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. 2019 Aug 26;20(10):e47734. doi: 10.15252/embr.201947734

Figure 1. Loss of autophagy perturbs EGFR endocytic trafficking.

Figure 1

The following experiments were performed in glial shNf‐1/shTp53 glial cells serum starved for 4 h before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2).
  1. Spinning disc confocal live cell imaging of Alexa 555‐EGF (555‐EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/ml 555‐EGF for the indicated durations. Scale bar: 10 μm.
  2. Immunofluorescence staining of EGFR following 5‐ or 15‐min stimulation with 20 ng/ml EGF. Scale bar: 20 μm.
  3. Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) (in B).
  4. Cells were stimulated with 20 ng/ml 555‐EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.
  5. Quantification of percentage of total EGFR vesicles that colocalise with 555‐EGF (in D).
  6. Cells stably expressing mCherry‐Rab5 were stimulated with 20 ng/ml EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.
  7. Pearson's colocalisation coefficient between mCherry‐Rab5 and EGFR (in F).
Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t‐test: NS P > 0.05, *P < 0.05, **P < 0.01.