Fig. 4: Activated STING is packaged into MVB-like structures whose formation is driven by RAB22A. | Cell Research

Fig. 4: Activated STING is packaged into MVB-like structures whose formation is driven by RAB22A.

From: Intercellular transfer of activated STING triggered by RAB22A-mediated non-canonical autophagy promotes antitumor immunity

Fig. 4

a Knockout efficiency of ATG5 shown by western blot analysis. b Immunofluorescence analysis of LC3 (red), Flag-RAB22AQ64L (magenta), and transiently transfected STINGV155M-GFP (green) in wild-type or ATG5-KO HeLa cells. Percentages of MVB-like structures containing LC3 and STINGV155M were quantified respectively on the right. P values were calculated by Student’s t-test. n = 6 fields. Scale bar, 10 μm. c Immunofluorescence analysis of LC3 (magenta), HA (red), and GFP-RAB22AQ64L (green) in the indicated HeLa cells stably expressing STING-HA, STINGV155M-HA or STINGV155M/LIR467-HA. Scale bar, 10 μm. d Immunofluorescence analysis of LC3 (green) and Flag (red) in the indicated stable HeLa cells. Percentages of MVB-like structures containing LC3 (n = 10 fields) and diameters of RAB22A-positive MVBs (n = 150) were quantified on the right. P values were calculated by Student’s t-test. Scale bar, 10 μm. e Intracellular structures were observed by electron microscopy in the indicated stable HeLa cells. Scale bar, 500 nm. f Western blot analysis of WCL and EVPs derived from the 3× Flag-RAB22AWT and 3× Flag-RAB22AQ64L stable HeLa cells. Relative protein expression was quantified on the right. P values were calculated by Student’s t-test. g Western blot analysis of WCL and EVPs derived from the RAB22A-KO HeLa cells reintroduced with 3× Flag-RAB22AWT, 3× Flag-RAB22AQ64L, or 3× Flag-RAB22AS19N as indicated. * represents the bands of RAB22A; ※ represents the bands of 3× Flag-RAB22A. h Immunofluorescence analysis of LC3 (red), Flag (magenta), and STINGV155M-GFP (green) in the indicated stable HeLa cells transiently expressing STINGV155M-GFP. Scale bar, 10 μm. i Western blot analysis of WCL and immunoprecipitated RAB22A-positive sub-organelles derived from the indicated stable HeLa cells. j Immunofluorescence analysis of GFP-RAB22AQ64L (green) and LC3 (red) in GFP-RAB22AQ64L stable HeLa cells using SIM. Scale bar, 5 μm. k Immunoelectron microscopy showing LC3 localization in the indicated stable HeLa cells. Scale bar, 200 nm. l The diameters of LC3-positive ILVs were calculated. n = 156.

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