Research Paper Volume 5, Issue 1 pp 3—17

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 [44] to measure mRNA levels using sequence specific primers for GAPDH and PSA as described previously [86]. (C), Cells were stained with antibodies to 53BP1 (red) and γH2AX (green). (D), Cells were stained with antibodies to γH2AX (green) and TIN2 (red) in order to evaluate the TIF response to AR knockdown. (E), Higher magnification images of a representative AR-siRNA-treated cell stained with antibodies to γH2AX and TIN2. (F), Quantitation of dysfunctional telomeres following AR knockdown or control SC-siRNA, based on the percentage of cells with >5 53BP1 foci/cell.