Ad 293

293T (ATCC ref. CRL-3216) and Ad-293 (Agilent Technologies ref. 240,085) cells were grown in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. For immunofluorescence experiments, 293T cells were grown on collagen-coated coverslips and transfected with 1 μg/ml plasmid DNA (expressing N- or C-terminally Flag-tagged, or untagged Mustn1) using 5 μg/ml polyethylenimine (PEI MAX, Polysciences ref. 24,765). 24 h after transfection, cells were fixed with 4% paraformaldehyde in 0.1M phosphate buffer (PBS) for 15 min, washed three times with PBS, permeabilized for 15 min (0.25% Triton X-100 in PBS), and incubated with blocking solution (10% goat normal serum in PBS) for 15 min, all at room temperature. Cells were then incubated with rabbit anti-Mustn1 antibody (Merck Millipore, ABD115, 1:500) overnight at 4 °C, washed three times with PBS, followed by incubation with AlexaFluor 594 goat anti-rabbit antibody (Invitrogen, A11037, 1:500) for 1 h at room temperature. Both primary and fluorophore-conjugated secondary antibodies were diluted in blocking solution. Cells were washed three times with PBS, counter-stained with DAPI and mounted on a microscopy slide using Vectashield for imaging. Images were captured using a Zeiss Axio Vert.A1 widefield fluorescence microscope with a LD Plan-Neofluar 40x/0.6 Korr M27 objective.

AD-293 (Agilent), 293T (ATCC), and HeLa (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) with 10% FBS (Hyclone). DBTRG-05MG cells (DSMZ) were cultured in RPMI-1640 with 10% FBS. AD-293 and HeLa cells were authenticated by STR testing (ATCC). Cells were routinely tested for mycoplasma and found negative (Universal mycoplasma detection kit, ATCC). For transient transfections, cells were seeded in either 10 cm or 6 well plates at 70% confluence and transfected with 10 µg or 3 µg of plasmid respectively, with polyethylenimine (Polysciences) for AD-293 or Lipofectamine 2000 (ThermoFisher) for HeLa cells. The medium was changed 8 h post-transfection and cells were collected after 48 h.

A549 (ATCC–CCL-185), HCT-116 (ATCC–CCL-247), SKOV (ATCC–HTB-77), HT-29 (Ark Therapeutics), DLD-1 (ATCC-CCL-221), AD-293 (Agilent Technologies), MRC-5 (ATCC–CCL-171), and Wi38 (ATCC–CCL-75) cell lines were maintained in DMEM high glucose with glutamine (Lonza) supplemented with 2mM L-glutamine (Gibco), 1mM Sodium pyruvate (Gibco) and 1mM non-essential amino acids (Gibco). For routine cell culture media was supplemented with 10% FBS (Gibco) and for assays with 2% FBS and 1mM Pen/Strep (Gibco).