FIG. 8.

Inhibition of HDAC activity increases DR5 expression. (A) (i) HEK293 cells were untreated (C) or treated with TSA (45 nM), VPA (500 μg/ml), or etoposide (Eto; 100 μM) for 24 h. An RNase protection assay was performed by using the hAPO3d probe set (Pharmingen) as described in Materials and Methods. A similar trend was observed in three different experiments. (ii) The amount of DR5 mRNA was quantified by a phosphorimager as the fold increase normalized to loading control (L32). (B) HEK293 cells were untreated, treated with EGF (1 μg/ml) alone or TSA (45 nM) alone or EGF and TSA in combination for 24 h. The cells were lysed and Western blotted with antibodies against DR5 or DR4. The blots were stripped and reprobed with antibodies against actin (loading control). (C) Cells were transfected with luciferase reporter gene constructs containing a promoter with NF-κB binding sites (NFκB-Luc) or a promoter with p53 binding sites (p53-Luc). The cells were then treated with TSA (45 nM) for 16 h and the increase in luciferase activity was determined. (D) The cells were also treated with TSA (45 nM) for 16 h and lysed. A ChIP (i.p.) assay was performed using antibodies against p65, p53, or HDAC1 as described in Materials and Methods. Sample with no antibody added was used as a negative control; genomic DNA was used as the input control.