Figure 3

Certain PRG/IRGs are significantly expressed in the presence of FVP during a mitogenic response. (A) Effects of FVP in the cell cycle were determined by FACS of Propidium Iodide(PI)stained cells. The percent of cells in S phase is indicated at the top of each panel. BJ-TERT fibroblasts were treated with either DMSO or 300 nM FVP followed by serum stimulation for 0, 14, 22 and 30 h and stained with PI. (B) Western blot analysis of cell cycle progression, transcription markers and MCL1. BJ-TERT fibroblasts were harvested and lysed. Ten microgram of protein was resolved by 8% polyacrylamide/SDS gel electrophoresis, transferred to a PVDF membrane and specific antibodies were used to detect proteins, as indicated above. (C) 300 nM FVP inhibits phosphorylation of the CTD of RNAPII on Ser-2 and Ser-5 without affecting the phosphorylation state of pocket proteins. Exponentially growing BJ-TERT fibroblasts were treated with FVP at 10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 10 μM for 2 and 4 h. Whole cell lysates were resolved by 6% SDS/PAGE and immunoblotted with antibodies to the indicated proteins/phosphorylation sites. Solid and dashed arrows at the bottom indicate the concentration of FVP leading to dephosphorylation of RNAPII and pocket proteins, respectively. The asterisk indicates a crossreacting band recognized by the anti-p130 antibody. (D) mRNA levels of selected genes at the indicated time points were detected by Q-RT-PCR. The results of three different experiments are shown. Data are represented as a fold change value of levels normalized to 1 at time zero. The results of three different experiments are shown.