Fig. 2
TRX1 suppression inhibits CRPC cell proliferation and promotes AD-associated cell death. a Western blot validation of shRNA constructs in LNAI. Blot was run using 10 μg of total protein lysate. b TRX1 suppression-mediated effects on cell growth in LNAI, under androgen-replete (FBS) or androgen-deprived (CSS) conditions. Cells were plated at an initial density of 5 × 104 and counted on the indicated days. c Relative change in LNAI cell numbers following 7 days of culture. Numbers from the indicated samples were normalized to shGFP values (=1) to show the relative degree of growth defect by TRX1 suppression under FBS or CSS culture. d TRX1 suppression-mediated effects on cell growth in the 22Rv1 CRPC line. Cells were plated and processed as in (b). e TRX1 suppression produces a lesser degree of growth inhibition in the androgen-responsive LNCaP SB0 line relative to LNAI. Cells were plated at an initial density of 1.5 × 105. f Progression to CRPC sensitizes cells to TRX1 suppression-induced growth defects. LNCaP SB5 (early CRPC) were plated and processed as in (b). These cells are derived from LNCaP SB0 cells in (e). g LNAI cells transduced with shGFP or shTRX1-259 were plated at 5 × 104 cells and cultured for 7 days under either FBS or CSS conditions. Total cell numbers in each category and the corresponding percent total cells stained with Trypan blue are shown. h Annexin V staining to detect apoptotic cells. Staining was carried out in LNAI cells, transduced with either shGFP or shTRX1-259, following 48 h of culture under denoted conditions. A rightward shift and increased peak height (indicated by the arrow) show elevated staining. The flow cytometric profile is representative of two independent experiments. i Western blot of p53 and cl-PARP protein levels. Blots were run using 10 μg of total protein from LNAI cells, following 3 days of culture under the indicated conditions. Relative changes in protein expression from n = 2 blots, from independently established samples, were normalized to shGFP levels under FBS conditions (right). Note that all error bars in this figure represent ± SD
