Culturing and Transfecting Human Cell Lines

HeLa cells were purchased from American Type Culture Collection (ATCC). Primary human foreskin keratinocytes (PHK) were prepared from neonatal foreskins obtained from the University of Massachusetts Memorial Hospital as described [38 (link)]. Immortalized human foreskin keratinocyte cells (NIKS) with stable expression of HPV E6, E6 mutant F2V or E7 were established using the pBabe retroviral system as described in our previous study [39 (link)]. Both PHK and NIKS cells were maintained on mitomycin C–treated J23T3 feeder cells in F-medium as described [39 (link)]. RPE1 is a human telomerase reverse transcriptase–expressing human retinal pigment epithelium cell line obtained from Clonetech Laboratories. RPE1 cells were cultured in 1:1 DME and Ham's F12 media (Sigma-Aldrich) as described [19 (link)]. miRNA mimics, siRNAs and negative control RNA (a random sequence) were purchased from Sigma-Aldrich. Cells were transfected with 60 nM of either miRNA or siRNA using Lipofectamine 2000 (Life Technologies) according to the manufacturer's recommended protocol.