Figure 4. TMPRSS2, ETVs and TMPRSS2:ETV5 gene fusions are involved in an IGF-1R-dependent growth stimulatory effect of E₂-ERβ2 axis in PC-3 cells.

(A) PC-3 cells were subjected to ERβ knockdown with shRNA, and then treated with 10 nM E₂ for 24 hours. Protein extracts were subjected to immunoblot analysis with antibodies against TMPRSS2, ERβ, ERβ2, or GAPDH. (B) PC-3 cells were incubated with 10 nM E₂, 10 nM E₂-BSA-FITC, or 10 nM of G-1, an agonist of the G protein-coupled estrogen receptor-1 (GPER-1) for 12 hours and TMPRSS2 and GAPDH mRNAs and protein levels were quantified by qRT-PCR (upper panel) and immunoblot analysis (lower panel). (C) Protein levels of TMPRSS2, IGF-1R or GAPDH were measured by Western blot in PC-3 cells transiently transfected with siRNA targeting IGF-IR siRNA or non-targeting control siRNA and then treated vehicle or 10 nM E₂ for 24 hours. (D) Assessment of TMPRSS2 protein expression in PC-3 cells treated with 10 nM E₂ in presence or absence of 5 μM of an IGF-1R inhibitor (AG1024). GAPDH protein expression was used as loading control. (E, F) TMPRSS2 transcripts quantified by qRT-PCR (E) and protein levels (F) as measured by Western blot analysis relative to their respective controls in PC-3 cells treated with vehicle, 10 nM E₂, 25 ng and 50 ng IGF-I or 50 ng IGF-II for 24 hours. (G) PC-3 cells were transiently transfected with non-targeting control siRNA or siRNAs targeting TMPRSS2, ETV5 or both and then treated with vehicle or 10 nM E₂ for 0, 24 or 48 hr. Cell proliferation was assessed by Cell Counting Kit-8. Data represent Mean ± SEM in triplicates. (H) Colony formation assay by PC-3 cells transiently transfected with non-targeting control siRNA or siRNAs targeting TMPRSS2, ETV5 or both and then treated with vehicle or 10 nM E₂. Bar graphs represent mean ± SEM values in triplicates. * denotes significant difference compared to controls at P < 0.05 (n = 3).