Figure 4

Increased levels of antisense intronic MYO5A transcript correlates to binding of androgen receptor to a putative upstream androgen response element. (A) Expression level of antisense intronic transcript of MYO5A was measured in androgen-treated (black bars) or untreated control (white bars) cells. Relative fold change (Y axis) shows the increase or decrease in transcript levels after androgen treatment relative to their levels in untreated control cells. Error bars refer to standard deviations relative to mean fold-change values. Differential expression of antisense MYO5A transcripts was statistically significant (p < 0.05) as indicated by asterisks. (B) Cellular lysates from androgen-treated (+) or control cells (-) were used in chromatin immunoprecipitation (ChIP) assays in the presence of an antibody reactive with the androgen receptor protein (anti-AR). PCR reactions were used for detection of immunoprecipitated DNA containing an androgen response element (ARE) located upstream to the MYO5A antisense intronic transcript (asMYO5A ARE) in androgen-treated (+) or untreated control cells (-). Prostate-specific antigen ARE (PSA ARE), or a non-related genomic sequence upstream of the tubulin α6 gene (TUBA6) were used as positive and negative PCR controls, respectively. An unrelated antibody (mouse IgG) was used as a control for nonspecific immunoprecipitation. Equal volumes of lysates from androgen-treated or control cells (1:100 of total lysate) were treated in parallel, and used as loading controls of input DNA for each ChIP assay (input). (C) Enrichment ratios of asMYO5A ARE bound to AR in androgen-treated cells relative to control cells. Enrichment ratios (Raw) represent mean ratio values calculated from the integrated band intensities obtained at two different PCR cycle numbers (25 and 30 cycles), in reactions similar to those of Figure 4B, as described in Methods. Band intensities from input DNA were used to obtain normalized enrichment ratios (normalized). Error bars show the standard deviation of mean ratios.