Fig 2: The N-terminal SKICH domain and NBR1-binding domain of TAX1BP1 are required for ATG7-independent flux of NBR1 (related to Fig 6) HEK293T cells were transfected with myc-TAX1BP1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP-conjugated dynabeads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies.Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. Ubiquitin, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.Wild-type K562 cells were nucleofected with tf-NBR1 (isoform 1) or tf-NBR1 (isoform 2; lacking the UBA domain) and analyzed for red:green ratio at 18 h post-nucleofection. Median values for each sample are identified by a black line within each violin. The red dotted line (max value) corresponds to the red:green ratio of tf-NBR1 isoform 1. The solid red line (min value) corresponds to the red:green ratio of tf-NBR1 isoform 1 upon Bafilomycin A1 (BafA1) treatment. n > 2,000 cells for each sample.Representative confocal micrographs (as maximum intensity projections) of ATG9AKO/TAX1BP1KO cells expressing tf-NBR1. Cells were transduced with the indicated BFP-tagged TAX1BP1 variants. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. BFP-TAX1BP1, magenta; NBR1, green; Scale bars: large panels, 5 µm; small panels, 1 µm.K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading.Clonal isolates of indicated knockout cells lines (from (E)) were treated with Bafilomycin A1 (BafA1) or DMSO for 18 h and analyzed for tf-NBR1 flux by red:green ratio as assessed by flow cytometry. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio of WT cells (normal flux). The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n > 5,000 cells.HEK293T cells were transfected with indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP conjugated dynabeads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies.HEK293T cells were transfected with HA-FIP200 and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP conjugated dynabeads. Input and eluates were resolved by SDS–PAGE followed by immunoblotting (IB) with indicated antibodies. Source data are available online for this figure.

Fig 3: FIP200 colocalizes with endosomal markers A–EHeLa cells were transfected with FLAG-tagged FIP200 alone (A) or together with Rabaptin5 (Rbpt5-wt) (B), GFP-Rab4 (C), RFP-Rab5 (D), or Citrine-Rab7 (E), fixed after 24 h, and subjected to immunofluorescence microscopy. Scale bar, 10 µm.

Fig 4: Comp 5 induces autophagy via AMPK-mTOR-ULK complex axis in GBM cells.a U87MG and T98G cells were transfected with GFP-mRFP-LC3 plasmid, after co-incubation with 10 μM Comp 5, the GFP-LC3 puncta were observed by fluorescence microscope. Scale bar = 20 μm. b U87MG and T98G cells were treated with 10 μM Comp 5 for 24 h, then the expression levels of Beclin-1, p62, and LC3 were determined by western blot. c U87MG and T98G cells were treated with Comp 5 in the presence or absence of BafA1, then the accumulation of p62 and LC3 were detected by western blot. d U87MG and T98G cells were treated with 10 μM Comp 5 for 24 h, then the expression levels of AMPKα, p-AMPKαthr172, mTOR, p-mTORser2448, ULK1, p-ULK1ser317, p-ULK1ser555, mATG13, FIP200, and ATG101 were determined by western blot. e U87MG and T98G cells were treated with Comp 5 in the presence or absence of Compound C, then the expression levels of SIRT1, AMPKα, p-AMPKαthr172, p62, Beclin-1, and LC3 were detected by western blot. f U87MG and T98G cells were transfected with SIRT1 siRNA for indicated time and treated with Comp 5 for additional 24 h. The expression levels of SIRT1, ULK1, p-ULK1ser317, p-ULK1ser555, and LC3 were determined by western blot. β-actin was used as a loading control. g U87MG and T98G cells were treated with 10 μM Comp 5 in the presence or absence of 3-MA (1 mM) for 24 h. Then, cell viabilities were detected by MTT assay. ***P < 0.001 vs. Comp 5-treated group

Fig 5: FIP200 deficiency increases host susceptibility to RNA virus infection.a RIG-I was transfected with vector, FIP200, or ULK1 into HEK293 cells. After 24 h, cells were infected with the VSV carrying a luciferase gene (VSV-Luc) for 16 h. Luciferase activity was measured to determine relative viral infection activity. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. (*P < 0.05, by two-tailed Student’s t test). b Fip200f/f and Fip200-/- MEFs were infected with VSV-Luc for 16 h. Luciferase activity was measured to determine relative viral infection activity. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. The P-value was calculated (two-tailed Student’s t test) by comparison with wild-type cells. *P < 0.05. c Wild type and FIP200 knockout RAW 264.7 macrophages were infected with 0.001 MOI of VSV. After the designated hour post-infection (h.p.i), virus titers were determined by TCID50 in Vero cells. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. The P-value was calculated (two-tailed Student’s t test) by comparison with the wild-type cells. *P < 0.05, **P < 0.01. d Fip200f/f and Fip200-/- MEFs were infected with the indicated MOI of HCoV OC43. After 24 h, the RNA levels of viral membrane gene were determined by qPCR. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. The P-value was calculated (two-tailed Student’s t test) by comparison with the wild-type cells. *P < 0.05, **P < 0.01. e Fip200f/f and Fip200-/- MEFs were infected with 0.1 MOI of HCoV OC43. After the designated day post-infection (d.p.i), virus titers were determined by TCID50 in Vero cells. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments. The P-value was calculated (two-tailed Student’s t test) by comparison with the wild-type cells. *P < 0.05, **P < 0.01. f Fip200f/f and Fip200-/- MEFs were infected with the VACV carrying a luciferase gene (VACV-Luc) for 16 h. Luciferase activity was measured to determine relative viral infection activity. All experiments were biologically repeated three times. Data represent means ± s.d. of three independent experiments.