Mccoy s 5a

DMEM (HyClone, Utah, USA) medium contained 10% fetal bovine serum (FBS, Gibco, NY, USA), penicillin (100 U/ L, Invitrogen, NY, USA), and streptomycin (100 mg/L, Invitrogen, NY, USA). McCoy's 5a (Invitrogen, NY, USA) medium contained 15% FBS, penicillin (100 U/L), and streptomycin (100 mg/L). DMEM/ F12 (HyClone, Utah, USA) medium contained 10% FBS, penicillin (100 U/L), and streptomycin (100 mg/L). U-2 OS cells were cultured in McCoy's 5a medium, MG-63 and 143B cells were cultured in DMEM, primary osteosarcoma cells were cultured in DMEM/F12, and all cells were placed in a humidified atmosphere with 5% CO₂ at 37°C.

All complete cell culture media were supplemented with 1% GlutaMAX (Gibco), 50 µg/mL Gentamicin (Gibco), and 10% fetal bovine serum (or 15% in the case of McCoy’s 5 A medium). HepG2 (cat. no. HB-8065), HEK293T (cat. no. CRL-3216), B16-F1 (cat. no. CRL-6323), SK-MEL-1 (cat. no. HTB-67), SK-MEL-3 (cat. no. HTB-69), SH-4 (cat. no. CRL-7724), and C32TG cells (cat. no. CRL-1579) were purchased from the American Type Culture Collection (ATCC). HEK293T, HepG2, human SH-4, and mouse B16-F1 melanoma cell lines were maintained in DMEM (Gibco, ThermoFisher Scientific, Waltham, MA, USA). C32TG and the non-adherent SK-MEL-1 cells were maintained in EMEM (Gibco), and SK-MEL-3 in McCoy’s 5a (Gibco). EMEM for the human C32TG melanoma cell line was supplemented with 50 µM of 6-thioguanine (Sigma-Aldrich, Missouri, USA). Human Me 204, Me 275, Me 343, and T672A cell lines from metastases of cutaneous melanoma patients were derived at the Ludwig Institute for Cancer Research (CHUV, Lausanne, CH) and maintained in RPMI (Gibco). YUMM3.3 cells were provided by Dr. Anna Obenauf (IMP, Vienna, AU) and maintained in DMEM/F12 (Gibco). The A375 cell line was provided by Dr. Mélanie Tichet (Hanahan lab, EPFL) and maintained in RPMI (Gibco). C8161 cells were provided by Dr. Mary Hendrix (Shepherd University, Shepherdstown, USA). Human SCC-12 and SCC-13 squamous cell carcinoma cells (provided by Dr. Freddy Radtke, EPFL) were maintained in keratinocyte Serum-Free Medium (SFM) (Gibco, 17005042) supplemented with 25 µg/mL Bovine Pituitary Extract (BPE) (Gibco, 13028014) and 0.2 ng/mL human EGF (Gibco, PHG0311). SSC-12, and SCC-13 were detached using accutase (CELLnTECH) 1x for 5 min at 37 °C. All other adherent cell lines were detached using Trypsin-EDTA 1x (Biowest, Nuaillé, FR). SK-MEL-1 are non-adherent cells. They were collected by centrifugation at 1000 x g for 5 min. Three B16F1 FurKO clones and one DKO CRISPR clone were generated by CRISPR/Cas9 editing single guide RNAs^{15 (link)}. B16-F1, FurKO, DKO, and YUMM3.3 cells that stably overexpress myc-tagged INHβA (βA) or an empty vector Ctrl were previously generated by stable transduction with lentivirus^{10 (link)}. HepG2 reporter cells stably transduced with lentiviral CAGA-Luc reporter and Renilla luciferase for signal normalization have been described previously^{19 (link)}. The HepG2.α1-PDX reporter cell line was derived by transducing HepG2 CAGA-Luc cells with α1-PDX-IRES-GFP lentivirus encoding the PC-inhibitory antitrypsin variant α1-PDX. A clonal cell line was obtained by serial dilution following the sorting of GFP⁺ cells on a FACSAria Fusion machine (BD Biosciences, San Jose, USA). All cell lines were cultured for maximally 6-8 weeks after de-freezing, and only after testing negative for Mycoplasma infections (Mycospy, Biontex, Munich, DE).