The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). I: Untreated control or sgAtg7 cells, or control cells pre-treated with 100 μM monensin 1 hr, stably expressing Flag-S-ATG16L1 were stimulated 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM) and images were reconstructed in Nikon Elements software. Scale bar: 10 μm. J: Quantification of the percentage of EGF-EEA1 colocalised vesicles that stained triple-positive with ATG16L1 by SIM (in I). Due to the low throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sgAtg7#1 (10 cells), sgAtg7#2 (9 cells), and control + monensin: (11 cells). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001. 