Chelex treated fetal calf serum

Primary mouse keratinocytes isolated from newborn Balb/C pups were cultured in modified Eagle’s medium (S-MEM, Thermo Fisher Scientific), 8% Chelex-treated fetal calf serum (Gemini Bio Products), and 0.05 mmol/L calcium unless otherwise indicated.^{11 (link)} ROCK inhibitors Y-27632, SR 3677, GSK-429286A, and TC-S 7001 were purchased from Tocris Bioscience. Y-27632 was reconstituted in DI water, while SR 3677, GSK-429286A, and TC-S 7001 were reconstituted in DMSO. Each of these inhibitors were further diluted in cell culture media to reach working concentrations. Live cell images were taken and confluence scores were calculated using the IncuCyte S3 Live-Cell Analysis System (Sartorius). Total cell counts were determined by trypsinizing attached keratinocytes and using the Cellometer Auto 2000 cell counter (Nexcelom Bioscience) after Trypan blue (Thermo Fisher) staining.

Primary mouse keratinocytes isolated from newborn C57BL/6J pups were cultured in modified Eagle’s medium (S-MEM, Lonza), 8% Chelex-treated fetal calf serum (Gemini Bio Products), and 0.05 mmol/L calcium unless otherwise specified.^{77 (link)} To induce OIS, cultured primary keratinocytes were transduced with v-Ras^Ha retrovirus (referred to as RAS) on Day 2 at a multiplicity of infection (MOI) of 1 in medium containing polybrene (4 μg/mL; Sigma-Aldrich). The v-Ras^Ha replication-defective ectopic retrovirus was prepared using Psi2 producer cells; retrovirus titers were routinely 1 × 10⁷ virus/ml^{78 (link)} These cells were transfected with teton-MYCT58A two days after V-RAS transduction as described in Cell line generation. Similarly as above, primary mouse dermal fibroblasts were isolated and cultured in phenol red DMEM (Invitrogen, #11–995-073) supplemented with 10% fetal bovine serum (FBS, R&D Systems, #S11150), 100 I.U./mL penicillin and 100 μg/mL streptomycin (pen-strep, Gibco, #15–140-163). OIS was induced as described above.