EMBO/soda-nel huggingface dataset:
CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 1

Figure 1. Endogenous NICD levels increase in HEK293 cells following treatment with Roscovitine, DRB and XAV939.

HEK293 cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO served as vehicle control. Transfection with pCS2-NICD vector served as positive control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform. β-Actin served as loading control.

Quantification of the density of western blot bands in (A) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01 and ns=not significant.

iPS cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO has been used as vehicle control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following Inhibitor treatment and the top band disappears following λ phosphatase treatment indicative that this top band is a phosphorylated isoform. β-Actin served as loading control.

Quantification of the density of western blot bands in (C) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01, ***p≤0.001 and ns= not significant.

HEK293 cells were treated with the same inhibitors as described in (A). NICD phosphorylation status was analysed by a Phos-tag assay. DMSO served as vehicle control. NICD phosphorylation profile varies following Roscovitine, DRB, XAV939 or MLN4924 treatment. Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms.
CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 2

Figure 2. NICD and FBXW7 interact directly at endogenous levels.

NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.

Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.

Roscovitine treatment reduced the NICD-FBXW7 interaction. 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.

Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.
CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 3

Figure 3. Mass spectrometry analysis of phosphorylated residues in hNICD.

LC-MS-MS analysis of in-gel-digested HEK293 cells transfected with hNICD-GFP, and subjected to immunoprecipitation using GFP antibody, identified multiple phosphorylation sites in NICD, highlighted in green.

Phosphorylation of serine 2513, but not serine 2516, appears to be essential for the NICD-FBXW7 interaction. hNICD-GFP phospho-mutant peptides encoding non-phosphorylatable residues at S2513 and/or 2516 (serine to alanine) were expressed in HEK293 cells. The exogenously expressed protein was subsequently immunoprecipitated with anti-GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody. Wild-type hNICD-GFP and GFP only vectors were included as positive and negative controls, respectively. Western blot using GFP antibody served as immunoprecipitation efficiency control. β-Actin has been used as loading control for the input lanes.
CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 4

Figure 4. Depletion or pharmacological inhibition of CDK1 or CDK2 increased endogenous levels of NICD in HEK293 cells.

HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK2 (+) followed by western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β-Actin served as loading control.

Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01.

HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK1 (+) followed by western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β-Actin has been used as loading control.

Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.

Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor Database (http://www.kinase-screen.mrc.ac.uk/kinase-inhibitors).

HEK293 cells were treated with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.

Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed, with *p≤0.05, **p≤0.01, and ns= not significant.

HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.

Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.
CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 5

Figure 5. NICD levels fluctuate during the cell cycle.

Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown.

Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments.

Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01 (ns=not significant).

Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05 and **p≤0.01.
CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 6

Figure 6. NICD levels peak in G1 and G2 phases of the cell cycle for iPS cells.

iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm.

G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells (p≤0.001). All groups were compared against each other with a one-way ANOVA analysis, followed by Turkey test. M phase cells have the lowest NICD. Each point in the box-plot represents the normalized intensity of a single cell. Data from three different experiments (n=3) was randomised collated and plotted.
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus (2022)
Figure 1

Figure 1. TNF induces M1-linked ubiquitination at mitochondria

A, B. A fraction of OTULIN is localized at mitochondria where it suppresses M1-linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1-linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.

C. TNF induces M1-ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in A.

D. TNF-induced mitochondrial M1-ubiquitination does not occur in HOIP-deficient cells. Wildtype (WT) and HOIP-KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.

E. Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red) and analyzed by SR-SIM. Scale bar, 5 µm.

F. TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.

G. TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies.
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus (2022)
Figure 2

Figure 2. PINK1 is stabilized at mitochondria by LUBAC-mediated ubiquitination

A, B. PINK1 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 4 independent experiments. *p < 0.05. A two-tailed non-parametric Mann-Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.

C. TNF-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 of lysosomal and neutral mt-mKeima.

D. Catalytically 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.

E. Recombinant 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.

F. Endogenous 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.
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus (2022)
Figure 3

Figure 3. PINK1 stabilizes M1-linked ubiquitin by phosphorylation and counteracts OTULIN activity

A. PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L, were transfected with PINK1, WT OTULIN or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.

B. Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. 48 h after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).

C. The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.

D. TNF-induced M1 ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in A, followed by immunoblotting using M1-ubiquitin-specifc antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of 3 independent experiments; n.s. not significant, ***p < 0.001. A two-way ANOVA test was used to analyze statistical significance.
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus (2022)
Figure 5

Figure 5. Mitochondria serve as a signaling platform for TNF-induced NF-ĸB activation

A. NF-κB signaling components are recruited to mitochondria upon TNF treatment. A. HEK293T cells were treated with TNF (25 ng/ml, 15 min), harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated; p-p65: phospho-S536-p65; p-TBK1: phospho-S172-TBK1.

B. Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH-SY5Y cells using the proximity ligation assay (PLA) between phospho-S536-p65 (rabbit) and TOM20 (mouse) couple antibodies. One set of cells were treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTrackerTM Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p-p65 antibodies prior to the PLA assay. Scale bar, 10 µm.

C. Quantification of the phospho-S536-p65/TOM20 PLA foci per cells. Data represent the number of PLA foci per cell (mean ±SD); at least 84 cells in total were analyzed per condition. ****p < 0.0001. A two-tailed non-parametric Mann-Whitney test was used to analyze statistical significance.
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus (2022)
Figure 6

Figure 6. PINK1 promotes NF-κB activation and modulates NEMO ubiquitination.

A. Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH-SY5Y cells were transfected with control or PINK1-specific siRNAs. For rescue experiments, cells were co-transfected with wildtype PINK1, PINK1Δ77, or kinase-dead PINK1-K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of 3 independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition. ***p < 0.001, Student's t-test, two-tailed, n=3.

B. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.

C. Linear ubiquitination of NEMO is reduced in PINK1-deficient cells. HEK293T cells were co-transfected with HA-NEMO and control or PINK1-specific siRNAs. 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1-ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real-time RT-PCR. Bars represent mean± SD with three technical replicates.
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus (2022)
Figure 7

Figure 7. Mitochondria facilitate the transfer of p65 to the nucleus

A. TNF induces the movement of peripheral mitochondria towards the nucleus. Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF (25 ng/ml). The motility of mitochondria was analyzed using Imaris 9.8 spot function. The graphical representation of the top 25% peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment. Exemplified dataset shows the median distance with the upper and lower quartile (shaded area) of n=105-187 mitochondrial spots per time point.

B. Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1/2 expression. HeLa cells were transfected with control or Miro1 and Miro2 siRNAs. 48 h later, cells were treated with TNF (25 ng/ml, 15 min), fixed and stained using antibodies against p65 and tubulin and DAPI. Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4.2.1. Quantification is based on 3 biological replicates. Kruskal-Wallis test followed by Dunn's multiple comparison test, n=79-157, *p < 0.05.

C. Cartoon of newly developed SPLICS reporter to detect mitochondria-nucleus contact sites. Fluorescence reconstitution between the ONM-targeted β11 and the OMM-targeted GFP1-10 fragment occurs at the contact site.

D. Scheme of the plasmids encoding the SPLICSNU-MT reporter.

E, F, G. TNF increases mitochondria-nucleus contact sites. Representative immunofluorescence images (E, F) and quantification (G) of mitochondria-nucleus contact sites in HeLa cells transfected with the SPLICSNU-MT and treated with 25 ng/ml TNF or PBS for 15 min. TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation. Nuclei were stained with Hoechst33342 (ThermoFisher, 1 μg/ml). Unpaired two-tailed t-test, n=43 (control) or 51 (TNF), Bars represent mean ± SEM. ****p < 0.0001. Scale bar: 10 µm.

H, I, J. TNF increases mitochondria-nucleus contact sites in a HOIP-dependent manner. Representative immunofluorescence images (H, I) and quantification (J) of mitochondria-nucleus contact sites in control or HOIP-silenced (HOIP siRNA) HeLa cells transfected with the SPLICSNU-MT probe treated with 25 ng/ml TNF or PBS for 15 minutes. Mitochondria and nuclei were stained as described in E and F. Two-way ANOVA followed by Tukey's multiple comparison test, n=34-44 from 3 independent experiments, Bar represents mean ± SEM. ****p < 0.0001. Scale bar: 10 µm.