Fig. 5: Cell-based assays of the interactions of ATG16L1 with GABARAPL1 and FIP200 in autophagy.
From: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins
a Co-immunoprecipitation assays showing that point mutations of key interface residues of GABARAPL1 observed in the GABARAPL1/ATG16L1 FIR complex structure essentially disrupt their specific interaction in cells. “IB” means immunoblotting. b Co-immunoprecipitation assays showing that point mutations of both devised ATG16L1 mutant and key interface residues of FIP200 observed in the FIP200 Claw/ATG16L1 FIR complex structure decrease or essentially disrupt the specific interaction between FIP200 and ATG16L1 in cells. c Western blot-based measurements of the LC3B lipidation and p62 degradation levels in ATG16L1-knockout HeLa cells (16KO) as well as that rescued with mEGFP-tagged WT ATG16L1 (16KO + WT), ATG16L1 D239R/I240F mutant (16KO + DRIF), or ATG16L1 I240Q/I243Q mutant (16KO + IQIQ) treated for 4 h using D10 + PS normal medium (F), D10 + PS normal medium treated with bafilomycin A1 at 400 nM (F + B), amino acid starvation medium (S), or amino acid starvation medium treated with bafilomycin A1 at 400 nM (S + B). d The levels of p62 and β-actin in (c) were quantified in ImageJ and normalized to that of 16KO cells treated with S + B. The data is presented as means ± SEM from four independent experiments. e The levels of LC3B-II and β-actin in (c) were quantified in ImageJ and normalized to the 16KO + WT cells treated with S + B. The data is presented as means ± SEM from four independent experiments. f Western blot-based measurements of the LC3B lipidation in ATG16L1-knockout HeLa cells (16KO) as well as that rescued with mEGFP-tagged WT ATG16L1 (16KO + WT), ATG16L1 D239R/I240F mutant (16KO + DRIF), ATG16L1 I240Q/I243Q mutant (16KO + IQIQ), ATG16L1 K490A mutant (16KO + K490A), ATG16L1 K490A/D239R/I240F mutant (16KO + K490A/DRIF), or ATG16L1 K490A/I240Q/I243Q mutant (16KO + K490A/IQIQ) treated for 1 h using D10 + PS normal medium (F), D10 + PS normal medium treated with monensin at 100 μM (M), or D10 + PS normal medium treated with monensin at 100 μM and bafilomycin A1 at 400 nM (M + B). g The levels of LC3B-II and β-actin in (f) were quantified and normalized to that of 16KO + DRIF cells treated with monensin at 100 μM (M). The data are presented as means ± SEM from three independent experiments. Statistical analyses were all performed in GraphPad Prism 9 by two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test, and P value style is P = 0.1234 [not significant (ns)], *P = 0.0332, **P = 0.0021, ***P = 0.0002, and ****P < 0.0001. h A proposed model depicting the functions of the FIP200/ATG16L1 and ATG8s/ATG16L1 interactions in canonical autophagy.
