Ga3502

HaCaT cells were obtained from Cell Bank of the Chinese Academy of Sciences in Beijing, China. Primary mouse keratinocyte (MKs) were cultured using a described method before 23 (link). Briefly, neonatal Balb/c mice (postnatal day 1-3) mice were immersed in 75% alcohol for 2-3 minutes, and then rinsed with PBS for 2-3 times. Gradually blunt skin separation with scissors and tweezers. After repeated washed by PBS, adding neutral protease II (1x) until submerged the skin, 4°C for the night. After the epidermal and dermis were separated, the epidermis was cut in sterile PBS, digested with 0.25% trypsin/ 0.02% EDTA solution for 2-3 minutes, then neutrallized with 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, and filtered with a sieve after blowing, centrifugated for 5min. MKs can be inoculated after resuspending with 1640 medium. Both HaCaT and primary mouse keratinocytes were cultured in 1640 medium (SH30809.01B, Hyclone, USA) containing 10% fetal bovine serum (S-FBS-500, Scitecher, USA) and 1% penicillin/ streptomycin (GA3502, Genview, Australia), digested with 0.25% trypsin/0.02% EDTA solution (SH30042.01, Hyclone, America). Cell cultures were performed in a 5% CO2 atmosphere at 37°C. Cells were treated with 1mM AICAR (ab120358, Abcam, UK), 2μM Compound C (ab120843, Abcam, UK).

Normal human skin fibroblasts were obtained from Cell Bank of the Chinese Academy of Sciences in Beijing, China and were maintained in Dulbecco's modified Eagle's medium (C11995500BT, Gibco, Canada) including 10% fetal bovine serum (S-FBS-500, Scitecher, USA) and 1% penicillin streptomycin (GA3502, Genview, Australia). Cell cultures were performed in a 5% CO₂ atmosphere at 37°C. The medium was changed three times a week. When the culture reached 90% confluence, the cells were separated from the flask with 0.05% trypsin-0.1% ethylenediaminetetraacetic acid (EDTA) solution, washed twice, and then resuspended in DBS supplemented with FBS medium. In each experiment, fibroblasts were used between passages 4 and 5.