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Published byAtle Christensen Modified over 6 years ago
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A B D C E F Figure 1 + + + + + + - - + + - - - - - - + + - + - + - +
FLAG β-actin p53 SET9 150 50 40 M.w. kDa D β-actin p53 G9a 50 40 150 M.w. kDa C E F P21 expression H1299 (p53 -/-) cells p53 F-hG9a F-mG9a Normalized relative band intensity M.w. kDa Dox p21 20 Puma 20 M.w. kDa mG9a Puma expression hG9a 150 150 G9a p53 50 p53 50 Normalized relative band intensity b-actin β-actin 40 40 Figure 1
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mRNA Expression (fold)
C mRNA Expression (fold) pLKO M.w. kDa shG9a 150 G9a β-actin 40 shG9a#pool shG9a#2 D mRNA Expression (fold) B F-hG9a(S) F-hG9a(L) F-hGLP M.w. kDa Dox FLAG p53 β-actin 150 50 40 Figure 2
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A B Figure 3 p53 β-actin Fold activation, PUMA-luc
HCT116 (p53+/+) cells treated with BIX-01294 Fold activation, PUMA-luc M.w. kDa 50 40 Figure 3
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Dual cross-linking ChIP: HCT116 cells (α-CBP)
Relative Fold Change H1299 (p53 -/-) Cells DAPI (DNA) GFP (p53) Flag (G9a) merge H1299 (p53 -/-) cells p21 promoter Start Site IP: α-FLAG M.w. kDa INP α-IgG α-FLAG p53RE CBP -2250 +1 250 150 Flag-G9a (S) 50 p53 H460 (p53 +/+) cells PUMA promoter Start Site CBP G9a (S) p53RE DAPI (DNA) CBP -224 +1 Flag (G9a) merge p53 E Dual cross-linking ChIP: HCT116 cells (α-CBP) G H F M.w. kDa 250 150 50 40 M.w. kDa CBP 250 CBP 150 FLAG-G9a G9a β-actin p53 40 β-actin Figure 4
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A B -Dox D C Figure 5 + - - + + + + + mG9a pCDNA3 hG9a(S)
H460 (p53 +/+) cells -Dox +Dox D H1299 (p53 -/-) cells pLKO C pLKO-shG9a M.w. kDa p53 Dox G9a 150 PARP 100 Cleaved-PARP p53 50 β-actin 40 Figure 5
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A B Figure 6 GEO dataset ID: GSE31210 GEO dataset ID: GSE13213
GEO dataset Type: lung adenocarcinoma I-II stage group=1: low expression of G9A group=2: high expression of G9A N Observed Expected (O-E)^2/E (O-E)^2/V group= group= Chisq= 3.3 on 1 degrees of freedom, p= GEO dataset ID: GSE13213 GEO dataset Type: lung cancer A B Survival differences between Group=1 and Group=2 Group=1: green , strong positive correlation (0.79) between TP53 and G9A Group=2: red , no (or negative) correlation (-0.22) between TP53 and G9A N Observed Expected (O-E)^2/E (O-E)^2/V group= group= Chisq= 9.5 on 1 degrees of freedom, p= Figure 6
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p53 p53 Transcription NO transcription PCAF CBP Ac Ac Human G9a
CDP/Cut, Gfi1, UHRF1, WIZ PCAF CBP hG9a CBP mG9a Ac Ac Me p53 p53 Me TATA TATA Transcription NO transcription Human G9a Mouse G9a Figure 7
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Supplementary S1 + + + + Coomassie [H ]-Me M.w. kDa GST-p53300-393
Histones 6His-G9a 130 100 70 GST-p 55 35 25 Histones 15 KMT G9a 95 130 72 6His-Set7/9 55 GST-p 43 34 26 Histones 17 KMT Set7/9 130 95 72 Flag-Smyd2 55 43 GST-p 34 26 Histones 17 Coomassie [H ]-Me 3 KMT Smyd2 Supplementary S1
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M.w. kDa G9a 150 mp53 50 β-actin 40 Supplementary S2
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M.w. kDa β-actin p53 Flag-G9a 150 50 40 Supplementary S3
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Relative Fold Change Supplementary S4
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Supplementary S5 - + - - - + + + + H460 (p53 +/+) cells hG9a(S) M.w.
hG9a(S) M.w. kDa mG9a Dox PARP 100 Cleaved-PARP G9a 150 p53 50 β-actin 40 Supplementary S5
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Supplementary S6 GEO dataset ID: GSE36471
GEO dataset Type: lung cancer Survival differences between Group=1 and Group=2 Group=1: green , strong positive correlation (0.67) between TP53 and G9A Group=2: red , no (or negative) correlation (-0.64) between TP53 and G9A N Observed Expected (O-E)^2/E (O-E)^2/V group= group= Chisq= 6.2 on 1 degrees of freedom, p= 0.013 Supplementary S6
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Supplementary S7 GEO dataset ID: GSE39582 Cancer Type: Colon cancer
p-value: Positive effect: EHMT2__high|TP53__high N Observed Expected (O-E)^2/E (O-E)^2/V EHMT2__high|TP53__high; Other; Chisq= 5.2 on 1 degrees of freedom, p= Supplementary S7
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