Du145

All PCa cell lines, including 22Rv1, DU145, C4–2, PC-3, and LNCaP, were obtained from the National Collection of Authenticated Cell Cultures. DU145 and PC-3 were cultured in Dulbecco’s modified Eagle’s medium, and 22Rv1, C4–2, and LNCaP were cultivated in RPMI-1640 medium. Culture media contained 10% fetal bovine serum and 1% double antibiotics (Penicillin and Streptomycin). All cell lines were maintained at 37 °C and 5% CO₂. For treatment with EBSS (24010043, GIBCO), 22Rv1 and DU145 were maintained in EBSS with or without 50 nM CQ (C6628-25G, 10 Sigma) or 10 μM BafA1 (B1793, Sigma) for 6 h.

SW480 and SW620 (Human colon cancer cell lines), CT26 (Mouse colorectal cancer cell line), HepG2 (Human liver cancer cell line), Du145 (Human prostate cancer cell line), A549 (Human lung cancer cell line) and MCF-7 (Human breast cancer cell line) were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China).293T were purchased from the American Type Culture Collection (ATCC). All cell lines were received as early passages and cultured in DMEM or RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel). Cells were maintained at 37 °C in humidified atmosphere containing 5% CO₂. The medium was changed every other day, cells were passaged using 0.25% trypsin (Solarbio, China) and preserved at early passages. All cell lines were free of mycoplasma contamination.

DU145, PC3, BT549 and HEK293T cells were purchased from the National Collection of Authenticated Cell Cultures. Human dermal fibroblast (HDF) cells were gifted by Center for Molecular Medicine of Xiangya Hospital, Central South University. These cells were maintained in DMEM or 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. For synchronization in G1/S phase, cells were treated with 2 mM thymidine (Sigma-Aldrich, Shanghai) for 18 h, and then washed and cultured in fresh medium for 9 h. The 18 h thymidine treatment was repeated once. For synchronization in S phase, DU145 cells first were synchronized at G1/S phase using a double treatment of thymidine, and then were washed and cultured in fresh medium for 3 h. For synchronization in mitotic phase, DU145 cells were treated with nocodazole (50 ng/ml) (Chemicals Selleck, Selleck, Shanghai) for 16 h.

One million NFs and CAFs were seeded into 10 cm culture dishes and cultured in 10 ml of serum-free medium for 2 days. Supernatant was then collected. PCa cell lines PC-3 and DU145 were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). PC-3 cells were cultured in RPMI-1640 medium (Gibco, USA) and DU145 in DMEM medium (C11995500BT, Gibco). Cell culture medium was supplemented with 10% FBS (ExCell Bio, China) and 1% penicillin/streptomycin (C100C5, NCM Biotech, China). All cells were mycoplasma free and cultured at 37 °C in a humidified 5% CO₂ atmosphere. Supernatant from NF and CAF medium was added into plates where PC-3 and DU145 cells were incubated, and a final supernatant concentration was 50%.

The human prostate cancer cell lines, DU145 and PC3, were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). DU145 and PC3 were cultured in RPMI 1640 with 10% serum and incubated at 37°C, 5% CO2. The two Small interfering RNAs (JTSBIO) sequences used to reduce FTO expression sequences used to reduce FTO expression were as follows: GCAGUGUAUCUGAGGAGCUCCAUAA UUAUGGAGCUCCUCAGAUACACUGC; CAGGCUGCACCUACAAGUACCUGAA UUCAGGUACUUGUAGGUGCAGCCUG.