Bafilomycin a

NIH 3T3 cells were cultured for 4 hours in the presence of 100 nM Bafilomycin A (Alfa Aesar) to block autophagy and/or in DMEM media without glutamine and serum for autophagy induction prior to downstream analysis. For treatment with MOs, each MO (GeneTools) was added to culture media at a final concentration of 10 μM along with Endoporter (GeneTools) and incubated for 60 hours. MW150 treatment was carried out by culturing NIH 3T3 cells in the presence of 8 μM MW150 for 8 hours prior to addition of MOs. For immunofluorescence analysis following MO treatment, NIH 3T3 cells cultured on glass coverslips in 24-well plates were washed briefly with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature, and permeabilized with 0.5% Triton X-100–PBS for 10 minutes at room temperature. Blocking and incubation with both primary and secondary antibodies were performed using 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Images were collected with an SP5 Leica confocal microscope.