Fig. 4: ULK1 complex and PI3KC3–C1 complex are required downstream of WIPI-driven autophagosome biogenesis.
a, Representative maximum intensity projection images of WT HeLa cells stably expressing Fis1-FRB and FKBP–GFP–WIPI2. Cells were left untreated or treated with rapalog for 16 h and immunostained for ATG13. Scale bars, 20 µm and 10 µm (zooms). Results are representative of two biologically independent replicates. b, Immunoblotting for phosphorylated ATG13 in HeLa cells overexpressing Fis1-FRB and FKBP–EGFP–WIPI2d, treated with rapalog for the indicated time. Results are representative of three biologically independent replicates. c, Mitophagy flux measured by flow cytometry in WT HeLa cells transfected with siRNAs targeting FIP200 or ATG13, and expressing Fis1-FRB, FKBP–GFP–WIPI1/2/3 and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Data are presented as mean ± s.d. (n = 4 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. d,e, As in c, but with or without the addition of the ULK1/2 inhibitor MRT68921 (d) or the VPS34-inhibitor VPS34-IN1 (e). Data are presented as mean ± s.d. (n = 6 biologically independent experiments in d and n = 3 in e). Two-way ANOVA with Dunnett’s multiple comparisons test. f,g, As in c, but with WT HeLa cells expressing mt-mKeima and transfected with siRNAs targeting ATG13, FIP200 or ULK1, and treated with DFP for 24 h. Data are presented as mean ± s.d. (n = 3 biologically independent experiments). One-way ANOVA with Dunnett’s multiple comparisons test. h,i, As in f, but with the kinase inhibitors GSK8612 for TBK1, MRT68921 for ULK1/2, VPS34-IN1 for VPS34, or bafilomycin A1 (BafA1). Data are presented as mean ± s.d. (n = 3 biologically independent experiments in h and n = 4 in i). Two-way ANOVA with Dunnett’s multiple comparisons test (i) or one-way ANOVA with Dunnett’s multiple comparisons test (h). j, Immunofluorescence images of WIPI2 and mitochondrial HSP60 in WT or FIP200 KO HeLa cells, untreated or treated for 24 h with DFP. All cells were depleted for PPTC7, aiding the visualization of mitophagy events. Scale bars, 20 µm and 5 µm (zoom). Data are representative of two biologically independent experiments. k, Quantification of the percentage of cells in each field of view that contained autophagosome-like cup structures that colocalized with the mitochondrial marker HSP60. Data are presented as mean ± s.d. (n = 4 biologically independent biological samples). One-way ANOVA with Dunnett’s multiple comparisons test. **P < 0.005, ***P < 0.001, ****P < 0.0001. NS, not significant. Source numerical data, including exact P values, and unprocessed blots are available in the source data.
