Epilife culture medium

HPV16-containing plasmid DNA (ATCC:45113D) was used as the template for PCR amplification and cloning of the HPV16 E7 gene into vector pEGFP-C1 following protocols described previously. NHEKs (ScienCell, Carlsbad, CA, United States) were cultured in 6-well plates using EpiLife culture medium (Gibco, United States) with 10% fetal bovine serum (Gibco, United States) and Human Keratinocyte Growth Supplement (Gibco, United States). NHEKs at 80% confluence were transfected with pEGFP-16E7 and pEGFP-C1 using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, United States). Six hours after Transfection, culture medium was replaced with fresh culture medium.

Normal human epidermal keratinocytes (NHEKs) were cultured in EpiLife culture medium (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and Human Keratinocyte Growth Supplement (Gibco, USA). Siha cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). C33A cells were cultured in MEM (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% Penicillin-Streptomycin Solution (Gibco, USA). All cells were cultured at 37°C in the presence of 5% CO2. Cells at 80% confluence were transfected with GFP-LC3 or mCherry-GFP-LC3 plasmid using Lipofectamine 3000 reagent (Invitrogen, USA). The culture medium was replaced 6 h after transfection and the cells were cultured for another 48 h before analysis.