Keratinocyte basal medium

Normal human keratinocytes were obtained from foreskin and cultured as described previously (16 (link)). Briefly, normal human keratinocytes were amplified on mitomycin C (Sigma-Aldrich, St Louis, MO, USA)-treated 3T3 cells and cultivated in Dulbecco’s modified Eagle’s medium and Ham’s F12 medium. Subconfluent secondary cultures for experiments were plated in a defined serum-free medium (KGM, Lonza Walkersville Inc., Walkersville, MD, USA). When cells were confluent, Ca²⁺ concentration was increased to 1.8 mM for 24 h, to induce keratinocyte differentiation, before the addition of any stimulus. Either human recombinant soluble FasL (rFasL) (0.1, 10, or 50 ng/ml, Sigma) or PVIgG and NIgG (1.5 mg/ml) were diluted in keratinocyte basal medium (Lonza) plus 1.8 mM Ca²⁺ and cycloheximide (Sigma) 1 µg/ml, and incubated for a further 72 h. For in vitro inhibitory experiments, keratinocyte cultures were pretreated with either 1 or 15 µg/ml of purified mouse anti-human FasL monoclonal Ab (NOK-2; BD Biosciences Pharmingen, San Diego, CA, USA) for 1 h, which was also added when medium was provided with NIgG, PVIgG, or rFasL.

Lawn culture of P. aeruginosa was grown on tryptic soy agar plates at 37°C for 16 h. Starting inoculum at a concentration of 10⁸ CFU/ml was prepared by suspending the bacteria in serum-free Keratinocyte Basal Medium (Lonza) until OD₆₅₀ reading was approximately 0.1. The starting inoculum was diluted 100-fold with 10 mM NaCl or 5 mM HEPES (pH 7.5) resulting 10⁶ CFU/ml bacteria. Stock solution of peptides (or equivalent volume of distilled water as no peptide control) was then added to 10⁶ CFU/ml bacteria and the final concentration of peptides was 200 μg/ml. The mixtures were incubated at 37°C for 3 h. The experiment was run in triplicate. Serial dilutions of the samples at time 0 and 3 h were plated on tryptic soy agar plates and incubated at 37°C overnight for viable bacterial counts in CFU/ml. The percentage killing was determined by: (bacterial count without peptide - bacterial count with peptide) / bacterial count without peptide × 100%.

The ERα-positive MCF-7 human breast adenocarcinoma cell line, purchased from the American Type Culture Collection (No. HTB-22, ATCC-LGC Promochem, Teddington, UK), were cultured in DMEM (Invitrogen, Karlsruhe, Germany), supplemented with 10% foetal bovine serum (FBS; Invitrogen) and antibiotics. Primary cultures of human vaginal mucosa cells (HVMs) were established from 1-cm² full-thickness biopsy of the vaginal mucosa, as previously reported [25 (link)], and maintained in Keratinocyte Basal Medium (Lonza Milano S.r.l., Milano, Italy) supplemented with KGM single quotes (Lonza Milano S.r.l., Milano, Italy), with medium change twice a week. Cells were treated with KGF (Upstate Biotechnology, Lake Placid, NY, USA), 17β-estradiol (E2; Sigma-Aldrich, Milan, Italy) or a combination of them for 24 hrs in proliferation experiments or for 5–30 min. in pathway analyses. Where indicated, cells were pre-treated with tamoxifen (Sigma-Aldrich, 100 nM) for 30 min. and then treated with KGF or E2 in the presence or absence of tamoxifen for 24 hrs. Prior to treatments, MCF-7 cells were grown in phenol red-free DMEM (Invitrogen) supplemented with 10% dextran charcoal-treated FBS (Invitrogen), and HVMs were switched to a steroid-reduced medium (KGM without EGF and BPE). Both MCF-7 and HVMs were also serum starved for 4 hrs.