Extended Data Fig. 4: SNX5 is dispensable for general autophagy or non-canonical forms of autophagy that require LC3 recruitment to endolysosomal or phagosomal compartments in HeLa cells.

a, b, Representative fluorescent micrographs (a) and quantitation (b) of GFP–LC3 puncta in HeLa-GFP–LC3 cells that were treated with indicated siRNA (72 h) and then cultured in normal medium (1 h), starvation medium (EBSS; 1 h), or normal medium containing torin 1 (250 nM; 1 h). Scale bars, 20 μm. Arrows denote representative autophagosomes that would be scored as positive in b. c, Quantitation of GFP–LC3 puncta in wild-type HeLa-GFP–LC3 cells (WT), HeLa SNX5^KO/GFP–LC3 cells (KO), and two clones of reconstituted HeLa SNX5^KO/GFP–LC3/SNX5 cells that were cultured in normal medium (1 h), starvation medium (EBSS; 1 h), or normal medium containing torin 1 (250 nM; 1 h). d, e, Representative fluorescent micrographs (d) and quantitation (e) of wild-type HeLa-GFP–LC3 cells and HeLa SNX5^KO/GFP–LC3 cells bearing Group A Streptococcus-containing autophagosome-like vacuoles (GcAVs) at 2 h post bacterial infection (MOI = 100) with Group A Streptococcus strain JRS4 and the isogenic mutant JRS4ΔSLO (which does not induce formation of GcAVs due to defective bacterial escape from endosomes). Cellular and bacterial DNA was stained with DAPI. Boxed areas are magnified by fourfold to show micrographs of indicated channels and representative GcAVs (white puncta). Scale bars, 20 μm. f, g, Representative fluorescent micrographs (f) and quantitation (g) of wild-type HeLa-GFP–LC3 cells and HeLa SNX5^KO/GFP–LC3 cells undergoing non-canonical autophagy after being cultured in either normal medium or hypotonic medium for 1 h. Boxed areas are magnified by twofold to show micrographs of individual channels. Scale bars, 20 μm. h, i, Representative differential interference contrast (DIC) microscopy and fluorescence microscopy micrographs (h) and quantitation (i) of monensin-driven LC3-associated phagocytosis (LAP) of latex beads in wild-type HeLa-GFP–LC3 cells and HeLa SNX5^KO/GFP–LC3 cells that were treated with monensin (100 μM) and polybead microspheres (3 μm in diameter) for 1 h. Boxed areas are magnified by ninefold to show micrographs of DIC channel and GFP channel. Scale bars, 20 μm. In b, c, e, g and i, bars represent mean ± s.d. of triplicate samples (100-150 cells analysed per sample). P values were determined with one-way ANOVA with Dunnett’s test for multiple comparisons (b and c) or unpaired two-tailed t-test (e, g and i). For a, d, f, and h, similar results were observed in three independent experiments.

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