All experiments involving mice were approved by the Weill Cornell Medical College Institutional Animal Care and Use Committee and were performed in accordance with institutional guidelines. Immortalized mouse melanocytes were derived from 1- to 3-day-old Adcy10fl/fl agouti newborn mice as previously described (41). Briefly, newborn mice were euthanized, and the skin was removed from the back, placed in a petri dish epidermis side up, and incubated in 2.5 ml of dispase in Eagle’s minimal essential medium without calcium and magnesium overnight at 4°C. On the next day, the dermis was discarded, and the epidermis was incubated in trypsin solution until the cells dissociated. Cells were washed to remove the trypsin solution and then cultured in mouse melanocyte medium [Ham’s F12 plus glutamine, penicillin-streptomycin, horse serum (7%), fetal bovine serum (FBS; 7%), dibutyryl cAMP (dbcAMP; 500 μM), Na3VO4 (1 μM)]. Once the immortalized line was established, the medium was changed to normal mouse melanocytes culture media [Opti-MEM medium supplemented with 10% FBS, 7% horse serum, 1% penicillin-streptomycin, 400 μM dbcAMP, 0.3 nM cholera toxin (CT), and 1.6 μM 12-O-tetradecanoylphorbol-13-acetate (TPA)]. To generate Adcy10−/− melanocytes (sACKO), parental Adcy10fl/fl cells were infected with either Ad5-CMV-GFP or Ad5-CMV-CREGFP (Vector BioLabs, Malvern, PA, USA) at 200 multiplicity of infection. Forty-eight hours after infection, cells were fluorescence-activated cell sorted for GFP fluorescence, and only cells that were in the upper 25% of fluorescence were collected and cultured. Independent pairs of Adcy10fl/fl (sACFF) and Adcy10−/− (sACKO) cells were generated. Genetic deletion of sAC was confirmed by PCR and functional assay. All experiments using mouse melanocytes were performed between passages 15 and 28. Before the experiments, melanocytes were cultured in “cAMP starvation media” without dbcAMP and without CT for 48 to 96 hours. The cell growth rate of each cell line under different media conditions was measured using the CyQUANT assay (Thermo Fisher Scientific) at the time point indicated.

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