Extended Data Fig. 9: ATG2 transports PS onto mitochondria, which is then locally converted to PE.

a. The blots show the knockdown efficiency with two siRNA oligos targeting mitochondria specific form of PISD (mito-PISD) in SH-SY5Y cells (n = 3). Oligo 2 was used for the following experiments because it’s of better knockdown efficiency. b. The silencing efficiency of anti-mitoPISD siRNA in mitochondria fractions, post-mitochondria fractions, total lysates and nuclear pellets (NP) of HeLa cell lysates. HeLa were transfected with 100 nM of scramble or anti-mitoPISD (#2) siRNA oligos and grown for 48 hours before fractionation. TOMM20, the marker of the mitochondrial fraction; calnexin, the MAM enriched fraction. Datapoints represent the PISD protein levels normalized to the loading (α-tubulin) in each fraction (n = 3). c. The blots show the knockdown efficiency of WIPI4 and PISD in Fig. 7a (representative blots of 3 biological replicates). d. SH-SY5Y cells were transfected with scramble, WIPI4 or si-mitoPISD-2 siRNAs and grown for 48 hours before LDH (n = 3) was measured. Mean (normalized to siWIPI4) ± SD. Blots on the right show the knockdown efficiency of WIPI4 and PISD (representative blots of 3 biological replicates). e. Expression levels of mitoPISD-Myc-DDK measured by staining of anti-FLAG and anti-PISD antibodies (n = 3). HeLa cells transfected with CMV-myc-DDK or mitoPISD-myc-DDK were grown for 24 hours before lysis. f. Confocal images showing the colocalisation of mitoPISD-Myc-DDK with Mitotracker Red and BPC12 (neutral lipid/ lipid droplet probe). HeLa cells transfected with CMV-myc-DDK or mitoPISD-myc-DDK were grown for 24 hours before fixation and immunostaining. These images are from one representative experiment of 3 biological repeats. g. PISD levels relative to α-tubulin in HeLa cells lysates measured by western blotting (n = 3). h. PS levels of mitochondria purified from HeLa cells 48 hours after transfection of anti-WIPI4 and anti-mito-PISD-2 siRNAs (n = 3). i. Representative live-imaging experiment showing the colocalization of transiently overexpressed Lactadherin-C2 GFP and Mitotracker Far-red in HeLa cells (n = 1). Datapoints represent the percentage of Lactadherin C2 GFP area positive for Mitotracker Far-red as a function of total Lactadherin-C2 GFP area. Data and error bars in b, d & g: normalized mean ± SD. Two-tailed one sample t-test to scramble and two-tailed paired t-test between other samples; in h: Mean ± SD. Two-tailed paired t-test. Source numerical data and unprocessed blots are available in source data. N = number of biologically independent experiments.