Lncap human prostate cancer cell line

LNCaP human prostate cancer cell line was obtained from the American Type Culture Collection. Early-passage cells were used in all experiments. These cells were grown in T-medium (Invitrogen, Grand Island NY, USA) supplemented with 10% FBS, 100 units/ml penicillin, and 100 ug/ml streptomycin at 37°C with 5% CO₂. M12 cells are AR-negative and were obtained from Dr. J. Ware at the Medical College of Virginia. The generation and characterization of the M12 prostate cell line has been described previously [50 (link)–53 (link)]. M12 cells were cultured in RPMI 1640 medium (Invitrogen) containing 5% FBS, 10 ng/ml EGF, 0.02 mM dexamethasone, 5 ug/ml insulin, 5 ug/ml transfection, 5 ng/ml selenium, fungizone, and gentamicin at 37°C with 5% CO₂. cDNA of the entire AR^v567es variant and AR^fl were cloned into p3xFlag-CMV-9 vector as described previously [19 (link), 53 (link)]. The expression constructs were transfected into the human prostate cancer cell lines using TurboFect reagent according to the manufacturer's protocol (Thermo Scientific, Pittsburgh PA, USA).

The LNCaP human prostate cancer cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium with phenol red containing 10% fetal bovine serum (FBS) and 1% pen–strep at a concentration of 2.5 × 10⁵ cells/mL in 175 cm² flasks at 37 °C with 5% CO₂.

The LNCaP human prostate cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% CO₂. Apogossypol and gossypol were synthesized and extracted in our laboratory (25 (link)), dissolved in dimethyl sulfoxide (DMSO) and stored at ‑20°C. Working solutions were prepared by diluting the stock solution with culture medium before use. MTT was purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-Bcl-2, anti-caspase-3, and anti-caspase-8 antibodies were purchased from Maixin Biotechnology (Fuzhou, China), Zhongshan Golden Bridge Biotechnology (Beijing, China) and Boster Biological Engineering (Wuhan, China), respectively. Monkey anti-mouse immunoglobulin (Ig)G labeled with fluorescein isothiocyanate (FITC) and goat anti-rabbit IgG labeled with rhodamine were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

LNCaP human prostate cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with a 10% heat-inactivated fetal bovine serum, HEPES (10 mM), penicillin, and streptomycin in a humidified 5% CO₂ atmosphere at 37 °C. To examine the effects of TP, cells were cultured in phenol red-free RPMI 1640 containing 5% charcoal stripped serum (CSS) (TCB, Long Beach, CA, USA) for 24 h, and 100 nM TP was then added to the medium. STO-609 (a CaMKKβ inhibitor, Tocris Bioscience, Ellisville, MO, USA) was used at 30 μM and BAPTA-AM (a calcium chelator, Sigma-Aldrich, St. Louis, MO, USA) was used at 20 μM for the experiment.

DU145 (both variants herein termed DU-L [E-cadherin low expressing] and DU-H [E-cadherin high expressing]), PC3 (both variants herein termed PC3-L [E-cadherin low expressing] and PC3-H [E-cadherin high expressing]) and LnCaP human prostate cancer cell lines, and RWPE-1 normal prostate cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). DU-H/DU-L and PC3-H/PC3-L cells were maintained in DMEM and F-12 K media, respectively. LnCaP cells were maintained in RPMI 1640 media and RWPE-1 cells in Keratinocyte Serum Free Medium (K-SFM). Cells were seeded into 6-well plates and after 24 h treated with IFNγ (5 ng/mL) or control (PBS) for 48 h. Cells were harvested for immunoblot or flow cytometry, or fixed for immunofluorescence.