Ham s f 12k

HEK293T cells were grown in Dulbecco’s modified Eagle medium (DMEM, Welgene, Gyeongsan, Republic of Korea). HT29 cells were grown in McCoy’s 5A medium (Welgene). CCD-18Co cells were grown in minimum essential medium (MEM, Welgene). DLD-1 cells were grown in Roswell Park MEMorial Institute (RPMI) 1640 medium (Welgene). LoVo cells were grown in Ham’s F-12K (Kaighn’s) medium (Ham’s F-12K, Welgene). All cells were grown in media supplemented with 10% fetal bovine serum (FBS, Welgene) and 1% penicillin/streptomycin (P/S, Welgene). Cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.
Cells were treated with rapamycin (25 nM) or PF-4708671 (10 μM) to inhibit mTOR or S6K1 activities, respectively, or the same amount of DMSO as a control. Cells were treated with Wnt-3a (300 ng/mL) or LiCl (25 mM) for the indicated time points.

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.