Figure 5
From: HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses
Validation of vorinostat-induced transcriptional responses by qRT-PCR. Transcriptional responses of BJ and BJ LTSTERas fibroblasts following DMSO (black) and 25 μM vorinostat treatment (red) were validated by qRT-PCR using the same template RNA as that used in the microarray study. Log2 transformed expression intensities of HG U133 plus 2.0 probe sets corresponding to (a) BAK1, BAD, BIK and BCL2A1 and (b) BCL2L11 and BMF are shown. Gene names and probe set IDs are indicated in the title of each panel. Normalized probe set intensity values (Y-axis) are presented, which allows direct comparison of basal expression (time 0 h), and log2 transcriptional responses over time between cell types (BJ and BJ LTSTERas). Data represent the summarized probe set intensities from three independent microarray time course experiments. qRT-PCR verification of selected genes is shown. Transcript expression was calculated relative to the control gene RPL32 (that does not respond to vorinostat or DMSO treatment), and fold-change mRNA expression over time was calculated relative to the 0 h time point. Data are presented as the mean±S.E.M. of three independent experiments. For both Affymetrix and qRT-PCR results, data sets for BJ cells are in the left panels while data sets for BJ LTSTERas are in the right panels. (c) Whole cell protein extracts from BJ and BJ LTSTERas fibroblasts treated with DMSO or 25 μM vorinostat for 0–48 h were separated by SDS-PAGE and analyzed for the expression of BMF, BIK and BIM by western blotting. The expression of α-Tubulin was analyzed to confirm equal protein loading in each lane. The asterisk (*) denotes a non-specific band in the BIK western blot, which acts as a positive control for activity of the BIK antibody. The arrow denotes the location of BIK. Images are representative of two independent experiments
