HaCaT Cell Culture and Chronification

HaCaT cells [47 (link)], obtained from ATCC, were cultured in full medium (FM) comprising Dulbecco’s modified eagle high-glucose medium (Biowest, Nuaillé, France), completed with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 1000 units/mL penicillin, 1000 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 1% l-glutamine (Biowest, Nuaillé, France), at 37 °C in a 7.5% CO₂ controlled atmosphere. Chronification was as indicated in Liarte et al., 2020 [18 (link)]. Briefly, HaCaT cells at 50% subconfluency were changed to a serum-deprived medium (SS). While some of them were kept in this condition, others were stimulated with 2 ng/mL of TGF-β1 (PeproTech, Rocky Hill, NJ, USA). For samples maintained continuously in the absence or presence of TGF-β, a culture medium supplemented with 2 ng/mL of TGF-β1 was refreshed every 24 h. After 48 h of treatment, cells were denominated SS (serum-starved) or SSTC (serum-starved TGF-β chronic) cells [18 (link)], respectively. Then, cells were treated for the conditions stated in each experiment.