Figure 5.

Ectopic expression of UGT2B15 reduces sensitivity to ralaniten. (A) Real-time PCR of UGT2B11, UGT2B15, UGT2B17, and UGT2B28 mRNA transcript normalized to levels of SDHA transcript harvested from untreated clones isolated following lentiviral transduction, and LNCaP-RAL^R cells. Data presented as mean ± SEM and analyzed by one-way ANOVA with Dunnett’s test applied post hoc, n = 3 independent experiments. (B) Western blots of ectopic and endogenous UGT2B15/UGT2B17 in LNCaP-RAL^R and lentiviral transduced clones. (C) UGT2B enzymatic activity quantified using microsomes harvested from LNCaP-RAL^R, 2B15-HA (C2), 2B17-HA (C1), and GFP-HA (C1) cells. Data presented as mean ± SEM and analyzed by two-way ANOVA with Tukey’s test applied post hoc, n = 3 independent experiments. (D) Dose response curves showing androgen dependent growth of LNCaP-RAL^R and lentiviral transduced clones treated with ralaniten and stimulated with 0.1 nM R1881. IC₅₀ values were calculated using a linear regression and interpolating/extrapolating where the line crossed 50% growth. GFP-HA (C1), 18.15 μM; LNCaP-RAL^R, 39.68 μM; 2B15-HA (C2), 32.48 μM; 2B17-HA (C1), 28.14 μM. Data presented as mean ± SEM and analyzed by two-way ANOVA with Dunnett’s test applied post hoc, n = 5 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ^#p < 0.0001; n.s., not significant.