FIGURE 5.

Involvement of Src kinase pathway in LCoR-mediated repression of AR. A, C4-2 cells were transfected with MMTV-Luc, pSG5, or pSG5-LCoR and treated with R1881 and Src kinase inhibitor PP2. The graph represents the fold hormone induction. B, C4-2 cells transfected with MMTV-Luc, VP16, or VP16-cLCoR (1 μg) were treated with R1881 and PP2. The graph represents the fold hormone induction. The influence of PP2 on LCoR-mediated AR repression (A) and on LCoR binding to AR (B) was significant (Student's t test, p < 0.001). C, a ChIP analysis was performed with C4-2 cells. Before lysis, samples were treated with PP2 for 48 h and with 10⁻⁸ m R1881 for 1 h. The harvested lysate was diluted and immunoprecipitated with antibodies against AR, anti-LCoR, or nonspecific anti IgG antibody. The DNA was eluted from the immunoprecipitates and amplified by primers spanning the PSA enhancer. As equal starting material prior to immunoprecipitation, the input is shown. D, C4-2 cells were transfected with the p(UAS)₄TATA-Luc reporter along with 1 μg of the Gal-LCoR plasmid and treated with U0126, rapamycin, LY294002, and PP2, and data were plotted with respect to values obtained for each empty vector and represent fold reporter repression over galactosidase empty vector control. E, in total, 200,000 C4-2 cells/well were seeded out in hormone-depleted FBS containing T media in six-well tissue culture dishes. After 24 h, cells were treated with R1881 (10⁻¹⁰ m) for 48 h. Then, total cellular RNA was isolated, reverse-transcribed to cDNA, and amplified by light cycler using specific primers and control primers for actin. The graph represents the actin-normalized values of the PSA transcript.