Cb 160

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 skin amelanotic melanoma cell lines A375 and C32 were acquired from ATCC (CRL-1974™, USA). Both cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with inactivated fetal bovine serum to a final concentration of 10%, as well as the antibiotics penicillin (100 U/mL), neomycin (10 μg/mL) and amphotericin B (0.25 μg/mL). Cells were cultured in a 5% CO2 incubator CB 160 (BINDER, Tuttlingen, Germany) at 37 °C with 5% relative humidity. The treatment with doxycycline and minocycline was started 24 h after seeding for melanoma cells. Tested drug solutions were prepared using the culture medium. Cells were detached with trypsin both during cultivation and after treatment.

All in vitro studies were performed on human epidermal melanocytes, neonatal, lightly pigmented HEMn-LP (Cascade Biologics, Portland, OR, USA) from passages 6 to 10. M-254 growth medium was supplemented with a human melanocyte growth supplement-2 (HMGS-2) as well as antibiotics: penicillin (100 U/mL), neomycin (10 μg/mL), and amphotericin B (0.25 μg/mL). Melanocytes were cultured in a 5% CO₂ incubator CB 160 (BINDER, Tuttlingen, Germany) at 37 °C.

The human normal colon mucosal epithelial cell line NCM460 (C335) was obtained from the Shanghai Institute of Cell Biological,Chinese Academy of Sciences. The cells were incubated at 37°C in air and 5% CO₂ in sterile basal medium/DMEM-H (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Genview, Beijing, China), 1% L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. The culture medium was changed every 2 to 3 d. Prior to treatment, the cells (1 × 10⁶ cells/ml) were plated with fresh medium and cultured in 37°C in air and 5% CO₂ for 24 h. On the second day after plating, the cells were treated with N-acetyl-L-cysteine (NAC) or apocynin (APO) and/or 85% oxygen (three-gas incubator, CB160, BINDER, Neckarsulm, Germany) for 24 h. The control cells received no treatment. Next, we harvested the cultured cells and extracted RNA and protein from the cells. All experiments were repeated 6 to 8 times.

Both conventional and silk-VM organoids were cultured at 37 °C in 21% O₂ and 5% CO₂ in normoxia conditions. For hypoxia analysis, VM organoids were transferred to the hypoxia chamber (Binder CB160), which was filled with 1% O₂ and 5% CO₂ and mixed with N₂, and collected after 4, 8, and 16 h.