Fibronectin

HeLa cells (CCL-2, ATCC) were maintained in T-75 flasks (Falcon) at 37°C and 5% CO2 in DMEM (Gibco) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate, 1× non-essential amino acids (Gibco), 10% (vol/vol) FBS (Sigma) and 100 U/mL penicillin-streptomycin mix (Gibco). 100,000 cells were plated on glass-bottom dishes coated with 10 μg/mL fibronectin (MatTek Corporation) in complete DMEM media. The next day, cells were incubated for 2 h in serum-free and phenol-red free DMEM media containing 0.5% BSA. After starvation, cells were incubated with 1 μM C2-568 and 10 μg/mL transferrin-Alexa488 or 100 ng/mL EGF-Alexa488 for 30 min in starvation media to allow the labeled molecules to get internalized by the cells. Labeled cells were washed twice with PBS and fixed with 4% PFA for 10 min. Fixed cells were then washed with PBS before imaging.

Normal rat kidney (NRK) cells (ATCC CRL6509) and COS-7 cells (UVA Tissue Culture Facility) were maintained in DMEM with glucose and GlutaMAX (Gibco 10566-016) plus 10% fetal bovine serum (FBS) without antibiotics. All cells were incubated at 37°C and maintained at 5% CO₂. For fixed imaging, cells were plated on coverslips coated with 10 μg/mL fibronectin (Sigma) for 1 hour at 37°C. For live imaging, cells were plated on 35mm dishes with 14mm glass bottom inserts (MatTek) coated with 10μg/mL fibronectin for 1 hour at 37°C. Transfections into NRK and COS-7 cells for fixed or live imaging experiments and for GST-RILP Rab7 activity pulldowns were performed using Lipofectamine 2000 (Invitrogen) per manufacturer’s instructions. Downstream applications were conducted 24 hours post-transfection unless otherwise stated. For establishment of stable Emerald-Rab7-WT and Em-Rab7-L8A NRK cell lines, Em-Rab7^WT or Em-Rab7^L8A were transfected into NRK cells. 24 hours post transfection, cells were maintained on 100μg/mL geneticin (Gibco) in complete DMEM. After 5 days of selection, individual cell colonies were selected and expanded in geneticin-containing medium. Purity of clones was screened for homogenous Em-Rab7 expression by fluorescence microscopy. Pure clones were expanded, frozen, and maintained in geneticin-containing DMEM for downstream applications.