Metabolic profiling of prostate cancer cells

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)).