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Impact of G9A Expression on p53 Activation in Cancer Cells

Investigating the relationship between G9A expression and p53 activation in lung and colon cancer cells, highlighting potential survival differences based on G9A levels.

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Impact of G9A Expression on p53 Activation in Cancer Cells

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  1. A B D C M.w. kDa G9a 150 FLAG p53 β-actin 50 SET9 50 p53 β-actin 40 E F P21 expression H1299 (p53 -/-) cells p53 + + + + + + - - + + - - F-hG9a Normalized relative band intensity F-mG9a - - - - + + M.w. kDa - + - + - + Dox p21 20 Puma 20 M.w. kDa M.w. kDa mG9a Puma expression 150 hG9a 150 150 G9a 50 p53 50 p53 50 Normalized relative band intensity 40 b-actin 40 β-actin 40 Figure 1

  2. C A M.w. kDa + - pLKO + - - + shG9a - + 150 G9a β-actin 40 shG9a#pool shG9a#2 D mRNA Expression (fold) mRNA Expression (fold) B - - + + - - - - F-hG9a(S) F-hG9a(L) - - - - + + - - F-hGLP - - - - - - + + M.w. kDa - + - + - + - + Dox FLAG 150 p53 50 β-actin 40 Figure 2

  3. A B HCT116 (p53+/+) cells treated with BIX-01294 Fold activation, PUMA-luc M.w. kDa 50 p53 β-actin 40 Figure 3

  4. A B C D H1299 (p53 -/-) cells H1299 (p53 -/-) Cells p21 promoter Start Site IP: α-FLAG M.w. kDa DAPI (DNA) GFP (p53) α-IgG α-FLAG INP p53RE CBP -2250 +1 250 150 merge Flag (G9a) Flag-G9a (S) 50 p53 Relative Fold Change H460 (p53 +/+) cells CBP PUMA promoter Start Site G9a (S) p53RE CBP DAPI (DNA) +1 -224 Flag (G9a) merge p53 E Dual cross-linking ChIP: HCT116 cells (α-CBP) G H F M.w. kDa M.w. kDa CBP 250 CBP 250 150 G9a FLAG-G9a 150 β-actin 40 p53 50 β-actin 40 Figure 4

  5. A B H460 (p53 +/+) cells pCDNA3 hG9a(S) mG9a -Dox +Dox D H1299 (p53 -/-) cells pLKO + - C pLKO-shG9a - + + + M.w. kDa p53 + + Dox 150 G9a PARP 100 Cleaved-PARP p53 50 β-actin 40 Figure 5

  6. GEO dataset ID: GSE13213 GEO dataset Type: lung cancer GEO dataset ID: GSE31210 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=1 96 18.4 13.5 1.78 3.34 group=2 127 13.8 18.7 1.28 3.34 Chisq= 3.3 on 1 degrees of freedom, p= 0.0677 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=1 88 23.9 30.73 1.53 9.51 group=2 28 14.3 7.48 6.26 9.51 Chisq= 9.5 on 1 degrees of freedom, p= 0.00204 Figure 6

  7. Me Me CDP/Cut, Gfi1, UHRF1, WIZ PCAF CBP CBP hG9a mG9a Ac Ac p53 p53 TATA TATA Transcription NO transcription Human G9a Mouse G9a Figure 7

  8. 3 + + [H ]-Me M.w. kDa Coomassie GST-p53300-393 + + Histones 6His-G9a 130 100 70 GST-p53300-393 55 35 25 Histones Histones Histones 15 KMT G9a 130 95 72 6His-Set7/9 55 GST-p53300-393 43 34 26 17 KMT Set7/9 130 95 72 Flag-Smyd2 55 GST-p53300-393 43 34 26 17 KMT Smyd2 Supplementary S1

  9. M.w. kDa 150 G9a 50 mp53 β-actin 40 Supplementary S2

  10. M.w. kDa 150 50 40 Flag-G9a p53 β-actin Supplementary S3

  11. Relative Fold Change Supplementary S4

  12. H460 (p53 +/+) cells - + - hG9a(S) M.w. kDa - - + mG9a + + + Dox PARP 100 Cleaved-PARP G9a 150 p53 50 β-actin 40 Supplementary S5

  13. 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=1 28 8.17 10.73 0.611 6.17 group=2 7 4.01 1.45 4.512 6.17 Chisq= 6.2 on 1 degrees of freedom, p= 0.013 Supplementary S6

  14. GEO dataset ID: GSE39582 Cancer Type: Colon cancer p-value: 0.0227 Positive effect: EHMT2__high|TP53__high N Observed Expected (O-E)^2/E (O-E)^2/V EHMT2__high|TP53__high; 67 15.4 23.1 2.58 5.19 Other; 123 45.8 38.1 1.56 5.19 Chisq= 5.2 on 1 degrees of freedom, p= 0.0227 Supplementary S7

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