Figure 5
GCN5 inhibition induces Bim expression through transactivating Egr-1 and E2F1. (a) CGNs were exposed to CPTH2 or dimethyl sulphoxide (DMSO) in 25K media for 18 h, small mRNAs were extracted and reverse transcribed as templates for Q-PCR to detect miR-24 expression and U6 small nuclear RNA (snRNA) was amplified to normalize the miR24 outputs. (b) CGNs in 25K media exposed to CPTH2 alone or together with indirubin-3’-oxime (10 μM), flavopiridol (2 μM), mithramycin A (10 μM), chromomycin A3 (0.3 μM), olomoucine (200 μM), roscovitine (50 μM), IGF (100 ng/ml), SP600125 (10 μM), pifithrin-alpha (2 μM) or JSH-23 (5 μM) for 24 h were subjected to WB to detect Bim expression; tubulin was reprobed to verify equal loadings. (c–e) CGNs in 25K media treated with CPTH2 at the indicated doses for 24 h or treated with CPTH2 (50 μM) for the indicated time courses were subjected to WB or RT-PCR to detect E2F1 and Egr-1 expression in protein or mRNA. (f and g) The cleaved chromatin fragments were immunoprecipitated by anti-E2F1 or Egr-1 antibody and then purified as templates to amplify the region of bim promoter by PCR. The part of chromatin fragments before IP was purified as templates to amplify bim promoter as a reference. (h) Rat C6 glioma cells transfected with NC, siE2F1 and siEgr-1 were subjected to WB to detect the E2F1 or Egr-1 expression. Tubulin was reprobed to verify equal loadings. (i) DIV5 CGNs transfected with NC, siE2F1 and siEgr-1 together with pCMV-Gal plasmids for 36 h were exposed to CPTH2 (50 μM) in 25K media for 18 h and then were subjected to immunofluorescence to detect the Bim expression. The percentage of stained β-Gal-positive neuron population with Bim was calculated. (j) DIV5 CGNs transfected with NC, siE2F1 and siEgr-1 together with p-GFP plasmids for 36 h were exposed to CPTH2 (50 μM) in 25K media for 24 h and then were subjected to nucleic staining and apoptotic analysis as in Figure 2c. Mean±S.E.M., n=3, *P<0.05
