Fig 2: The BH3 domain promotes BNIP3 turnover in a manner inhibited by ULK1. (a) Western blot for different mutant forms of HA-BNIP3 (WT, S17A, S17E, DBH3, DPEST, G180A, DTMD) expressed in HEK-293 T cells in the presence (lanes 8–14) or absence (lanes 1–7) of exogenous FLAG-ULK1. Fold change in protein levels of BNIP3 dimer and BNIP3 monomer are shown relative to WT. (b) Western blot for HA-BNIP3 and different mutant forms (WT, S17A, S17E, DBH3, DBH3/S17A, DBH3/S17E) of HA-BNIP3 expressed in HEK-293 T cells in the presence (lanes 7–12) or absence (lanes 1–6) of exogenous FLAG-ULK1. Fold change in protein levels of BNIP3 dimer and BNIP3 monomer are shown relative to WT. (c) Pulldown of GFP-LC3 stably expressed in HEK-293 T cells with transiently expressed HA-BNIP3 (WT) and different HA-BNIP3 mutants (DBH3, S17A/DBH3, S17E/DBH3) in the presence or absence of 100 nM bafilomycin A1. Inputs to the pulldown are shown on the left and the result of the pulldown on the right. Fold change in protein levels of BNIP3 dimer and BNIP3 monomer are shown relative to WT. (d) Pulldown of FLAG-ULK1 stably expressed in HEK-293 T cells with transiently expressed HA-BNIP3 (WT) and different CTD mutant forms of FLAG-ULK1 (full-length, D829–1051, D1038–1051). Fold change in levels of BNIP3 dimer and BNIP3 monomer are shown relative to Full-length. (e) Immunofluorescent staining for TOMM20 (green, mitochondria), LC3B (magenta, autophagosomes), HA-BNIP3 (red) and DAPI (blue) in U2OS cells transiently expressing HA-BNIP3 (top panels) or HA-BNIP3DBH3 (bottom panels).

Fig 3: The effect of inhibiting ULK1 on TGFβ1-dependent autophagy in NSCLC cell lines. A549 (A) or H1299 (B) cells stably expressing GFP-LC3-RFP-LC3ΔG were treated with 10 μM of the ULK1/2 inhibitor, ULK-101, or DMSO (vehicle control) in the presence and absence of 250 pM TGFβ1 for 24 h. Cells were lysed and subjected to SDS-PAGE and immunoblotting for anti-ULK1, anti-ULK2, anti-LC3B and anti-GAPDH antibodies. Quantitative analysis of steady state ULK1, ULK2, and LC3B-II protein levels and the GFP/RFP ratio are shown graphically below representative immunoblots (n = 3 ± SD). Significance is indicated as * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. (C) A549 cells stably expressing GFP-LC3-RFP-LC3ΔG were treated as described above. Hoechst stain (blue) was added 10 min prior to imaging with a 63x objective using an Olympus IX 81 inverted fluorescence microscope. Bar = 10 μm. (D) ImageJ was used to quantify the green and red pixel intensity, and the GFP/RFP ratio is shown graphically below representative images (n = 3 ± SD). Significance is indicated as ** = P < 0.01. (E) Cells and number of puncta/cell were counted using ImageJ version 2.0 software. The data were graphed and shown below representative images (n = 3 ± SD). Significance is indicated as *** = P < 0.001 and **** = P < 0.0001.

Fig 4: The effect of TGFβ1 on mTOR and ULK1 activity in NSCLC cells. A549 (A) or H1299 (B) cells were treated with 250 pM TGFβ1 for 24 h. Cells were lysed and subjected to SDS-PAGE and immunoblotting for anti-mTOR, anti-phospho-S2448-mTOR, anti-ULK1, anti-phospho-S555-ULK, anti-phospho-S757-ULK1, or anti-GAPDH (loading control) antibodies. The steady-state levels of phospho-S555-ULK1, ULK1, phospho-S2448-mTOR and mTOR were quantified using QuantityOne software and the phospho-mTOR/mTOR and phospho-ULK1/ULK1 ratios were graphed (n = 3 ± SD). Significance is indicated as * = P < 0.05. (C) A549 cells were treated with 250 pM TGFβ1 for 24 h. Cells were fixed and stained with DAPI (blue), antibodies against ULK1 (green) and ULK2 (red), and imaged using an Olympus IX 81 inverted fluorescence microscope. Bar = 10 μm.

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.