Fig. 8: ATG9A transports RTN4 vesicles to promote the formation of RTN4 noncanonical autophagosomes.

a WT or ATG9A^–/– HeLa cells were transfected with RTN4B-mCherry for 36 h, after which the formation of RTN4B-mCherry noncanonical autophagosomes colocalized with endogenous LC3 and RTN4B-mCherry vesicles were monitored. Scale bar, 10 μm. The data are presented as means ± SEMs. n = 22, 29 cells from three independent experiments. p values were calculated by Student’s t-test. **p < 0.01, ****p < 0.0001. b Colocalization analysis of GFP-ATG9A and endogenous LC3 on RTN4B-mCherry noncanonical autophagosomes (upper lane) or RTN4B-mCherry vesicles (lower lane) in WT or ATG5^–/– HeLa cells co-transfected with RTN4B-mCherry and GFP-ATG9A. Scale bar, 10 μm. Quantification of colocalization was presented as Pearson’s correlation coefficient (r). n = 36, 39 cells from three independent experiments. c LC3-II levels in WT and ATG9A^–/– HeLa cells with or without FLAG-RAB22A^Q64L overexpression were measured by western blotting. The data are presented as means ± SEMs. n = 3. P values were calculated by Student’s t-test. *p < 0.05, ns indicates not significant. d The formation of Rafeesomes containing endogenous LC3 was assessed in WT and ATG9A^–/– HeLa cells stably expressing FLAG-RAB22A^Q64L. Scale bar, 10 μm. The data are presented as means ± SEMs. n = 30, 33 cells from three independent experiments. p values were calculated by Student’s t-test. ****p < 0.0001. The number of Rafeesomes containing endogenous LC3 was counted.