Lentiviral transfer vector

U87-MG and U373 glioma cells (ATCC, Manassas, VA) and 293TN cells (System Biosciences, Palo Alto, CA) were cultured in DMEM (VWR, Radnor, PA) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA). Cells were maintained in a humidified environment at 37°C, 5% CO₂. U373 cells were stably transfected with EGFP as described previously.50 (link), 51 (link), 52 (link) In brief, cells were transfected with a murine stem cell virus (MSCV) retroviral vector encoding an EGFP-Firefly luciferase fusion protein and sorted for EGFP positivity. U87-MG cells were stably transfected with Rab5-GFP or LAMP1-GFP using standard procedures. In brief, Rab5-GFP (Addgene # 56530) or LAMP1-GFP (Addgene # 34831) was cloned into a lentiviral transfer vector (System Biosciences, Palo Alto, CA) by restriction enzyme digest. Lentiviral particles were produced by triple-transfecting (TransIT-Lenti transfection reagent; Mirus Bio, Madison, WI) 293TN cells with either transfer vector and lentiviral packaging and envelope plasmids (Addgene #12260,12259). Lentivirus was harvested, filtered, and diluted in cell culture medium to transduce U87-MG cells. Cells stably expressing the desired fusion protein were selected with 1 μg/mL puromycin (VWR, Radnor, PA).