Fig. 2: SPATA33 interacts with ATG16L1 to form autophagic puncta upon starvation induction.

a Coimmunoprecipitation of MYC-SPATA33 with Cherry-FLAG-ATG16L1. HEK293T cells were transiently transfected with pMYC-SPATA33 and pCherry-FLAG-ATG16L1. After 48 h, the whole cell lysate was extracted for coimmunoprecipitation with anti-MYC, or anti-FLAG. Anti-FLAG or anti-MYC was also used for western blotting. Arrowheads indicate the immunoprecipitated bands. b Coimmunoprecipitation between MYC-SPATA33 and deletion mutants of ATG16L1. pMYC-SPATA33 was transiently co-transfected with pGFP-N-ATG16L1 or pGFP-C-ATG16L1 in HEK293T cells. Cell lysates were examined by western blotting using the anti-MYC or anti-GFP antibody. For coimmunoprecipitation, the lysates were immunoprecipitated with anti-MYC, followed by immunoblotting with the anti-GFP antibody. Arrowheads indicate the immunoprecipitated bands. c Endogenous ATG16L1 interacted with FLAG-SPATA33. HEK293T cells were transiently transfected with p3xFLAG-SPATA33, and after 48 h, the whole cell lysate was extracted for coimmunoprecipitation with anti-ATG16L1 or anti-FLAG. Anti-ATG16L1 or anti-FLAG antibody was also used for western blotting. d Colocalization of SPATA33 with ATG16L1 in HeLa cells under starvation condition. HeLa cells were transiently co-transfected with pCherry-SPATA33 and pGFP-ATG16L1. After cultured in normal medium for 24 h, the cells were starved in EBSS medium for 2 h, and analyzed by confocal microscopy. The nuclei were stained by Hoechst (blue). The insets showed an enlarged view of the indicated squares. Yellow puncta in merged panels are the colocalized puncta. Scale bar: 25 μm. e Statistical analysis of colocalized puncta between SPATA33 and ATG16L1. f Colocalization of SPATA33 with LC3B in HeLa cells under starvation condition. HeLa cells were transiently co-transfected with pCherry-SPATA33 and pGFP-LC3B and cultured in normal medium for 24 h. The cells were starved in EBSS medium for 2 h, and analyzed by confocal microscopy. The nuclei were stained by Hoechst (blue). The insets showed an enlarged view of the indicated squares. Yellow puncta in merged panel are the colocalized puncta. Scale bar: 25 μm. g Statistical analysis of colocalized puncta between SPATA33 and LC3B. Data are presented as means ± S.D. **p < 0.01 (n = 3 independent experiments).