Dld 1

Human cancer cell lines: A375 (CRL-1619), C32 (CRL-1585), MDA-MB-231 (HTB-26), MCF-7 (HTB-22), DLD-1 (CCL-221), HT-29 (HTB-38), A549 (CCL-185) were purchased from ATCC (Manassas, VA, USA). Normal human fibroblasts were obtained from Sigma Aldrich Inc. (St. Louis, MO, USA). HT-29 cells were cultured in McCoy’s 5a medium (Sigma Aldrich Inc., St. Louis, MO, USA), DLD-1 cells were cultured in RPMI 1640 medium (PAN-Biotech, Aidenbach, Germany), and normal human fibroblasts were cultured in Fibroblast Growth Medium, all-in-one ready-to-use (Sigma Aldrich Inc., St. Louis, MO, USA). The remaining cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA, USA). Culture media, except from the one for normal fibroblasts, were supplemented with 10% (v/v) fetal bovine serum (Gibco, Waltham, MA, USA) as well as penicillin (100 U/mL), neomycin (10 µg/mL) and amphotericin B (0.25 µg/mL) obtained from Sigma Aldrich Inc. (St. Louis, MO, USA). Cells were maintained at 37 °C in a CO₂ incubator CB 160 (BINDER, Tuttlingen, Germany) in humidified atmosphere with 5% CO₂. The novel betulin derivatives EB366 and EB367 were dissolved in dimethyl sulfoxide (Sigma Aldrich Inc., St. Louis, MO, USA) to prepare 10 mg/mL stock solutions, which were used to prepare dilutions in culture media.

Human colon cancer cell lines SW480 (CCL228) and DLD-1 (CCL221) were purchased from the American Type Culture Collection (ATCC). The DLD-1 cells were grown in McCoy’s 5A medium (PAN-Biotech GmbH, Aidenbach, Germany), and the SW480 cells were grown in MEM medium (PAN-Biotech GmbH). All the media were supplemented with 10% fetal bovine serum (PAN Biotech), 1000 U penicillin/mL, and 10 mg streptomycin/ml (PAN-Biotech GmbH). The cells were cultured at 37 °C in humidified 5% CO2 incubator (HERA cell 150; Thermo Electron Corporation, Waltham, MA, USA).