Figure EV3. (related to Fig 5): Effects of EGFR on STING trafficking.

1. STING^−/− MEFs expressing GFP‐STING (Green) were incubated with cGAMP for 2.5 h; cells were fixed and observed under confocal microscope. In the images from cGAMP‐treated cells, the STING‐enriched regions were expanded and shown in the right panels (red border). Scale bars represent 10 μm.
2. STING translocation in cells was quantitated by ImageJ. At least 40 cells were randomly picked. The green dots/clusters were highlighted, and the area of each dots/clusters and the total area were calculated. The top 5 individuals in each cell were picked, and the sum of their total area was calculated as Area10. In case, Area10 was bigger than 30% of the area of all green dots/clusters in the cell; the cell was considered as cells with clusters. Otherwise, the cell would be considered as cells with dots. The percentage of cells with clusters or punctate dots in total of cells with translocated STING were calculated, and the number of cells in each part is labeled in each column. All values are means ± SD, Student's t‐test (unpaired); ***P < 0.001.
3. Pearson's co‐localization coefficient between STING and LC3 from Fig 5D and E. At least 25 cells were quantified from each independent experiment, which was repeated for three times with similar results. Values are presented as mean ± SD, ***P < 0.001, two‐tailed Student's t‐test (unpaired).
4. Pearson's co‐localization coefficient between STING and CD63 from Fig 5F. At least 25 cells were quantified from each independent experiment, which was repeated for three times with similar results. Values are presented as mean ± SD, ***P < 0.001, two‐tailed Student's t‐test (unpaired).
5. Hela cells were transfected with STING‐GFP (Green) for 24 h and then transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, red) antibody. Co‐localization (Col) is indicated by white dots. Scale bars represent 10 μm.
6. EGFR^−/− Hela cells were transfected with STING‐GFP (Green) for 24 h and transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, red) antibody. Co‐localization (Col) is indicated by white dots. Scale bars represent 10 μm.
7. Same treatments as in (E), but labeled with CD63 (late endosome marker, red) antibody. Scale bars represent 10 μm.
8. Same treatments as in (F), but labeled with CD63 antibody (red). Scale bars represent 10 μm.