Cell culture, retroviral infection and organotypic cultures

NHK were isolated from foreskin tissue (Saarland University Medial Center) and cultured in supplemented KBM-Gold medium (Lonza, Basel, Switzerland). NHK and the spontaneously immortalized keratinocyte cell line HaCaT (a gift from Dr. P. Boukamp, German Cancer Research Center, Heidelberg, Germany) [37] were retrovirally engineered to express HPV8E6, E7 or E6/E7 using the pLXSN vector system (BD Biosciences, Heidelberg, Germany) as described [5]. HaCaT and the HPV-negative cervical carcinoma cell line C33A (HTB-31; American Type Culture Collection, Manassas, VA) were cultured in supplemented DMEM [70]. For differentiation-inducing experiments 6x10⁵ or 1x10⁵ cells were seeded onto 10 cm dishes or 6-well plates, respectively, treated with 1.2 mM CaCl₂ (Sigma Aldrich, Steinheim, Germany) and harvested after 72 h. To generate organotypic cultures, 3x10⁵ foreskin fibroblasts, passage 3–5, were seeded in 4 mg/ml rat-tail collagen (as described in [71]) on a collagen-fleece (MedSkin Solutions, Billerbeck, Germany) in 24-well plates and cultured in DMEM medium. Next day, 6x10⁵ HaCaT-pLXSN or 8E6 expressing HaCaT were seeded onto the collagen-fibroblasts in DMEM containing 25% Ham's F-12 medium with supplements as described in [69]. 100 U/ml penicillin/streptomycin was used as antibiotic. 24 h later, cultures were transferred on a metal grid in 6-well plates to culture them at the liquid-air interface. Two weeks later the cultures were fixed in 4% formaldehyde and embedded in paraffin. To verify C/EBPα knock-down at the protein level, HaCaT cells were transfected with 10 nM siRNA or control siRNA 16 h before seeding on collagen/fibroblasts and cultured for 6 d at the liquid-air interface.