Epidermal keratinocytes were isolated from human foreskin as previously described (Halbert et al., 1992 (link)). The cells were propagated in medium 154 supplemented with human keratinocyte growth supplement, 1,000× gentamycin/amphotericin B solution (Invitrogen), and 0.07 or 0.2 mM CaCl2.
Keratinocytes were transduced with retroviral supernatants produced from Phoenix cells (provided by G. Nolan, Stanford University, Stanford, CA) as previously described (Getsios et al., 2004 (link)). For differentiation of submerged cultures, cells were grown to confluence and switched to E-medium containing 1.8 mM Ca2+ for 1–6 d (Meyers and Laimins, 1994 (link)). For raft cultures, transduced cells were expanded and grown at an air–medium interface according to published protocols (Meyers and Laimins, 1994 (link)). Organotypic cultures were grown for 3–10 d, at which time they were lysed for RNA/protein analysis, embedded in optimal cutting temperature compound for frozen sections, fixed in 10% neutral-buffered formalin, and embedded in paraffin for histology or fixed in 2% paraformaldehyde/2% glutaraldehyde in cacodylate buffer for EM analysis. For some experiments, cultures were treated with 2–5 µg/ml ETA, DMSO (Thermo Fisher Scientific), 10 µM PKI166 (Novartis), 5 µM U0126 (Cell Signaling Technology), or 10 µM SB203580 (EMD).
Keratinocytes were transduced with retroviral supernatants produced from Phoenix cells (provided by G. Nolan, Stanford University, Stanford, CA) as previously described (Getsios et al., 2004 (link)). For differentiation of submerged cultures, cells were grown to confluence and switched to E-medium containing 1.8 mM Ca2+ for 1–6 d (Meyers and Laimins, 1994 (link)). For raft cultures, transduced cells were expanded and grown at an air–medium interface according to published protocols (Meyers and Laimins, 1994 (link)). Organotypic cultures were grown for 3–10 d, at which time they were lysed for RNA/protein analysis, embedded in optimal cutting temperature compound for frozen sections, fixed in 10% neutral-buffered formalin, and embedded in paraffin for histology or fixed in 2% paraformaldehyde/2% glutaraldehyde in cacodylate buffer for EM analysis. For some experiments, cultures were treated with 2–5 µg/ml ETA, DMSO (Thermo Fisher Scientific), 10 µM PKI166 (Novartis), 5 µM U0126 (Cell Signaling Technology), or 10 µM SB203580 (EMD).
