Effects of HIF-1β and AR on miR-101 expression. A. Schematic diagram of HIF-1β and AR binding elements upstream of miR-101 coding region. B. Protein and miR-101 expression of DFO-treated PC-3 cells. Nuclear proteins of PC-3 cells treated with 100 μM DFO or mock were analyzed by Western blot (left panel) and Real-Time RT-PCR (right panel, average of two individual experiments with triplicated samples). C and D. LNCaP cells were treated by R1881 and bicalutamide as indicated and analyzed by (C) Real-Time RT-PCR for miR-101 (average of three individual experiments) and (D) Western blot for Ezh2. E, F and G. Effects of R1881 on cell growth, miR-101 levels and gene expression in LNCaP cells. In E, cells were seeded at 1 × 105 cells/well on 6-well plates in phenol red-free RPMI medium with 1% charcoal-stripped FBS, followed by treatment of 0, 0.01, 0.1, 1.0, and 10 nM of R1881. At days 0 and 6, cells in triplicates were counted. Data are the averages of three or more individual experiments. In F, miR-101 levels in the extracted RNA at day 6 were determined by Real-Time RT-PCR with each sample analyzed in triplicate. Data are the average of four independent experiments. Student t-test was used to determine statistical significance (* indicates p < 0.05). In G, whole cell lysates at day 6 of the treatment were analyzed by Western blot using the antibodies labeled on the left.