Figure 3
Inhibition of GCN5 transcriptionally upregulates Bim. (a) CGNs treated with CPTH2 and MB-3 in 25K media at the indicated doses for 24 h and Bim was detected by WB. 5K treatment causes a Bim induction as a positive control. GAPDH was reprobed to verify equal loadings. (b) CGNs treated with CPTH2 (50 μM) and MB-3 (150 μM) in 25K media for the indicated time course were subjected to WB to detect Bim expression. (c) CGNs treated with CPTH2 (50 μM) and MB-3 (150 μM) in 25K media for 12 h were subjected to RT-PCR to detect bim mRNA level. The actin was amplified to verify equal loadings. (d) Pulse chase assay: CGNs treated with CPTH2 (50 μM) in 25K media for 18 h were subjected to further co-treatment of CPTH2 with Actinomycin D (2 μM) for 2, 4 and 8 h, and then to RT-PCR. Changes in bim mRNA levels were determined as changes in band intensity after normalization to actin by the ImageJ software. (e) DIV5 CGNs transfected with bim-luc reporter for 12 h were administered with CPTH2 (50 μM) in 25K media for 24 h and subjected to dual reporter analysis. (f) CGNs transfected with NC, siGCN5a and siGCN5b were subjected to immunofluorescence to detect Bim expression (scale bar=10 μm). (g) CGNs transfected with bim-luc reporter together with NC, siGCN5a and siGCN5b for 48 h were subjected to dual reporter analysis. Mean±S.E.M., n=3, *P<0.05. DMSO, dimethyl sulphoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase
