Fig. 5: The STX13-SNAP23-VAMP3 SNARE complex is involved in the fusion of ATG9A vesicles with PM in protection against PM damage.

a Confocal microscopy analysis of plasma membrane permeabilization after knockdown of STX1, STX2, STX3, STX4, STX5, STX13, or VTI1A in HeLa cells. Cells were stained with propidium iodide (PI) following plasma membrane damage induced by digitonin (Dig). Scale bars, 50 μm. Representative data from 6 independent experiments are shown. b Quantification of plasma membrane permeabilization induced by digitonin after knockdown of the indicated genes. The percentage of PI⁺ (nuclei) cells in (a) is shown as mean ± SEM and analyzed using Student’s t-test and Welch’s t-test (n = 6 biologically independent experiments). ns not significant; ***p < 0.001. c Confocal microscopy analysis of plasma membrane permeabilization after knockdown of VTI1B, GOSR1, GOSR2, STX8, STX10, SNAP23, or SNAP29. Cells were stained with propidium iodide (PI) following plasma membrane damage induced by digitonin (Dig). Scale bars, 50 μm. Representative data from 6 independent experiments are shown. d Quantification of plasma membrane permeabilization induced by digitonin after knockdown of the indicated genes. The percentage of PI⁺ (nuclei) cells in (c) is shown as mean ± SEM and analyzed using Student’s t-test, Welch’s t-test, and Wilcoxon rank sum test (n = 6 biologically independent experiments). ns not significant; **p < 0.01; ***p < 0.001. e Confocal microscopy analysis of plasma membrane permeabilization after knockdown of VAMP2, VAMP3, VAMP4, VAMP7, SEC22A, SEC22B#1, or YKT6. Cells were stained with propidium iodide (PI) following plasma membrane damage induced by digitonin (Dig). Scale bars, 50 μm. Representative data from 6 independent experiments are shown. f Quantification of plasma membrane permeabilization induced by digitonin after knockdown of the indicated genes. The percentage of PI⁺ (nuclei) cells in (e) is shown as mean ± SEM and analyzed using Student’s t-test (n = 6 biologically independent experiments). ns not significant; *p < 0.05; ***p < 0.001. g Cell surface biotinylation analysis of ATG9A in control (NC), STX13-depleted (siSTX13), SNAP23-depleted (siSNAP23), or VAMP3-depleted (siVAMP3) HeLa cells expressing ATG9A-GFP (under normal conditions or after plasma membrane damage induced by Dig). ATG9A-GFP was immunoprecipitated with an anti-GFP antibody and analyzed by Western blot using Streptavidin-HRP antibody. Representative data from 4 independent experiments are shown. h Quantification of the levels of biotinylated ATG9A (normalized to ATG9A-GFP levels) is shown as means ± SEM and analyzed using Student’s t-test (n = 4 biologically independent experiments). *p < 0.05; **p < 0.01.