Pc3 cells

PC3 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured in a Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. Formononetin (cat. no. 20100311) was purchased from Jubang Biotechnology Co., Ltd. (Chengdu, China), and Calycosin (20575-57-9) was purchased from Sigma-Aldrich (St. Louis, MO, USA), both of which were dissolved with dimethyl sulfoxide (DMSO). The cells in the experimental groups were treated with formononetin (10 µM) or Calycosin (20 µM), and the control cells were treated with DMSO.

Prostate carcinoma PC-3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). PC-3 cells were maintained in RPMI-1640 medium, supplemented with 10% heat-inactivated foetal bovine serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin sulphate from Thermo Fisher Scientific (Waltham, MA, USA). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂. Cells were initially plated and treated when they reached a confluency level of 70% to 80%. Cells exposed to varying doses of silybin or its derivatives (5 or 10 µM) were dissolved initially in DMSO. These treatments were administered for specific time intervals as described for each experiment. The concentration of DMSO in all treatments did not exceed 0.1% (v/v) in the medium.
The antibodies for cleaved caspase 3 (Asp175) (#9661) and β-Actin (#3700) were from Cell Signaling Technology (Beverly, MA, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Amersham™ ECL™ Western blotting detection reagents were from Fisher Scientific (Hampton, NH, USA).

Human embryonic kidney (HEK).TPα, HEK.TPβ, and HEK.β-Gal cells, stably overexpressing haemagglutinin (HA)-tagged forms of TPα, TPβ, and β-galactosidase (β-Gal), respectively, have been previously described [35 (link)] and were grown in minimal essential medium (MEM), 2 mM L-glutamine and 10% (v/v) fetal bovine serum (FBS). Routinely, approx. 48 hr prior to transfection, cells were plated at a density of 2 × 10⁶ cells/10 cm dish in growth medium (8 ml). Thereafter, cells were transiently transfected with 400 ng of pADVA and 1.6 μg of pCMV-based vectors using Effectene and routinely harvested 48–72 hr post-transfection. The prostate carcinoma PC-3 cells were obtained from the American Type Culture Collection (ATCC) and were cultured in RPMI 1640, 2 mM L-glutamine, and 10% FBS (complete medium). For serum-free media conditions, cells were cultured in their respective growth medium in the complete absence of FBS (0%) for times specified for the given study. All cells were grown at 37°C in a humid environment with 5% CO₂ and were confirmed mycoplasma free.

PC3 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Burlington, Ontario, Canada) at 37°C in a 5% CO₂ atmosphere.

PC‐3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone/Cytiva) and 50 U/ml penicillin/50 μg/ml streptomycin. The cells were grown in an incubator at 37°C with 5% CO₂ on standard tissue culture plastic.