Fig 2: FIP200 interacts with TBK1. Flp-In T-REx 293 cells inducibly expressing GFP or GFP-FIP200 were treated with 0.1 µg/ml doxycycline for 16 h to induce expression. Afterwards, the GFP proteins were purified overnight using GFP-trap beads. Cleared cellular lysates (input) and purified proteins were immunoblotted for the indicated proteins. The full-length blots are presented in Figure S5.

Fig 3: Loss of FIP200 further enhances TBK1 accumulation and activation caused by defective autophagy. (A) Atg3 WT and atg3 KO MEFs were transfected with either 20 nM negative control siRNA or 20 nM Fip200 siRNA for 72 h, and the cleared cellular lysates were immunoblotted for the indicated proteins. The asterisk indicates a nonspecific band. The full-length blots are presented in Figure S9. The densities of the bands on immunoblots from three independent experiments were quantified and normalized to those of Actin. For each protein, the normalized densities of atg3 KO samples were normalized to that of the respective untransfected sample. The boxes represent the highest and the lowest value, while the centerline shows the mean. Two-way ANOVA with Tukey's multiple comparisons test was used to determine differences between treatments. n.s. = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (B + C) atg3 KO MEFs were transfected with either 20 nM negative control siRNA or 20 nM Fip200 siRNA for 72 h and immunostained for TBK1 pS172 (B; fixed in 4% PFA) or TBK1 in combination with either FIP200 or SQSTM1/p62 pS403 (C; fixed in 100% MeOH). For (B), the number, area, and intensity of TBK1 pS172-positive structures of at least 232 cells per cell line were quantified using ImageJ 1.53c and normalized to those of untransfected atg3 KO cells. The boxes represent the highest and the lowest value, while the centerline shows the mean from four independent experiments. For IF imaging, DAPI was used to stain nuclei.

Fig 4: Loss of components of the ULK1 complex leads to aberrant accumulation and increased activation of TBK1. (A) fip200 KO MEFs were transfected with an empty vector or cDNA encoding full-length FIP200 (FIP200 WT) or C-terminally truncated FIP200 (amino acids 1369–1594, FIP200 ?CT). These transfectants, atg3 KO and Atg3 WT MEFs were fixed in 4% PFA and immunostained for TBK1 pS172. Representative sections are depicted. (B) atg13 KO MEFs transfected with an empty vector or cDNA encoding WT ATG13 were fixed in 4% PFA and immunostained for TBK1 pS172 and FIP200. Representative sections are depicted. (C) For costaining of TBK1 and FIP200, atg13 KO MEFs were transiently transfected with cDNA encoding GST-TBK1, fixed in 4% PFA, and immunostained for FIP200 and GST. Representative sections are depicted. For IF imaging, DAPI was used to stain nuclei. (D) The number, area, and intensity of TBK1 pS172-positive structures in at least 198 cells per cell line were quantified using ImageJ 1.53c and normalized to the values in FIP200 WT cells. The means + SDs of three independent experiments are shown. (E) Cleared cellular lysates of MEF ulk1/2 DKO, fip200 KO and atg13 KO MEFs transfected with an empty vector or the respective WT cDNA (encoding FIP200, ATG13, ULK1, or ULK2) were immunoblotted for the indicated proteins. The full-length blots are presented in Figure S6. The densities of the bands on immunoblots from three independent experiments were quantified and normalized to those of Vinculin. The normalized densities of all samples of each cell line were then normalized to that of the respective reconstituted cell line. Only the relative levels of TBK1 pS172 are shown. The boxes represent the highest and the lowest value, while the centerline shows the mean. Statistical analysis was performed using two-way ANOVA with uncorrected Fisher's LSD test. n.s. = not significant; *p < 0.05; ***p < 0.001.

Fig 5: ULK1 interacts directly with the PRMT5 complex independent of its role in autophagy. (A) Flp-In T-REx 293-GFP-ULK1 and Flp-In T-REx 293-GFP cells were stimulated with 0.1 μg/ml doxycycline for 18 h, followed by 1 h of starvation treatment with EBSS. After cytoplasm extraction (S100) GFP-IP was performed and analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against GFP, PRMT5, WD45, and pICln. (B) Flp-In T-REx 293-GFP-ULK1 and Flp-In T-REx 293-GFP cells were stimulated with 0.1 μg/ml doxycycline for 18 h, followed by treatment with full or starvation medium (EBSS) in the absence or presence of bafilomycin A1 (BafA1; 10 nM) for 1 h. Afterward, cells were harvested, lysed, and cleared cellular lysates were subjected to Tris/Glycine-SDS-PAGE and immunoblotting for GFP, ULK1 pS757, ATG14, ATG14 pS29, p62, LC3, GAPDH, and Actin. (C) Flp-In T-REx 293-GFP-ULK1, -GFP-ULK1/ΔCTD and -GFP cells were stimulated with 0.1 μg/ml doxycycline for 18 h. After cytoplasm extraction (S100) GFP-IP was performed and analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against FIP200, GFP, PRMT5, ATG13, WD45, pICln and ATG101. (D) HEK293T cells were stably transfected with pMSCVbsd-GFP-ULK1 kinase domain (k. dom.) and pMSCVbsd-GFP constructs. Additionally, HEK293T cells were transiently transfected with pcDNA5-FRT-TO-GFP-ULK1 GABARAP domain (GABARAP dom.) and -ULK1 C-terminal domain (CTD) constructs. After cytoplasm extraction (S100) GFP-IP was performed and analyzed by Tris/Glycine-SDS-PAGE and western blotting using antibodies against GFP, FIP200, ATG13, ATG101, PRMT5, WD45, and pICln. (E) S100 extract was generated of HEK293T cells and applied to a Superdex 200 increase column. Fractions were analyzed by Tris/Glycine-SDS-PAGE and immunoblotting using antibodies against ULK1, ATG13, ATG101, PRMT5, WD45 and pICln. (F) S100 extract of HEK293T cells was applied to a Superdex 200 increase column and subsequent immunoprecipitation of endogenous was performed with antibody against ULK1. Immunoprecipitation was analyzed by Tris/Glycine-SDS-PAGE using antibodies against ULK1 and pICln.