Fig 2: The ATG14 LIR motif is important for binding of the PtdIns3K-C1 complex to GABARAP and GABARAPL1, and for activation of the complex by ULK1-mediated Ser29 phosphorylation of ATG14. (a) GST affinity isolation using bead-bound GST-Atg8-family fusion proteins and lysates from HCT116 cells knocked out for ATG14 and reconstituted with WT or LIR mutated MYC-ATG14. Bead bound proteins were detected by western blots using the indicated antibodies. (b) MYC-Trap experiments performed with the indicated cell lines. Bound proteins were detected by western blots using the denoted antibodies. (c) MYC-Trap experiments performed with the indicated cell lines, incubated in starvation medium (HBSS) for 6 h or not. Bound proteins were detected by western blots using the indicated antibodies. (d-g) Relative quantifications of the data in (c), showing the relative amount of BECN1, p-S93 BECN1 and p-S29 MYC-ATG14 in the MYC precipitates. Data information: Means ± SD of 3 independent experiments. Significant P values are indicated (Student´s two-tailed, unpaired t-test). ns, not significant.

Fig 3: HUNK binds the Beclin-1 complex members. HA-HUNK and either (A) Flag-Beclin-1, (B) GFP-Atg14L, (C) Flag-Vps34, (D) Flag-UVRAG, or (E) Flag-Rubicon were co-transfected into 293T cells. HUNK was immunoprecipitated and binding of individual proteins was assessed via immunoblotting for each Beclin-1 complex member. Each member of the Beclin complex co-immunoprecipitated with HUNK. HUNK was immunoprecipitated and detected with anti-HA antibody and Beclin-1 complex members were detected with anti-Flag or anti-GFP antibodies. Interactions were also detected by individual antibodies to each Beclin-1 complex member.

Fig 4: Verification of functional LIR motifs in PIK3C3, BECN1 and ATG14. The putative core LIR motifs, identified using peptide array analysis, were mutated by site-directed mutagenesis in the context of the full-length protein. Point mutations of both the aromatic residue and the conserved hydrophobic position were introduced in order to replace these residues with alanine. The impact of mutating the LIR motifs was then assessed in GST-pulldown analyzes with recombinant GST-Atg8 homologs and in vitro-translated 35S-labelled Myc-tagged PIK3C3, BECN1 and ATG14. Phosphomimetic mutations of the previously identified important serines (S90E, S93E and S96E) of BECN1 were made and tested for functionality with the identified LIR motif FTLI or a LIR mutant. (a) Autoradiographs (AR, top panels) and Coomassie Brilliant Blue-stained immobilized GST or GST-fusion proteins (bottom panel) are shown. (b-d) Quantifications of the binding of wild-type and mutant constructs to the GST-Atg8 proteins is presented as percentage binding relative to the 10% input. There is a significant increase in binding of the phosphomimetic mutant of BECN1 in (c). Data information: Means ± SD of 3 independent experiments. Significant P value is indicated as * = P value is 0.017 (Student´s two-tailed, unpaired t-test). ns, not significant.

Fig 5: Circ_0005567 promoted autophagy and inhibited chondrocyte apoptosis by upregulating ATG14 expression. (A) Immunofluorescence results of chondrocytes transfected with GFP-LC3, (B) protein levels of Beclin-1 and LC3-II/LC3-I examined by western blot, (C) cell apoptosis rate determined by flow cytometry after Annexin V-FITC/PI staining, and (D) protein levels of caspase-3, Bax, and Bcl-2 examined by western blot in chondrocytes co-transfected with si-ATG14/si-Ctrl and circ_0005567 overexpression vector/empty vector in the presence of IL-1ß. The data are expressed as mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.01, versus the si-Ctrl + Vector group; #P < 0.05, ##P < 0.01, versus the si-Ctrl + circ_0005567 or Vector + si-ATG14 group.