Human osm

Human OSM (R&D systems, Minneapolis, MN) was added to HUVECs, HAECs and HMEC-1 cells in a concentration range from 0–20 ng/mL. After 3 or 6 hours, RNA was isolated with the NucleoSpin^® RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Isolated RNA (500 ng) was reverse transcribed into cDNA with the qSCript™ cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA) and analyzed by real-time fluorescence assessment of SYBR Green signal (iQ™ SYBR^® Green Supermix, Bio-Rad, Hercules, CA) in the CFX96™ Real-Time Detection System (Bio-Rad, Hercules, CA). Each sample was measured in duplicates. Primers were designed for the human genes of interest, sequences are listed in Table 1. MRNA levels were analyzed and corrected for the housekeeping gene ACTB. Experiments were repeated 4–7 times.

Dermal fibroblasts were obtained from surgical samples of healthy breast skin or from hypertrophic or keloid scars. Dermal tissues were cut into 1–2 mm³ pieces and incubated in a culture plate with Dulbecco’s-modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 10% foetal calf serum (FCS), 100 u/ml penicillin and 100 µg/ml streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA) until the fibroblasts migrated out from the pieces of tissue. For all experiments, cells were used at the 3^rd passage.
For qRT-PCR and western blot analyses, cells were starved for 24 h in DMEM supplemented with 0.5% FCS before being treated with or without 10 ng/ml recombinant human TGFβ1 (PeproTech, Rocky Hill, NJ, USA) and human OSM (R&D Systems Europe, Lille, France) alone or in combination for 24 h for mRNA quantification or 48 h for western blot.