Figure 3. Androgen-depletion or AR knockdown triggers a TIF response.
(A), Androgen -depletion triggers a TIF response. Exponentially growing LNCaP cells in complete medium were either left untreated (bar labeled FBS), or were treated with 100 μM Casodex (FBS+Casodex) or Casodex + 10 nM R1881, for 24 hr. To determine the effect of androgen depletion, LNCaP cells growing in complete medium (FBS) were switched to steroid-free medium (CSS, phenol red-free medium containing 10% charcoal-stripped serum) or CSS medium + 10 nM R1881, and incubated for 24 hr. Data are presented as the percentage of cells with >5 53BP1 foci/cell. Bars represent the mean ± standard deviation. (B, C, D), AR knockdown triggers a TIF response. Exponentially growing LNCaP cells were treated with AR-siRNA (AR) or scrambled (SC)-siRNA for 48 hr. (B), Cell extracts were subjected to Western blot to confirm knockdown of AR protein and PSA, an AR target gene. Actin was used as a loading control. Total RNA was extracted from siRNA-treated cells using Trizol (Invitrogen) and RT-PCR was performed as described previously [