Fig 2: VPS34 is still active after ULK1 inhibition.(A) MEF cells were pre-treated for 15 min with either 2 µM MRT68921, or 1 µM VPS34-IN1, or 1 µM ULK-101 followed by treatment with EBSS for 1 h in the presence or absence of the inhibitors at the above concentrations. After fixation, the cells were stained for endogenous ULK1 (red), WIPI2 (green) and DAPI (blue) and imaged using a Nikon Eclipse Ti wide-field microscope. Scale bar, 10 µm. (B) Quantitation of data shown in A representing mean of n = 3 ± SEM. Statistical analysis was performed with a two-way ANOVA and a Sidak’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 and ns, nonsignificant. Comparisons of each treatment are with the relevant DMSO control.

Fig 3: ATG9 promotes the formation of ATG13 puncta.(a) HEK293 cells were either fed or starved for 1 h, immunolabelled for ATG13 and ATG9 and imaged by wide-field microscopy. Arrowheads in inserts point at ATG13 particles associating with ATG9. Bar corresponds to 10 µm. (b) Values are ATG13 particles in c,d associating with ATG9 particles in the first two frames from their emergence. From analysis of 75 montages. (c,d) Wide-field live-cell imaging of starved HEK293 cells stably expressing GFP-ATG13 and mRFP-ATG9. Representative montages of ATG13 particles forming in association with ATG9 (c) or not (d) are shown. Arrowheads point at the ATG13 particles in the first two frames from their emergence, the same that were used for the analysis in b. (e) Wide-field live-cell imaging of starved HEK293 cells expressing stably GFP-ATG13 and mRFP-ATG9, and transiently CFP-ER. Representative montage of ATG13 particle forming on a tubular extension of ER previously hosting an ATG9 vesicle is shown. Arrowheads point at the ATG13 particle in the first two frames from its emergence and at the associating extension of ER and ATG9 vesicle. (f,g) HEK293 cells were transfected with non-targeted (siNT) or ATG9 (siATG9) siRNA, starved for 1 h, immunolabelled for ATG13 or WIPI2 and imaged by confocal laser scanning microscopy. Values are means±s.e.m. puncta of ATG13 (f) or WIPI2 (g) per cell, for at least five different fields with 15–30 cells each. Significance levels were determined with unpaired t-tests. Bar in c–e corresponds to 300 nm. **P=0.01%; ***P=0.001%.

Fig 4: GLIPR2 inhibits PtdIns3K-C1 complex lipid kinase activity. (A and B) PtdIns3P probe staining (A) and quantification (B) of PtdIns3P puncta in indicated cells. Bars represent mean ± SEM for triplicate samples (50 cells analyzed per sample). Similar results were observed in three independent experiments. n.s., not significant, ***p < 0.01; one-way ANOVA with Dunnett’s test for multiple comparisons. Scale bars, 10 µm. (C and D) Immunostaining (C) and quantification (D) of WIPI2 puncta in indicated cells. Bars represent mean ± SEM for triplicate samples (100–150 cells analyzed per sample). Similar results were observed in three independent experiments. n.s., not significant, ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons. Scale bars, 10 µm. (E) In vitro pull-down assays of purified Strep-tagged PtdIns3K-C1 with wild-type GLIPR2 (WT GLIPR2) or mutant GLIPR2 (H54A E86A G102K H103A N138G) (mt GLIPR2) analyzed by SDS-PAGE and stained with Coomassie Blue. (F) In vitro pull-down assays of Strep-tagged PtdIns3K-C1 with WT GLIPR2 or mt GLIPR2, analyzed by western blotting. The left and right panels were cropped from the same gel. (G) Effect of PIK-III and CHAPS on in vitro lipid kinase activity of PtdIns3K-C1 as measured by mass spectrometry analyses. Bars represent mean ± SEM from five independent experiments. ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons. (H) Effect of GLIPR2 on in vitro lipid kinase activity of PtdIns3K-C1 as measured by mass spectrometry analyses. Bars represent mean ± SEM from five independent experiments. n.s., not significant, *p < 0.05; one-way ANOVA with Dunnett’s test for multiple comparisons. (I) Effect of Tat-BECN1 on in vitro lipid kinase activity of PtdIns3K-C1 as measured by mass spectrometry analyses. Bars represent mean ± SEM from six independent experiments. n.s., not significant, ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons. (J and K) Effects of Tat-BECN1 and GLIPR2 on PtdIns3K-C1 membrane association assessed using liposome flotation assays. Four fractions were collected from each sample and subjected to SDS-PAGE and immunodetection of BECN1 and PIK3C3/VPS34. Representative western blots (J) of BECN1 and PIK3C3/VPS34 in fractions with indicated treatments. Similar results were observed in four independent experiments and the relative quantification (K) of BECN1 and PIK3C3/VPS34 in liposome-containing Fraction 1. Bars represent mean ± SEM. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Dunnett’s test for multiple comparisons

Fig 5: Reduced DFP-induced mitolysosomes upon DGAT1 inhibition Schematic outlining the mito-QC mitophagy reporter system. mCherry-GFP is targeted to the mitochondrial outer membrane via the specific targeting sequence of FIS1. Cytosolic mitochondrial network appears as yellow, due to red–green fluorescence. Upon mitophagy, mitochondria delivered to endolysosomes are distinguished by mCherry-only puncta, whereas GFP does not fluoresce in the acidic microenvironment.Representative photomicrographs of mitophagy reporter cells (human ARPE19 cells with stable expression of mito-QC) treated with DFP (1 mM) for 7 h followed by treatment with LD inhibitors (5 µM) as indicated on the images (I denotes the presence of inhibitor, for example, DGAT1i = DGAT1 inhibition). Cells were fixed after 24 h of DFP treatment. Mitochondrial networks are visible in yellow and mitolysosomes as red-only puncta signifying mitophagy. Nuclei are counterstained with Hoescht 33342. Scale bar = 5 µm.Mitophagy quantification for experiments in 5a. Mitophagy index per cell is measured as a ratio of the number of mitolysosomes per mitochondrial content. Graph generated using the mean of each biological replicate ± SEM (n = 7 with at least 70 cells quantified per biological replicate for each condition). Mean area of mitolysosomes per cell and mean number of mitolysosomes per cell for experiments in 5A.Representative flow cytometry assay of mito-QC SH-SY5Y cells treated with DMSO or LD inhibitors (DGAT1/2i, 5 µM) for 24 h, in the presence or absence of DFP. A decrease/increase in GFP/mCherry expression is quantified to measure the per cent of cells undergoing mitophagy (indicated by number in each panel).Combined quantitation of mito-QC flow data is represented by mean percentage of cells undergoing mitophagy from three independent experiments ± SEM. Statistical analysis was performed with one-way ANOVA and Tukey’s multiple comparisons test. ****P < 0.0001; **P < 0.01.Enlarged regions from representative confocal photomicrographs showing the pattern of endogenous mitochondrial NIX in human ARPE19 cells treated with DFP ±DGAT1i/2i. Samples were immunolabelled with primary antibodies to NIX (green) and ATPB (red). Scale bar = 5 µm.Representative confocal photomicrographs of cathepsin-reactive endolysosomes in human ARPE19 cells treated with DFP ± DGAT1i/2i. Inset shows examples of lysosomal phenotypes. Scale bar = 5 µm.Colocalisation quantitation for experiments shown in 6g. One-way ANOVA with Bonferroni’s post-hoc test. ****P < 0.0001. Median represented by dashed line, quartiles by dotted lines n = 2 biological replicates.Colocalisation quantitation for ULK1/WIPI2 immunolabelling in human ARPE19 cells treated with DFP ± DGAT1i/2i. Median represented by dashed line, quartiles by dotted lines n = 2 biological replicates.Quantitation of lysosome positioning in DFP ± DGAT1i/2i. One-way ANOVA with Bonferroni’s post-hoc test. ****P < 0.0001. Median represented by dashed line, quartiles by dotted lines n = 2 biological replicates.