTCC/GGGCATATGT * FAS GGACAAGCCC/TGACAAGCCA Class I * MASPIN GAACATGTTG/G/AGGCCTTTTG p53 activated (SERPINB5) * 14-3-3σ AGCATTAGCCC/AGACATGTCC (SFN) * APAF1 * AGACATGTCT/GGAGACCCTAGGA/CGACAAGCCC NOXA GAGCGTGTCC/GGGCAGGTCG (PMAIP1) * FDXR GGGCAGGAGC/GGGCTTGCCC * TCACAAGTTA/G/AGACAAGCCT BAX GGGCAGGCCC/GGGCTTGTCG * * TLR3 AGGCATGCAC/CAACATGCCC * TMEM27 AGACATGTTG/TGGCTTGAAA * PLAC8 TTACAAGTAA/GAACTTGTTT * repressed GCTCATGCTC/C/CATCTAGCCT α GJB4 AGACAAGCCC/AA/GGACAAGGAA Class II * * TCACATGCAG/C/CTCCTTGGCC p53 activated, GGT6 Np63 Δ * BLNK TGACAAGTGA/GAACTTGTCT TCACATGATT/ATGTAG/AAACATGAAA ** EDN2 CTGCAAGCCC/GGGCATGCCC * NTN4 TTCCTAGTAA/GGACTTGTTG * ST14 TTTCATGACC/AGACAAGTCC * ZHX2 GGCCTTGGAA/TTTAAAAAATCAC/TATCAAGCCT
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