Fig 2: Linear ubiquitination promotes the formation of NEMO assemblies and IKK complex activation upon IL-1β receptor activation.(A) In contrast to WT NEMO, Q330X and K285/309R NEMO do not form foci upon IL-1β treatment. NEMO KO MEFs were reconstituted with HA-tagged WT NEMO, Q330X NEMO or K285/309R NEMO. 24 h later, the cells were stimulated with murine IL-1β (20 ng/ml) for 15 min or left untreated and then analyzed after saponin extraction by immunocytochemistry using an antibody against NEMO (scale bar, 10 µm). Right Panel: IKK complex activation was analyzed by immunoblotting using an antibody against phospho-IKKα/β. Antibodies against IKKβ and NEMO were used to control expression levels. GAPDH was immunoblotted as input control. (B) IL-1β–induced nuclear translocation of p65 is impaired by OTULIN. The cells were treated as described in (A) and analyzed by immunocytochemistry using antibodies against NEMO, OTULIN, and p65 (scale bar, 10 µm). Right panel: Data are displayed as mean ± SD and were analyzed by Mann–Whitney test, n = 4, *P ≤ 0.05. Expression levels of HA-tagged WT NEMO and OTULIN were analyzed by immunoblotting using antibodies against NEMO and OTULIN. GAPDH was immunoblotted as input control. (C) IL-1β–induced formation of NEMO assemblies and IKK complex activation are compromised in HOIP-deficient cells. Control SH-SY5Y cells and HOIP KO SH-SY5Y cells were stimulated with human IL-1β (20 ng/ml) for 10 min and then analyzed by immunocytochemistry after saponin extraction using an antibody against NEMO (scale bar, 10 µm). Right Panel: IKK complex activation was analyzed by immunoblotting using an antibody against phospho-IKKα/β. Antibodies against IKKβ, HOIP, and NEMO were used to control expression. GAPDH was immunoblotted as input control.Source data are available for this figure.

Fig 3: M1-ubiquitination at mitochondria protects from apoptosis ASTS-induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 µM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1-ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax-specific signal intensities were quantified and normalized to TIM23-specific signals (lower panel). Data represent the mean with standard error of eight independent experiments. *P < 0.05, ***P < 0.001. Kruskal–Wallis test followed by Dunn's multiple comparison test, n = 8.BSTS-induced cytochrome c release is decreased by TNF. HeLa cells were treated as described in (A). The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of five biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. *P < 0.05. Kolmogorow–Smirnov normality test, paired t-test, two-tailed, n = 5.C, DTNF treatment prevents damage of mitochondrial cristae under proapoptotic conditions. (C) SH-SY5Y cells were treated as described in (A), fixed and embedded and the mitochondrial ultrastructure was imaged by electron microscopy. Scale bar: 400 nm. (D) Cristae abundance (average cristae number per mitochondrial area) and cristae length (average cristae length per mitochondrium) were analyzed by Imaris 9.8. *P < 0.05, **P < 0.01, ***P < 0.01. Cristae abundance: Shapiro–Wilk normality test, One-way ANOVA followed by Tukey's multiple comparison test, n = 27. Cristae length: Kruskal–Wallis test followed by Dunn's multiple comparison test, n = 24–27. Bars represent mean ± SEM.EThe fast anti-apoptotic effect of TNF is not affected by the NF-?B inhibitor I?Ba. SH-SY5Y cells were transiently transfected with the NF-?B super-repressor I?Ba-2S or luciferase as a control. Twenty-four hours later, the cells were treated with STS (5 µM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase-3. Signal intensities were quantified by immunocytochemistry ancf I?Ba-2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. *P < 0.05, **P < 0.01. Student's t-test, two-tailed, n = 3.FThe protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. Forty-eight hours after transfection, cells were treated with STS (1 µM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls.GThe protective effect of TNF is dependent on PINK1 expression. HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in (F). The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies. GAPDH was immunoblotted as input control. Source data are available online for this figure.

Fig 4: PINK1 is stabilized at mitochondria by LUBAC-mediated ubiquitination A, BPINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) before harvesting or incubated with OTULIN-specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1-specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of four independent experiments. *P < 0.05. A two-tailed nonparametric Mann–Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.CTNF-induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt-mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt-mKeima -expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 µM each) for 1 h. The analysis was done by flow cytometry gating lysosomal and neutral mt-mKeima.DCatalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin.ERecombinant HOIP ubiquitinates PINK1 in vitro. V5-tagged-PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C-terminal HOIP, and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies.FEndogenous PINK1 is modified with M1-ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1-V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1-linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6–8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1-ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1. Source data are available online for this figure.

Fig 5: WT NEMO undergoes phase separation in an M1-ubiquitin-dependent manner.(A) In contrast to Q330X NEMO, WT NEMO undergoes LLPS upon linear ubiquitination by HOIP. The samples from the in vitro ubiquitination assay described in (C) were analyzed by bright-field microscopy. Representative images are shown (scale bar, 20 µm). (B) Liquid droplets formed by ubiquitinated NEMO are dynamic. WT NEMO ubiquitinated by HOIP as described in (C) were analyzed by bright-field microscopy to follow up fusion events. The images were taken every 5 s over a period of 10 s (scale bar, 20 µm). (C) WT but not Q330X NEMO is modified by M1-linked ubiquitin in vitro. Untagged WT NEMO and Q330X NEMO were subjected to an in vitro linear (M1-linked) ubiquitination assay by incubating the recombinant proteins (5 µM) with 50 µM mono-ubiquitin (ub), 1.5 µM ubiquitin-activating enzyme UBe1, 4 µM E2 ubiquitin-conjugating enzyme UbcH5c, 4 µM C-terminal HOIP comprising the RBR and LDD domains, ATP, and MgCl2 in 50 mM HEPES buffer (pH 7.4) containing 0.5 mM TCEP (tris(2-carboxyethyl)phosphine) for 2 h at RT. As a control, ATP was omitted (−ATP). The samples were analyzed by immunoblotting using antibodies against NEMO and M1-ubiquitin.Source data are available for this figure.