Immunofluorescence staining and confocal microscopy

The number of LC3 puncta and colocalization of LC3 with acidified lysosomes was determined by confocal microscopy as previously described⁶¹. Briefly, BMM cultured on cover slips were infected with IOE at MOI of 5 or left uninfected. Cells were then washed 3X with PBS, fixed with 2% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After blocking with 5% BSA (Sigma-Aldrich, A2153) for 60 min, the primary antibodies; anti-LC3 (Sigma, 50 ug/mL) was added for 1 h at room temperature. Cells were washed and then incubated with fluorescent labeled anti-rabbit secondary antibody DyLight (VectaFluor, 1:500) for 1 h. Nuclei were stained with DAPI and cells were analyzed by confocal microscopy (Olympus Flouview 1000). Analysis of acidified lysosome was performed using LysoTracker Red (cat. L-12492, Thermofisher) at 37 °C for 1 h and assessed with a confocal microscope (Olympus flouview 1000).