Cell Culture and Transfection
We used primary skin fibroblasts from individuals with HS and primary skin fibroblast cell lines completely deficient of PEX1 (compound heterozygous for p.[Thr263Ilefs∗6];[Ile700Tyrfs∗42], c.[788_789del];[2097dup])19 or PEX6 (homozygous for p.Gly135Aspfs∗23 [c.402del]).20 Cells were cultured in DMEM with L-glutamine (Bio-Whittaker) supplemented with 10% fetal bovine serum (Bio-Whittaker), 25 mM HEPES buffer (BioWhittaker), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies), and 250 ng/ml Fungizone (Life Technologies) in a humidified atmosphere of 5% CO2 at 37°C or 40°C. Transfections were performed with the AMAXA NHDF Nucleofector Kit (Lonza) according to the manufacturer’s instructions (program U23). The medium was changed 24 hr after transfection, and the cells were imaged 72 hr after transfection.