Rat tail collagen

Collagen gels were prepared using rat-tail collagen (#354236, BD Biosciences). Old cardiac fibroblasts (4×10⁵) were mixed with the neutralized collagen solution. Aliquots of mixture were placed in a 35 mm culture dish, and allowed to gel at room temperature. The gel was then discharged from the culture plate, and incubated with different CM for 48 hours. Gel size was measured using AlphaImager 2200 software, and presented as gel area.

After viral infection of HSCs for 48 h, HSCs were induced by 10 ng/mL TGF-β1 for 72 h, and then, the cells were harvested for contraction assay on collagen gel. Rat-tail collagen (4 g/L; BD Biosciences) was preplated in 12-well plates and cultured for 1 h at 37°C to allow for gelatinization. Next, HSCs were plated on top of the gel (the density of cells and volume of gel were 1 × 10⁶ cells/well and 50 μL, respectively) and co-cultured for 48 h. Experiments were terminated by adding 53% ethanol. Finally, gels were imaged, and the collagen gel contraction was observed based on the area of the gels.

Human N/TERT keratinocyte cell line N/TERT-2G, purchased from J. Rheinwald laboratory (Harvard Medical School, Boston, MA, USA), was cultured in Epilife medium (MEPI500CA, ThermoFisher Scientific, Waltham, MA, USA), complemented with human keratinocyte growth supplement (S0015, ThermoFisher Scientific) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich, Saint-Louis, MO, USA). Human epidermal equivalents (HEEs) were generated as previously described^{62 (link)}, with minor adjustments. Briefly, inert Nunc cell culture inserts (141002, ThermoFisher Scientific) were coated with rat tail collagen (100 μg/mL, BD Biosciences, Bedford, USA) at 4°C for 1 hour. 1.5×10⁵ N/TERT-2G keratinocytes (either wildtype, ΔAHR, or ΔTFAP2A keratinocytes) were seeded on the transwells in 150 μL Epilife medium (ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) in a 24 wells format. After 48 h, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 (v/v)) without penicillin/streptomycin for 24 h and then cultured at the air-liquid interface for an additional ten days. Culture medium was refreshed every other day until harvesting at day ten of the air-exposed phase.