Figure 5A) Pulldown assay was performed incubating purified GST-tagged LC3 (A, B and C) isoforms and HA-TFG transfected HeLa cell lysates. HA-TFG protein levels were analysed by WB; LC3 isoforms and GFP are shown in the Ponceau staining. Densitometry analysis of TFG over GST-tagged LC3 isoforms is reported in the graph (right). Data are expressed as mean ± SEM. Statistical analysis were performed by one-way ANOVA followed by Tukey's multiple comparison test. ***, P<0.001. (n=3 independent experiments). B) The model illustrates the domain composition of TFG, including a PB1 domain (residues 10-91), a coiled-coil motif (CC, residues 97-124) and a C-terminal region including a proline-glutamine-rich mostly disordered region. The location of the predicted LIR motifs is also reported, along with the associated PSSM scores from i-LIR. The model of the 3D structure of the PB1 domain is colored according to the solvent accessibility of each residue as derived by Chimera and the residues for the predicted LIR motif 31-36 for HP1 and HP2 hydrophobic pockets of LC3 proteins are highlighted as sticks. C) Pulldown assays were performed by using purified GST- LC3 (A, B and C) isoforms, each incubated together with empty vector (EV) or HA-TFGwtLIR or HA-TFGmutLIR3 transfected HeLa cell lysates respectively. Ponceau staining reports GST-LC3 isoforms and GST-GFP used as a negative control. WB analyses show HA-TFGwtLIR or HA-TFGmutLIR3 in the pulldown and in total extracts (Input). Densitometry analyses of HA-tagged TFG normalized over GST-tagged LC3 isoforms (Ponceau) were performed and reported (right). Data are mean value ± SEM. Statistical analysis was performed by multiple t-test. *, P<0.05; ns, not significant. (n=3 independent experiments). D) HeLa cells were co-transfected with HA-LC3C together with HA-TFGwtLIR or HA-TFGmutLIR3 plasmids. After 30' of starvation, protein extracts were immunoprecipitated using an anti-TFG antibody or IgG as a negative control. LC3C, TFG were analysed by WB. E) HeLa cells were co-transfected with 3'UTR TFG siRNA together with HA-TFGwtLIR or an empty vector (EV) or TFGmutLIR3 plasmids, expressed at near endogenous levels. Cells were cultured with complete (FED) or starvation (STV) media and grown in presence or absence of CLQ for 1h. ACTIN and LC3B-II protein levels are shown. Densitometry analysis of LC3B-II over actin for indicated conditions is shown in the graph (right). Data are mean value ± SEM. Statistical analysis was performed one-way ANOVA followed by Dunnett's multiple comparison test. *, P<0.05. (n=3 independent experiments). F) HeLa cells were co-transfected with unrelated oligonucleotides as a negative control (siCTR) and empty vector (EV) or 3'UTR siRNA TFG siRNA together with EV or HA-TFGwtLIR or TFGmutLIR3 plasmids, expressed at near endogenous levels. Protein extracts were analysed by WB to detect ULK1, TFG, and ACTIN as indicated. Densitometry analysis of ULK1 over ACTIN is shown (right). Data are mean value ± SEM. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test. *, P<0.05; **, P<0.01, ns, not significant. (n=4 independent experiments). HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) Hoechst was used to stain nuclei. ULK1 puncta number (G) was analysed and reported as mean ± SEM in the graph (right) (n=3 independent experiments, n=24 fields analysed). Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm. HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) alone or ULK1 (red) and ERGIC53 (green) (H). Hoechst was used to stain nuclei. analysed). Co-localization analyses (H) were performed by Jacop plugin. Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm. I) HeLa cells stably expressing GFP-ATG13 together with BFP-TFGwtLIR or BFP-TFGmutLIR3 were co-transfected with a siRNA targeting endogenous TFG (siTFG 3'UTR) and mCherry-SEC16, then cultured for 40' with starvation medium. Cells were visualised with cellSens microscope for live-cell imaging. Analysis of the percentage of GFP-ATG13 lifetime colocalizing with mCherry-SEC16 in the presence of TFGwtLIR or TFGmutLIR3 is reported in the cumulative relative frequency plot. Statistical analysis was performed by Kolmogorov-Smirnov test. P <0.0001. (n=3 independent experiments, n≥995 number of colocalization events analysed). J) HeLa cells stably expressing GFP-ATG13 together with BFP-TFGwtLIR or BFP-TFGmutLIR3 were transfected with 3'UTR TFG siRNA to downregulate endogenous TFG. Cells were cultured for 40' with starvation medium and visualised with cellSens microscope for live-cell imaging. Analysis of the percentage of GFP-ATG13 lifetime colocalizing with BFP-TFGwtLIR or BFP-TFGmutLIR3 is reported in the cumulative relative frequency plot. Statistical analysis was performed by Kolmogorov-Smirnov test. P=0,0268. (n=3 independent experiments, n≥379 number of colocalization events analysed). K) HeLa cells harboring endogenously HA-tagged LC3C were transfected with specific RNAi oligonucleotides (siLC3C) or unrelated oligonucleotides as a negative control (siCTR) and cultured in starvation medium for 30'. Protein extracts were analysed by WB to detect ULK1, ACTIN and HA-LC3C as indicated. Densitometry analysis of ULK1 over ACTIN is reported (right). Data are expressed as mean ± SEM. Statistical analysis was performed by unpaired Student's t‐test. *, P<0,05. (n=3 independent experiments). L) HeLa cells harboring endogenously HA-tagged LC3C were transfected with a siRNA targeting TFG (siTFG) or with a non-targeting control (siCTR). After 30' of starvation, cells were lysed, and protein extracts were immunoprecipitated by using an anti-HA antibody or IgG as negative control. Protein extracts were analysed by WB to detect ULK1, HA-LC3C as indicated. M) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co-transfected with HA-LC3C and Myc-ULK1 plasmids as indicated. Cells were starved for 30' and protein extracts were immunoprecipitated using an anti-HA antibody or IgG as negative control. Protein extracts were analysed by WB to detect ULK1, LC3C, TFG and ACTIN as indicated.