Du145

PCa cell lines (PC-3 and DU145) and normal human prostatic epithelial cells RWPE-1 were bought from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma, St Louis, MO, USA) supplemented with fetal bovine serum (10%, Gibco, Rockville, MD, USA) and penicillin/streptomycin (100 U/mL, Gibco) in an incubator with 5% CO₂ at 37 °C. DTX-resistant PCa cell lines (PC-3/DTX and DU145/DTX) were obtained by gradually exposing the parental cell lines (PC-3 and DU145 cells) to a medium with increasing DTX doses (Sigma) for 6 months [21 (link)]. The starting concentration of DTX was 5 nM and the final concentration was 160 nM. The medium containing DTX was changed every 2–3 days. Moreover, 10 nM DTX was used for subsequent analysis.

22Rv1 (hormone-dependent prostate cancer cell line), DU145 and PC-3 (hormone-refractory prostate cancer cell line) were purchased from American Type Culture Collection and used in the study. 22Rv1 was cultured in RPMI1640 (Welgene) medium, DU145 was cultured in MEM (Welgene) medium and PC-3 was cultured in Ham’s F-12 K (Welgene) medium, and 10% FBS and Antibiotic & Antimycotic solution (Welgene) were added in those media. The incubator was maintained at 5% of CO₂, proper humidity and 37°C.
The expression of KLOTHO mRNA was monitored by treating DU145 and PC-3 cell lines with 0.5, 1, 2, 5, and 10 µM of 2′-deoxy-5-azacytidine (DAC) (Invivogen), a DNA methyltransferase (DNMT) inhibitor, for 72 h or with 1 µM for 12, 24, 28,72, and 96 h. In a separate experiment, we added trichostatin A (TSA) (Sigma), a histone deacetylase, to the DU145 and PC-3 cell lines at a concentration of 25, 50, 100, 250, or 500 nM for 24 h and with a concentration of 100 nM for 6, 12, 24, and 48 h. To monitor the changes in KLOTHO expression when each cell line was treated with DAC and TSA, 1 µM DAC was applied for 72 h and 100 nM TSA was applied for 24 h to the DU145 cell line and 1 µM DAC was applied for 96 h and 100 nM TSA was applied for 24 h to PC-3 cell line. Gene expression in each cell line was investigated. The DU145 and PC-3 cell lines without DAC and TSA treatment were used as the control group.

HeLa, MCF-7 and MDA-MB-231 cells were obtained from Sigma-Aldrich and maintained as reported previously [22 (link)]. MRC-5, PC-3, DU 145 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). PC-3 and DU 145 were maintained in RPMI medium (Sigma-Aldrich) whereas MRC-5 cells were maintained in DMEM medium (Sigma-Aldrich). All media were complemented with 10% Fetal bovine serum (FBS, Sigma-Aldrich) 2 mM L-glutamine, 0.01% streptomycin and 0.005% ampicillin (Sigma-Aldrich). Cells were cultured under standard conditions in a 37 °C incubator containing 5% CO₂ in 95% humidity.