Human mesencult proliferation kit

The human prostatic carcinoma cell lines, PC-3 and LNCaP, were purchased from American Type Culture Collection and cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, HyClone, USA), 100 Units/ml penicillin and 0.1 mg/ml streptomycin. Human primary cultures of bone marrow-derived MSCs (BM-MSCs) were purchased from Stemcell Technologies (Vancouver, BC) and cultured using the Human MesenCult® Proliferation Kit (Stemcell Technologies Inc). The adherent cells were cultured in a 5% CO₂ humidified environment at 37 °C and the medium was changed every 2–3 days. Only cells from passages three to six were used for the experiments.
An antagonist of CCR5 (Maraviroc) was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA), and another antagonist of CCR5 (TAK-779) was synthesized by Takeda Chemical Industries (Osaka). Notch1 inhibitors LY3039478 and IMR-1 were obtained from Selleck (Houston, TX, USA).

LNCaP, C4-2, and CWR22RV1 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 with 10% FBS. Human BM-MSCs were purchased from Stemcell Technologies (Vancouver, BC) and cultured in Human MesenCult^® Proliferation Kit (Stemcell Technologies Inc). All cells were maintained in a humidified 5% CO₂ environment at 37°C.

Human ASCs (hASCs) were purchased (PT-5006, Lonza, Walkersville, MD, USA) and cultured using the human MesenCult proliferation kit (#05411, STEMCELL Technologies, Vancouver, BC, Canada) containing 100 U/mL penicillin/streptomycin (P/S; 15140, Gibco, Waltham, MA, USA). Cells were incubated in a humidified chamber with 5% CO₂ at 37 °C. hASCs were treated with recombinant human Tβ4 (#140-14, Peprotech, Rocky Hill, NJ, USA) in Dulbecco’s modified Eagle’s medium (DMEM) with low glucose (SH30021, Hyclone) containing 2% fetal bovine serum (FBS; #16000-044, Invitrogen, Waltham, MA, USA) and P/S. Phase-contrast images were obtained using an upright fluorescence microscope (DMI 300B, Leica Microsystems, Wetzlar, Germany). Cell length along the long axis was evaluated using the ImageJ software (v1.32, National Institute of Health, Bethesda, MO, USA).