Concentration- and time-dependent response of hTERT expression by androgens. LNCaP (A,B) or C4-2 (C,D) cells were treated with the indicated concentrations of R1881 for 3 days and were analyzed by qRT-PCR for KLK3 mRNA, encoding PSA, as a positive androgen-responsive gene (A,C), or for the hTERT mRNA levels (B,D). β-Actin was used for normalization. The values were calculated relative to DMSO, which was set arbitrarily as 1. Error bars indicate SEM. (E) hTERT expression was analyzed at an mRNA level by qRT-PCR of LNCaP cells treated for the indicated time periods with 1nM R1881. Indicated are the relative values normalized to house-keeping gene β-Actin and RPL13a. (F) Western blotting indicates the protein level of the two hTERT isoforms (arrows) at the indicated treatment times. The treatment with second-generation AR antagonist Enzalutamide (Enz; 10 µM) was included. β-Actin was used for normalization. DMSO served as the solvent control. p-values: * p ≤ 0.05, ** p ≤ 0.01.