Fig. 3.

GPI expression is oppositely regulated by AR and androgen/AR-targeted therapy in hypoxia. a The change of GPI mRNA in LNCaP clones in response to androgen (1 nM R1881) and enzalutamide (10 µM) in normoxia or hypoxia. All cells were treated with the indicated conditions for 48 h; the basal condition was CSS media without androgen. GPI mRNA was determined by qRT-PCR. All values were mean ± s.d. relative to the basal-solvent condition, n = 3, *, ^#P < 0.05, **, ***P < 0.01, repeated measures ANOVA. b LNCaP clones were cultured with CSS + 1 nM R1881, and treated by enzalutamide (Enza) or solvent control (R1881) in 20% or 1% O₂ for 48 h. The enzymatic activity of GPI was measured and expressed as mean ± s.d. relative to solvent control, n = 3, *P < 0.05, two-sided t-test. c Representative western blots with LNCaP-AdtHs cells cultured with basal (CSS media), hypoxia (1% O₂), androgen (1 nM R1881), or hypoxia + androgen conditions for 48 h. Tubulin (Tub) was used as loading control. d Representative western blots with LNCaP-AdtHs cells, cultured in CSS + 1 nM R1881 media, and treated with 10 µM enzalutamide (Enza) in normoxia (20%) or hypoxia (1%) for 48 h. e GPI mRNA levels in LNCaP-AdtHs cells in the presence of siRNA silencing HIF1α, HIF2α, AR, or non-targeting control (siNT). Cells were transfected with siRNA, and cultured in the indicated conditions for 72 h. All values are mean ± s.d. relative to the basal condition, n = 3, **P < 0.01, two-sided t-test and/or repeated measures ANOVA. f Representative western blots with DU145+Ev or DU145+AR cells treated by ADT (basal), 1 nM R1881, 1% O₂, or combination of hypoxia + R1881 for 48 h. g Representative western blots with DU145+Ev/AR cells treated by siNT or siAR for 72 h in 20% or 1% O₂. h Schematic representations of GPI promoter-driven luciferase plasmids, wild-type (wt) or mutants without the hypoxia-response elements (ΔHRE) or androgen response/AR-binding element half-site (ΔAREhs). i Relative luciferase activity driven by wild-type (wt) or mutant (ΔAREhs or ΔHRE) GPI promoter in LNCaP-AdtHs cells. Cells were first transfected with siRNA (siNT or siAR). Then, wt or mutant reporter plasmids were co-transfected with constitutive GFP plasmids. The transfected cells were cultured in basal, androgen, hypoxia or combination condition for 48 h. The luciferase values were determined, adjusted by GFP, and expressed as mean ± s.d. relative to basal condition, n = 3, *P < 0.05, NSD, two-sided t-test. j Relative luciferase activity driven by wild-type (wt) or mutant (ΔAREhs or ΔHRE) GPI promoters in DU145+Ev (Ev) or DU145+AR (AR) cells in normoxia (basal) or hypoxia condition. All values are means ± s.d. n = 3, *P < 0.05, two-sided t-test