Ccd25sk

The study was performed on the primary skin fibroblast line obtained from an OI type I patient carrying an out of frame deletion in exon 5 of the COL1A1 gene (g.2674 del T, c.459delT, p.Gly145Alafs * 111, unpublished data) in accordance with the Declaration of Helsinki and was approved by the Bioethical Committee of the Jagiellonian University in Kraków, Poland (KBET/108/B/2007, 31 May 2011). Normal human skin fibroblast line CCD25Sk was purchased from the American Type Culture Collection and used as control. Fibroblasts were used between passages 2–6 and cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin in an incubator at 37 °C and humidified atmosphere containing 5% CO₂. For the experiments, cells were grown to 90% confluence and 2 h before treatment, culture medium was replaced with fresh serum-free DMEM supplemented with magnesium ascorbate (25 µg/mL). Compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C as the concentrated stock solutions. Fresh dilutions in DMEM were made prior to adding them to cell cultures for the final concentrations: RA (0.1–100 µM), RE (0.1–100 µg/mL) and LBE (0.1–100 µg/mL), with DMSO not exceeding 0.1%.

Tongue squamous cell carcinoma cells (SCC-25), melanoma cells (C32, A375), colorectal adenocarcinoma cells (DLD-1), gastric adenocarcinoma cells (AGS) and normal human skin fibroblasts (CCD25Sk) were purchased from the American Type Culture Collection (ATCC, Manassas, MD, USA). SCC-25 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM):Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% foetal bovine serum, 1% penicillin/streptomycin (all reagents obtained from Thermo Fisher Scientific, Waltham, MA, USA) and 400 ng mL⁻¹ hydrocortisone (Sigma-Aldrich, Saint Louis, MO, USA). The other cell lines were cultured in DMEM (Gibco) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin. Cell culture was maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

The human glioblastoma cell line, T98G, the breast cancer cell lines, MCF7 and MDA-MB-231, human embryonic kidney cell line, HEK293, human endothelial cell line, HUVEC, and human skin fibroblast cell line, CCD-25Sk, were purchased from the American Type Culture Collection (ATCC) (VA, USA). The lung cancer line A549 was purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were grown as a monolayer culture in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, MO, USA) or RPMI 1640 (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Lonza, Basel, Switzerland) solution at 37°C in an incubator containing 5% CO2.

HaCaT keratinocytes were purchased from AddexBio (San Diego, CA, USA). Melanoma cells A375 were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Melanoma cells C32 and normal human skin fibroblasts CCD25Sk were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in DMEM (PAN-Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Gibco FBS; ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA, USA) at 37 °C in a 5% CO₂ incubator.

HDF cells (CCD-25SK; American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) Supplemented with 10% foetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, at 37 °C in a humidified incubator with a 5% CO₂ atmosphere^{45 (link)}. In addition, an established mouse fibroblast cell line (L929, catalogue code CCL-1; American Type Culture Collection) was cultured in minimal essential medium Supplemented with 5% foetal calf serum, 100 U/mL penicillin, 100 μL/mL streptomycin, and 2 mmol/L l-glutamine at 37 °C in a humidified incubator with a 5% CO₂ atmosphere^{46 (link)}.

Colorectal adenocarcinoma cells (DLD‐1), gastric adenocarcinoma cells (AGS) and normal human skin fibroblasts (CCD25Sk) were purchased from the American Type Culture Collection (Manassas, VA, USA). Hepatocellular carcinoma cells (HepG2) were obtained from Sigma‐Aldrich (USA). Cells were grown in Dulbecco's Modified Eagle Medium (PAN‐Biotech, Germany) supplemented with 10% foetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) in 95% air and 5% carbon dioxide at 37°C.