Fig. 7: TMEM33 promotes the formation of RTN4 noncanonical autophagosomes.

a ATG16L1^–/– and ATG5^–/– HeLa cells with or without TMEM33 knockdown were transfected with RTN4B-mCherry, after which the number of RTN4B-mCherry vesicles was counted. Scale bar, 10 μm. The data are presented as means ± SEMs. n > 15 cells from three independent experiments. p values were calculated by Student’s t-test. ****p < 0.0001. b HeLa cells were transfected with Vector or mCherry-TMEM33, and the number of endogenous RTN4B noncanonical autophagosomes colocalized with endogenous LC3 denoted by white arrows was quantified. Scale bar, 10 μm. The data are presented as means ± SEMs. n = 40, 45 cells from three independent experiments. p values were calculated by Student’s t-test. ****p < 0.0001. c Colocalization analysis of GFP-TMEM33 and endogenous RAB22A on RTN4B-mCherry noncanonical autophagosomes (upper lane) and RTN4B-mCherry vesicles (lower lane) in WT and ATG5^–/– HeLa cells co-transfected with RTN4B-mCherry and GFP-TMEM33. Scale bar, 10 μm. Quantification of colocalization was presented as Pearson’s correlation coefficient (r). n = 32, 30 cells from three independent experiments. d Working model for the in vitro fusion assay between GFP-labeled RAB22A^Q64L early endosomes and mCherry-labeled RTN4B noncanonical autophagosomes. e Fusion puncta (yellow), which indicate Rafeesomes, were quantified after captured using a super-resolution confocal microscopy. The represented merged puncta were shown on the xy, xz and yz axis. Scale bar, 10 μm. The data are presented as means ± SEMs. n = 11, 19 fields from three independent experiments. p values were calculated by Student’s t-test. ****p < 0.0001.