Cell Culture and Transfection

Human embryonic kidney 293 cells (ThermoFisher, {"type":"entrez-nucleotide","attrs":{"text":"R70507","term_id":"844024","term_text":"R70507"}}R70507) were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin. All of the cell lines used in this study are free of mycoplasma contamination.

For transfection, cells were seeded at 4 × 10⁵ cells per well in a six-well dish 16 h before transfection. Plasmid transfections were performed in OPTI-MEM (Invitrogen) with 2 µl Lipofectamine 2000 per well for 6 h, followed by trypsinization and replating onto glass-bottom confocal dishes at 3.5 × 10⁵ cells per well. Cells were imaged in live-cell medium (DMEM with 10% FBS and 20 mM Hepes no antibiotics) 16–24 h after transfection. For all transfection experiments in this study, the following amounts of DNA were used per 3.5 cm well (individually or combined for cotransfection): 1000 ng for VPS13B^sfGFP; 500 ng for truncated VPS13B mutations; 500 ng for pAcGFP-Golgi; 50 ng for Halo-Rab6 or Rab6 mutants; and 500 ng for mito-BFP.