Dihydrotestosterone

LNCaP, 22RV1, and C4-2 cell lines were obtained from the American Tissue Culture Collection. Cells were maintained in 10% fetal bovine serum (FBS) in RPMI medium. RNA isolation was performed using the illustraMiniSpin-kit (GE Healthcare) according to manufacturer’s instructions, and cDNA was synthesized using the qScript cDNA Synthesis Kit (Quantabio). For androgen-starvation experiments, cells were maintained in phenol red-free RPMI medium supplemented with charcoal-stripped FBS. AT7519 was purchased from Selleckchem for reverse-phase protein array and metabolite profiling experiments, and for the other experiments, from MedChemExpress. OSMI-2 was synthesized in Professor Suzanne Walker’s laboratory (Harvard Medical School), while OSMI-4 (Martin et al. 2018 (link)) and dihydrotestosterone were purchased from MedChemExpress. Cell lysates for western blotting were prepared as previously described (Itkonen and Mills 2013 (link)), and antibodies used are from Cell Signaling technology: p-Ser2-RNA Pol II (13499) and OGT (24083); from Santa Cruz Biotechnology: p-H2AX (517348); and from Abcam: RL2 (ab2739) and Actin (ab49900). Signal intensity of western blot (densitometry) was determined using Image Lab version 6.0 (Bio-Rad). Cell number was assessed using crystal violet staining assay as previously described (Barkovskaya et al. 2020 (link)).

Human PCa cell lines LNCaP, C4–2, 22Rv1 and PC3 were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China) and maintained in RPMI-1640 (Invitrogen Grand Island, NY, USA). Human embryonic kidney cell HEK-293 T and human cervical cancer cell line HeLa were purchased from American Type Culture Collection (Manassas, VA, USA), and maintained in DMEM (Invitrogen, Grand Island, NY). All mediums were supplemented with 10% fetal bovine serum (FBS, Gibco, Australia), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All cell lines were cultured in incubators with humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Anti-MIIP and anti-FLAG antibody were from Sigma-Aldrich (St. Louis, MO, USA). Anti-PP1α was from Sata Cruz Biotechnology (Dallas, Texas, USA). Anti-HA was from Abcam (Cambridge, MA,USA). Anti-PI3K(p110), anti-AKT, anti-p-AKT (Ser473), anti-mTOR, anti-p-mTOR (Ser2448), anti-p-mTOR (Ser2448), anti-AR, anti-GAPDH and anti-α-tubulin antibodies were from Cell Signaling Technology (Beverly, MA, USA). Dihydrotestosterone, Enzalutamide and BKM120 were commercially purchased (MedChemExpress, Princeton, NJ).