Article Figures & Data
Figures
- Figure S1. Immunoblot of cell extracts from cell line used in along with WT MEFs.
Fig 1
Source data are available for this figure.
Source Data for Figure S1[LSA-2020-00815_SdataFS1.pdf]
- Figure 1. 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.
Source Data for Figure 1[LSA-2020-00815_SdataF1.pdf]
- Figure 2. Expression of kinase-dead ULK1 results in aberrant autophagosomal structures.
(A) WT MEF cells or ULK1/2 double KO (DKO) expressing Flag-ULK1 WT or Flag-ULK1 K46I (kinase-dead mutant) were treated with EBSS or EBSS and 2 μΜ MRT68921 for 1 h as indicated. Cells were subsequently fixed and stained with antibodies against ULK1 (green) and LC3 (red). Arrows mark conventional phagophores, whereas arrowheads mark abnormal structures. Scale bar, 10 μm. (B, C) Quantitation of LC3 puncta number (B) and size (C) in ULK1/2 DKO cells and ULK1/2 DKO cells expressing Flag-ULK1 WT or Flag-ULK1 K46I, treated with EBSS for 60 min. Data represent mean of n = 3 ± SEM. Statistical analysis was performed with one-way ANOVA and a Tukey’s multiple comparisons test.* = P < 0.05 and ** = P < 0.01. LC3 puncta size was calculated manually using the “area” tool in NIS Elements. (D) Immunoblot of lysates from cells used in (A) showing ULK1 expression levels.
Source data are available for this figure.
Source Data for Figure 2[LSA-2020-00815_SdataF2.pdf]
- Figure 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.
Source Data for Figure 3[LSA-2020-00815_SdataF3.pdf]
- Figure S2. Structures of MRT68921, ULK-101, and SBI0206965.
- Figure S3. Upstream regulation of ULK1 upon inhibitor treatment.
(A, B) Quantification of pS757 ULK1 levels (A) and pS555 ULK1 levels (B) normalised to total ULK1. Bars represent mean of n = 4 ± SEM. Statistical analysis was performed with a two-way ANOVA and a Tukey’s multiple comparisons test. The presence of the inhibitors resulted in no significant changes among the different conditions. This figure is complementary to Fig 3. (C) Co-immunoprecipitation of ULK1 with FIP200 and ATG13 from cells growing in Fed or EBSS conditions treated with 2 μM MRT68921, or 1 μM ULK-101, or 5 μM SBI0206965 or DMSO for 1 h, as indicated.
Source data are available for this figure.
Source Data for Figure S3[LSA-2020-00815_SdataFS3.pdf]
- Figure S4. Autophagosomal structures under fed conditions.
MEF cells were pre-treated for 15 min with either 2 μM MRT68921, or 1 μM ULK-101, or 5 μM SBI0206965 followed by incubation for a further 1 h in complete medium in the presence or absence of inhibitors, as indicated. (A, B) Cells were subsequently fixed and stained for (A) endogenous ULK1 (red) and LC3 (green) or (B) WIPI2 (green) and GABARAPL1 (red). Scale bar, 10 μm. This figure is complementary to Fig 4.
- Figure 4. Distinct ULK1 inhibitors lead to aberrant autophagosomal structures.
(A) MEF cells were pretreated for 15 min with either 2 μM MRT68921, or 1 μM ULK-101, or 5 μM SBI0206965 followed by treatment with EBSS for 1 h in the presence or absence of the inhibitors at the above concentrations. Cells were fixed and stained for endogenous ULK1 (red), LC3 (green), and DAPI (blue) and imaged with a ZEISS LSM 880 confocal microscope. Scale bar, 10 μm. Boxed area is shown enlarged in zoom panels and dotted line is shown as a line scan below to highlight colocalization and puncta intensity. (B) Quantitation of indicated data from (A) shown as mean of n = 3 ± SEM. Statistical analysis was performed with a two-way ANOVA and a Tukey’s multiple comparisons test (or a two-way ANOVA and a Sidak’s multiple comparisons test for puncta size) Note, method of quantitation of LC3 mean size was distinct from that used in Fig 2 (see the Materials and Methods section for more detail). (C, D) as in (A, B) but cells were stained for endogenous WIPI2 (green) and GABARAPL1 (red). Arrowheads mark examples of abnormal WIPI2-GABARAPL1 autophagic structures. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 and ns, nonsignificant.
- Figure S5. LC3 structures following autophagy induction and ULK1 inhibition.
MEF cells were co-treated with EBSS and either DMSO, 2 μM MRT68921, 1 μM ULK-101, or 5 μM SBI0206965 as indicated. Cells were subsequently fixed and stained for endogenous LC3 (green) and DAPI (blue) and imaged using a Nikon Eclipse Ti wide-field microscope. Scale bar, 10 μm.
- Figure 5. 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.
- Figure S6. VPS34 activity is required for ULK1-inhibited LC3 puncta formation.
(A) MEF cells were pretreated for 15 min with either 2 μM MRT68921 or 1 μM VPS34-IN1, followed by incubation for a further 1 h in complete medium or EBSS in the presence or absence of inhibitors. Cells were fixed and stained for endogenous ULK1 (red), LC3 (green) and DAPI (blue) and imaged using a Nikon Eclipse Ti wide-field microscope. Scale bar, 10 μm. (A, B) Quantitation of data from (A), representing mean of n = 3 ± SEM. Statistical analysis was performed with a two-way ANOVA and a Tukey’s multiple comparisons test. ** = P < 0.01, *** = P < 0.001 and ns, nonsignificant.
Supplementary Materials
- Video 1
Autophagosome formation under control conditions (associated with Fig 1).Download video
- Video 2
Autophagosome formation under ULK1-inhibited conditions (associated with Fig 1).Download video
