Fig 2: Stalled autophagosomes upon inhibition of ULK1 with MRT68921 are positive for DFCP1 and LC3.(A) MEF cells expressing GFP-LC3 and mCherry-DFCP1 were pretreated for 15 min with 1 μM MRT68921 or DMSO followed by treatment with EBSS or EBSS and 1 μM MRT69821. The cells were then immediately subjected to live imaging using a Nikon Eclipse Ti wide-field microscope. (B) Representative images of cells are shown at 30 min in and asterisk marks the section highlighted in panel (B). The movies are provided as supplementary information (Videos 1 and 2). Scale bar, 10 μm. (A, B) Montage of the marked areas from the movies shown in (A) from minutes 22–41. Arrowheads indicate colocalisation between GFP-LC3 and mCherry-DFCP1. (B, C) Graphical representation of the maximum intensity of the mCherry-DFCP1 (red) and GFP-LC3 (green) puncta indicated with arrows in (B). (D) Quantitation of the mean time for which GFP-LC3 and mCherry-DFCP1 signals colocalised on the same punctate structure (from n = 4 events, bars represent mean with SEM). (E) Immunoblot of WT MEF cells treated with EBSS and 1 μM MRT68921 as indicated. (F) Quantitation of (E). pS318-ATG13 levels were normalised to total ATG13 (left) and LC3-II levels were normalised to Tubulin (right). Data represent mean of n = 4 ± SEM. Statistical analysis was performed with two-way ANOVA and a Tukey’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, **** = P < 0.0001 and ns, not significant.Source data are available for this figure.

Fig 3: Distinct ULK1 inhibitors impair autophagy.(A) Immunoblot of lysates from MEF cells treated with EBSS, 50 nM BafA1, 1 μM MRT68921, 10 μM SBI0206965, and 1 μM ULK-101 for 1 h as indicated. (B) Quantitation of pS318-ATG13 levels normalised to total ATG13 (top) and LC3-II levels normalised to α-Tubulin (bottom). Data represent mean of n = 4 ± SEM. Statistical analysis was performed with two-way ANOVA and a Tukey’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 and ns, nonsignificant (unless indicated comparisons of each treatment are with the relevant DMSO control). (C) Representative flow cytometry assay of MEF cells expressing mCherry-GFP-LC3 treated with either DMSO or 50 nM BafA1, or 4 μM MRT68921, or 20 μM SBI0206965, or 2 μM ULK-101 in EBSS for 4 h as indicated, where a decrease/increase in GFP and mCherry expression can be observed and quantified to measure the percent of cells undergoing autophagy. The number in each panel indicates % of cells undergoing autophagy. Quantitation of the flow data is shown on the right and represents mean of n = 3 ± SEM. Statistical analysis was performed with one-way ANOVA and a Tukey’s multiple comparisons test. **** = P < 0.0001.Source data are available for this figure.

Fig 4: HaloPROTAC-E L-AdPROM-mediated GFP-ULK1 Degradation Inhibits Starvation-Induced Autophagy(A) ARPE-19 ULK1GFP/GFP FLAG-empty and FLAG-aGFP6M-Halo-expressing cells were treated with 250 nM HaloPROTAC-E for 24 h and subjected to ATG13 or IgG IP.(B) ARPE-19 ULK1GFP/GFP FLAG-empty and FLAG-aGFP6M-Halo-expressing cells were pre-treated with 250 nM HaloPROTAC-E for 24 h followed by either EBSS or 50 nM Bafilomycin-A1 (Baf-A1) for 2 h.(C and D) Quantification of (C) p-S318 ATG13 normalized to total ATG13 protein levels and (D) LC3-II protein levels normalized to a-tubulin from (B) ± SD of n = 3 independent experiments. +AA indicates amino acid-rich conditions.(E) ARPE-19 ULK1GFP/GFP FLAG-aGFP6M-Halo-expressing cells were pre-treated with 250 nM HaloPROTAC-E for 24 h or with the ULK1 inhibitors MRT68921 (2 µM) or SBI-0206965 (5 µM) for 2 h followed by either EBSS or 50 nM Baf-A1 for 2 h.(F and G) Quantification of (F) p-S318 ATG13 normalized to total ATG13 protein levels and (G) LC3-II protein levels normalized to a-tubulin from (E) ± SD of n = 3 independent experiments.Statistical analyses were carried out by one-way analysis of variance using Tukey’s post-test. For (A), (B), and (E), extracts and IPs were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies.