Fig. 2: GR is not essential for prostate fibroblast survival but is functionally active.

A, B AR/GR mRNA and protein expression in benign and malign immortalized stromal prostate cell lines and in primary isolated and cultured NAFs and CAFs. C Representative Western blot bands, as well as quantification of reduced GR expression after 6 d transient GR knockdown with specific doxycycline-inducible shRNA sequences shGR-1 and shGR-2 in PF179T-CAF-shGR-1/shGR-2 and DU145-shGR-1 cells. Data represent mean + SEM from at least 3 independent experiments (unpaired t-test; ***P < 0.001). D Measurement of altered cell proliferation, cell viability, as well as apoptosis with ³[H]-thymidine incorporation, WST uptake, and PI-staining after 6 d GR single knockdown, pharmacological inhibition with RU486 or combination treatment of PF179T-CAF-shGR-1/shGR-2 and DU145-shGR-1 cells. Data represent mean + SEM from at least 3 independent experiments (one-way ANOVA and correction for multiple testing using Dunnett’s comparison test; *P < 0.05; ***P < 0.001). E Representative Western blot bands of altered GR and cPARP expression after single GR knockdown, pharmacological inhibition with RU486, or combination treatment of PF179T-CAF-shGR-1/shGR-2 and DU145-shGR-1 cells. F Representative immunofluorescence microscopy images of PF179T-CAF and primary isolated CAFs for cytoplasmatic and nuclear GR expression after 60 min 100 nM Dex treatment. Magnification: 40× (scale bar = 50 µm). G Dose-dependent altered GILZ and IGFBP1 mRNA expression after treatment with increasing concentrations of Dex and Pred (0, 1 nM, 10 nM, 100 nM, 1000 nM) for 1 d. Data represent mean + SEM from 3 independent experiments (one-way ANOVA and correction for multiple testing using Dunnett’s comparison test; **P < 0.01; ***P < 0.001).