Extended Data Fig. 9. Analysis of additional ATG9A interactors and ATG9A M33 mutant in defense against PM damage.

a, Confirmation by immunoblotting of CRISPR-Cas9 in ATG2A^U2OS-KO cells. One of 3 independent experiments. b, Confirmation by immunoblotting of CRISPR-Cas9 in ATG2B^U2OS-KO cells. One of 3 independent experiments. c, Graph, HCM quantification of PM permeabilization (PI, Dig) in ATG2A^U2OS-KO, ATG2B^U2OS-KO and ATG9A^U2OS-KO cells. Data, % of total cells that are PI⁺ cells (mean±SEM; n=3 biologically independent samples, two-way ANOVA Sidak’s test). d,e, HCM quantification of PM permeabilization (PI, d) and cell viability (LDH release in the supernatant, e) in ATG2A^U2OS-KO, ATG2B^U2OS-KO and ATG9A^U2OS-KO cells after LPS electroporation. Data, % of total cells that are PI⁺ cells (d, HCM; e, % LDH release quantified, mean±SEM; n=6 biologically independent samples, unpaired t test). f, Confirmation by immunoblotting of PI4KB KD in HeLa cells (one of 3 independent experiments). g, Graph, HCM quantification of PM permeabilization (PI, Dig) in PI4KB KD cells. Data, % of total cells that are PI⁺ cells (mean±SEM; n=5 biologically independent samples, two-way ANOVA Sidak’s test). h, HCM complementation analysis of PM permeabilization sensitivity (Dig) in ATG9A^Huh7-KO transfected with ATG9A-FLAG (WT or M33 scramblase mutant). PI⁺ cells quantified after gating on FLAG⁺ cells (HCM, mean± SEM, n=6 biologically independent samples, unpaired t test). i, Immunoblot confirmation of Rab7 KD in HeLa cells. One of 3 biologically independent experiments. j, HCM quantification of PM permeabilization (PI, Dig) in HeLa cells after Rab7 KD. Data, % of cells positive for PI (mean±SEM; n=6 biologically independent samples, two-way ANOVA Sidak’s test). k, CoIP (anti-GFP) analysis of FLAG-ATG9A and GFP-AP2M1 interaction during PM damage (Dig, HEK293T, one of 3 independent experiments). l, CoIP (anti-GFP) analysis of FLAG-ATG9A and GFP-AP4M1 interaction during PM damage (Dig, HEK293T, one of 3 independent experiments). m, Bimolecular fluorescence complementation assay schematic for analysis of ALIX and ATG9A association with split Venus fluorescent protein. ALIX and ATG9A were respectively fused with the N-terminal (VN^ALIX) and C-terminal (VC^ATG9A) fragments of Venus and expressed in HeLa cells. Venus fluorescence corresponding to ALIX and ATG9A association was then assessed by HCM and confocal microscopy. n, Confocal images of ATG9A and ALIX association using the bimolecular fluorescence complementation assay (VN^ALIX+VC^ATG9A=green) during PM damage (Dig). GM130 (Golgi staining, red). Scale bars, 10 µm. o, p, HCM quantification of (o) ATG9A and ALIX association and (p) their overlap with PM using the bimolecular fluorescence complementation assay, during PM damage (Dig). HeLa cells transiently expressing VN^ALIX and VC^ATG9A were labelled with cell outline/plasma membrane stain CellMask reagent. Data, HCM quantification of (o) Venus puncta and (p) the overlap area between Venus puncta and CellMask (mean±SEM; n=5 biologically independent samples, one-way ANOVA Dunnett’s test). q, HCM quantification of cytosolic PI staining in ATG9A^Huh7-WT and ATG9A^Huh7-KO cells without damage (mean±SEM; n=6 biologically independent samples, unpaired t test).

Source data