M154cf500

THEKs from original line N/TERT-2G (31 (link)) (a gift from James Rheinwald, Brigham and Women’s Hospital, Boston, Massachusetts, USA) were grown in keratinocyte serum-free medium (KSFM) ordered as a kit (Thermo Fisher Scientific catalog 37010022) with supplements of 0.2 ng/mL human epidermal growth factor and 30 μg/mL bovine pituitary extract. Other additives included 0.31 mM CaCl₂, 100 U/mL penicillin, and 100 μg/mL streptomycin.
NHEKs were procured as described below and grown in Medium 154 with 0.07 mM CaCl₂ (Thermo Fisher Scientific catalog M154CF500), 1× human keratinocyte growth supplement (Thermo Fisher Scientific catalog S0015), and 1× gentamicin/amphotericin (Thermo Fisher Scientific catalog R01510).
J2-3T3 immortalized murine fibroblasts (a gift from Kathleen Green, Northwestern University, Chicago, Illinois, USA) were grown in complete DMEM (Thermo Fisher Scientific catalog 11965092) supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific catalog SH3039603), 2 mM GlutaMAX (Thermo Fisher Scientific catalog 35050061), 100 U/mL penicillin, and 100 μg/mL streptomycin.
All cell lines were maintained at 37°C in 5% CO₂ in an air-jacketed, humidified incubator. Cells were grown on sterile cell culture dishes and passaged at subconfluence using 0.25% Trypsin-EDTA (Thermo Fisher Scientific catalog 15400054).

ASZ001 BCC cells were cultured in 154CF medium (ThermoFisher; M154CF500) supplemented with 2% chelated FBS and 0.05 mM CaCl₂ as previously indicated^{13 (link)}. Experiments were carried out using low serum conditions with 154CF medium containing 0.2% chelated FBS and 0.05 mM CaCl₂. ASZ_001 cells were derived from mouse basal cell carcinoma cells and previously authenticated^{52 (link)}. In our experiments, cells were monitored by their morphology, sequencing, and utilization of mouse-specific primers. No mycoplasma was detected through regular testing.