Fig. 5: RAB22A-mediated non-canonical autophagosomes originate from ER and fuse with RAB22A-positive early endosomes to generate Rafeesomes.

a Immunofluorescence analysis of the ER marker calnexin (red), Flag (magenta or pseudo-green as indicated) and GFP-LC3 (green) in the indicated stable HeLa cells transiently expressing GFP-LC3. Co-localization of GFP-LC3 with calnexin and the percentage of MVB-like structures containing calnexin were quantified. P values were calculated by Student’s t-test. n = 8 fields. Scale bar, 10 μm. b Immunofluorescence analysis of the early endosome marker EEA1 (red), Flag (magenta or pseudo-green as indicated), and GFP-LC3 (green) in the indicated stable HeLa cells transiently expressing GFP-LC3. Percentage of MVB-like structures containing GFP-LC3 and co-localization of EEA1 with RAB22A were quantified. n = 8 fields. Scale bar, 10 μm. c Immunofluorescence analysis of GFP-RAB22A^Q64L (green), EEA1 (magenta), and calnexin-mCherry (red) in GFP-RAB22A^Q64L stable HeLa cells transiently expressing calnexin-mCherry. Scale bar, 10 μm. d Immunofluorescence analysis of GFP-RAB22A^Q64L (green) and calnexin (red) in GFP-RAB22A^Q64L stable HeLa cells using SIM. Scale bar, 5 μm. e Time-lapse images showing GFP-RAB22A^Q64L (green) and calnexin-mCherry (red) in living tet-on GFP-RAB22A^Q64L stable HeLa cells transiently expressing calnexin-mCherry treated with 50 ng/mL doxycycline (dox) (Supplementary information, Video S1). Scale bar, 10 μm. f Time-lapse images showing GFP-RAB22A^Q64L (green) and STING^V155M-Halo (red) in living tet-on GFP-RAB22A^Q64L stable HeLa cells transiently expressing STING^V155M-Halo and treated with 50 ng/mL dox (Supplementary information, Video S2). Scale bar, 10 μm.