Extended Data Fig. 5: ATG2A mislocalises more onto mitochondria in WIPI4 depleted conditions.

a. After immunoprecipitation with endogenous ATG2A (ED Fig. 4e), proteins were eluted from the magnetic beads with Laemmli buffer. Sec23a is the marker for ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. Calreticulin is a marker for ER resident proteins (n = 2). b. Representative images from videos of RFP-LC3 stable HeLa cell line 24 hours after transfection with GFP-ATG2A. The images are from videos taken in one representative of 3 biologically independent experiments. c. HeLa cells transfected with pHAGE-GFP-ATG2AmLIR and pHAGE-GFP-ATG2A were treated with DMSO or Lip-1 for 24 hours in media containing Incucyte Cytotox Red Dye (n = 3). Images were taken 30 hours after first transfection. Dead cells were counted as red objects and the number of transfected cells were counted as green objects. Datapoints represent the ratio of red object count vs. the green object count normalized to the transfection efficiency of each sample. At least 30 images were taken per sample per experiment. Protein levels of overexpressed wildtype ATG2A and ATG2AmLIR are shown in the blots. d. Representative super-resolution structured illumination microscopy images of endogenous ATG2A co-stained with mitochondria marker TOMM20 and ER protein Sec61-BFP. Left hand panels are unprocessed and other panels are rendered using Imaris software. Scale bar in the left 4 panels = 0.4 µm, scale bar in the right hand panels = 0.2 µm. This shows increased ATG2 on TOMM20 (mitochondria) in WIPI4 knockout cells. In control cells, 471/2531 ATG2A structures colocalised with mitochondria, while in WIPI4 knockout cells, 407/1438 ATG2A structures colocalised with mitochondria (p = 1.52216E-12 by chi-squared test). 5 cells were scored per condition. e. Representative confocal images of endogenous ATG2A co-stained with Mitotracker Far-red and ER protein Sec61-BFP. f. Quantification of Pearson’s coefficient of ATG2A and Mitotracker Far-red from confocal images (n = 3, >= 20 images per experiment). Scale bar = 10 µm. g. Live imaging of HeLa cells transfected with GFP-ATG2A and stained with BodipyC12 (n = 3). Scale bar = 5 µm. Two-tailed paired t-test. h. The ATG2A levels in Fig. 4b were compared based on the proportion in mitochondrial fraction to post-mitochondrial fraction (n = 4). Two-tailed one sample t-test to control and two-tailed paired t-test between other samples. Graphs c, f, h present normalized mean ± SD. Graph in g presents mean ± SD. N represents biologically independent experiments. Source numerical data and unprocessed blots are provided.