Figure 4.

Effect of BIC, ENZA and APA on the expression of several apoptotic regulators in LNCaP-WT and LNCaP-STEAP1 knockdown cells. LNCaP cells were transfected with siRNA targeting STEAP1 or scramble siRNA. 24 h following transfection, LNCaP cells were treated with 100 µM of BIC, or 10 µM of ENZA or 10 µM of APA for 24 h. (A) Bax/Bcl-2 protein ratio, (B) p53 protein expression and (C) p21 mRNA expression were determined by western blotting and reverse transcription-quantitative PCR, respectively. (D) Caspase-3 activity was measured spectrophotometrically by the release of the product pNA and (E) immunofluorescence analysis of TUNEL-positive cells was determined by the TUNEL assay being the results expressed as the mean of TUNEL-positive cells (red staining) relatively to the total cell number [Hoechst 33342 (blue) staining]. (F) Relative protein expression was normalized with total protein load on gel as represented in and relative mRNA expression was normalized with the β2M housekeeping gene. Representative immunoblots are also shown. (G) Representative microscopy images showing TUNEL and Hoechst staining in the different groups were obtained in the AxioImager Z2 fluorescence microscope (magnification, ×400). Results are expressed as fold-variation relative to LNCaP-WT (control group). Error bars indicate mean ± standard error of the mean (n≥2). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. the LNCaP-WT condition; ^##P<0.01 and ^###P<0.001 vs. the LNCaP-STEAP1 knockdown condition; ^$$P<0.01 vs. LNCaP-WT plus respective drug. BIC, bicalutamide; ENZA, enzalutamide; APA, apalutamide; WT, wild type; siRNA, small interfering RNA; STEAP1, six transmembrane epithelial antigen of the prostate 1; β2M, β-2-microglobulin; WT, wild type.