Extended Data Fig. 6: ATG2A mislocalises more onto ER–mitochondria contact sites in WIPI4 depleted conditions.
From: Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis
a. Confocal images of endogenous ATG2A co-stained with MAM marker calnexin in wildtype and ATG16 knockout HeLa cells depleted of WIPI4. These images are from one representative experiment out of 3 biological repeats. b. N = 2 biologically independent experiments of proximity ligation assay for VDAC1 and IP3R1 proteins (>= 15 images were taken per sample per experiment). After fixation and blocking, HeLa cells were incubated overnight with rabbit anti-IP3R3 1:100 and mouse anti-VDAC1 1:100 primary antibodies. After PBS wash, PLA probes anti-rabbit PLUS and anti-mouse MINUS were added for 1 hour in the antibody diluent buffer and then stained with Duolink fluorophore red (excitation/emission = 594/624). As a negative control, one or both of the primary antibodies was/were omitted, and the PLA probe background staining was imaged. The background staining was limited for both probes. The average area of PLA signal in cells positive of GFP-spCas9 was quantified using ImageJ. Each datapoint represents the average area (in arbitrary units) of PLA signal per GFP-spCas9 positive cell in the given image. Source numerical data are provided.
