EMBO/soda-nel huggingface dataset:
Cyclin O (Ccno) functions during deuterosome‐mediated centriole amplification of multiciliated cells (2015)
Figure 5

Disturbed deuterosome formation and impaired centriole biogenesis in Ccno‐deficient MCCs

Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6.
IF staining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cell membrane in Ccno‐deficient MCCs as seen in z‐projection.
IF staining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.

Data information: Scale bars: 1 μm in all images. Error bars indicate the standard error of the mean (s.e.m.). P‐values were calculated by a two‐sided Student's t‐test.
Cyclin O (Ccno) functions during deuterosome‐mediated centriole amplification of multiciliated cells (2015)
Figure 6

Centriole maturation and localization to the apical cell membrane are defective in Ccno‐deficient MCCs

A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cell membrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software.
C, D Double IF staining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.

Data information: Scale bars: 1 μm in all images. Error bars indicate the standard error of the mean (s.e.m.). P‐values were calculated by a two‐sided Student's t‐test.
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 1

Figure 1. CHCHD10 resides in the same complex than Mitofilin, CHCHD3 and CHCHD6. A. Second dimension of the Blue-Native (BN) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin, CHCHD3 and CHCHD6 in isolated mitochondria from mouse brain. B. BN-PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P1, P2) fibroblasts. Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody (CI), 70kDa subunit antibody (CII), core I antibody (CIII) and cytochrome c oxidase subunit I antibody (CIV). MICOS complex was detected with an anti-Mitofilin antibody. C. Second dimension of the BN-PAGE showing that the steady-state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts. D. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. The dividing lines correspond to gels sections visible in the raw data file.
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 2

Figure 2. Interaction of CHCHD10 with components of MICOS complex. A. Co-immunonoprecipitation (IP) of endogenous mitofilin, CHCHD10 and CHCHD3 in control and patient fibroblasts (1mg of total extracts were used for each IP (left lower panel) and 200µg were used for the input (left upper panel)). The same results were found in P2 fibroblasts (not shown). Reverse co-IP experiment in HeLa cells with an antibody against CHCHD10 (right lower panel). 1mg of total extracts were used for each IP and 200µg were used for the input (right upper panel). The dividing lines correspond to gels sections visible in the raw data file. B. Duolink proximity ligation assay between Mitofilin and CHCHD10 (upper panels) and, CHCHD10 and CHCHD6 (lower panels) in control and patient fibroblasts observed by confocal microscopy. Mitochondria were stained with Mitotracker. Duolink spots per cell were quantified for 30 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student’s t-test (two-sided): extremely significant (***: p=0.0001). Scale bar = 10 µm.
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 4

Figure 4. Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles. A. Transfections were performed with empty vector (EV) or vectors encoding wild-type CHCHD10-FLAG (WT), CHCHD10-FLAG (S59L) or CHCHD10-FLAG (P34S) mutants. Mitochondria were stained with Mitotracker. Cells overexpressing wild-type and mutant CHCHD10 were labeled with FLAG antibodies. Nucleoids were visualized with an antiserum against DNA. Image analysis was performed by confocal microscopy. Scale bar: 10 µm. B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student’s t-test (two-sided): very significant (**: p=0.0280) or extremely significant (***: p=0.0001). D. Mitochondrial DNA content was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nDNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student’s t-test (two-sided).
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 5

Figure 5. Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress. A. Total extracts from control (C) fibroblasts and intact isolated mitochondria from control and patient fibroblasts (P1, P2) were analyzed by immunoblotting using antibodies against PCNA (nuclear protein), GAPDH (cytosolic protein) or SMAC (mitochondrial intermembrane space protein) (upper panel). Mitochondria from patient and control fibroblasts were incubated with NP-40 and separated into pellets (P) and supernatants (S). The fractions of each extraction were subjected to western blot analysis. VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins, respectively (middle panel). Ratio of mtDNA amplified from supernatant/mtDNA amplified from pellet, by qPCR, was quantified in control and patient fibroblasts (lower panel). B. Determination of the mtDNA copy number after H2O2 treatment. mtDNA/nDNA values in control, patient 1 and patient 2 fibroblasts. mtDNA : mitochondrial DNA, nDNA: nuclear DNA. C. mtDNA repair after H2O2 treatment. A long-range PCR was used to evaluate the oxidative damage, induced by H2O2 treatment, in mtDNA. The relative PCR amplification of a 15.6kb mtDNA fragment was normalized to mtDNA copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment. mtDNA repair activity in control, patient 1 and patient 2 fibroblasts. B-C. Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times. Untreated control cultures were incubated in serum-free medium alone. Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed. Values were normalized to untreated cells and differences were analyzed by Student’s t-test (two-sided): significant (*:0.05>p>0.01), very significant (**:0.01>p>0.001). Patient 1 versus control: *: p=0.015 (recovery 1h), *: p=0.022 (recovery 2h), **: p=0.003 (recovery 4h). Patient 2 versus control: *: p=0.0230 (recovery 2h), *: p=0.041 (recovery 4h). NT: cells not treated with H2O2.
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 6

Figure 6. No autophagy inhibition (A-C) and no mitophagy defect (D-E) in patient fibroblasts. A. Immunoblot of LC3B-I/-II expression in fibroblasts of patient 2 (1,2), patient 1 (3,4) and control individual (5,6) in absence (-) or presence (+) of chloroquine (CLQ). B. Quantitative analysis of LC3B-II/β-tubulin ratio from immunoblots (n=3). C. Quantitative analysis of LC3B-II + CLQ/ LC3B-II - CLQ ratio from immunoblots (n=3). D. Confocal images of fibroblasts derived from a control, patient 1 and patient 2. Boxed regions are enlarged. Scale bar: 20µm. E. Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol (FL lyso/FL mito ). Statistical analysis were performed on mean ± SEM using Student’s t-test (two-sided).
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 7

Figure 7. CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts. A. Control (C1, C2) and patient (P1, P2) fibroblasts were treated with 1 μM of staurosporine (STS) for 16 h. Cell death was determined by flow cytometry using Annexin V/DAPI staining. Three independent experiments were performed per condition with two points analyzed per experiment. Differences between the control and patient fibroblasts were analyzed by Student’s t-test (two-sided): significant (*: p=0.0116) or extremely significant (***: p=0.0001). B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) or by DEVDase activity measurement (C). Two (B) or three (C) independent experiments were performed per condition. Differences between the control and patient fibroblasts were analyzed by Student’s t-test (two-sided): extremely significant (***: p=0.0001). D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student’s t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).
CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis (2015)
Figure 8

Figure 8. Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death. Transfections were performed with empty vector (EV) or vectors encoding either wild-type CHCHD10-FLAG (WT) or mutant CHCHD10-FLAG (P34S and S59L). A. Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90. B-D. HeLa cells transiently expressing the WT or mutated forms of CHCHD10-FLAG (P34S or S59L) were treated with 1 μM Actinomycine D (ActD) for 4, 6 or 8 h with measurement of Annexin V/DAPI staining (B), DEVD-ase activity (C) from three independent experiments. Differences between the mutated and non mutated alleles were analyzed by Student’s t-test (two-sided): significant (*:0.05>p>0.01) or very significant (**:0.01>p>0.001). P34S versus WT: **: p=0.0055 (4h), *: p=0.0103 (8h). S59L versus WT: *: p=0.0304 (4h), *: p=0.0129 (8h). D. Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac. Hsp90 was used as a loading control.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf1

(a) p35 interacts with EndoB1 in a GST pulldown assay. (b,c) EndoB1 co-immunoprecipitates (IP) with p35 in COS-7 cell lysates expressing p35 and full-length EndoB1 (b) and adult rat brain lysate (c). (d) Putative Cdk5 phosphorylation site(s) in the EndoB1 sequence. Two proline-directed threonine residues (arrows) are present with a Cdk5 consensus site found at Thr 145 (underlined). (e) Dose-dependent phosphorylation of EndoB1 by Cdk5/p35 in an in vitro kinase assay. (f) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay. (g) Cdk5/p35 phosphorylates EndoB1 at Thr 145 in COS-7 cells. (h) Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5+/+, but not
Cdk5−/− mouse brains. Quantification of p-Thr145-EndoB1 level is shown in the right panel. Data are means ± s.e.m.;
n=3. Uncropped images of blots are shown in Supplementary Fig. S8.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf2

(a) Left: starvation increases the level of RFP–LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP–LC3 puncta. Right: pretreatment with 10 mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown. (b) Knockdown of EndoB1 abolishes the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP–LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=3. (c) Starvation enhances Cdk5 activity (top) and increases the level of Thr 145-phosphorylated EndoB1 in neurons (top and bottom). Histone H1 was used as a Cdk5 substrate. Data are means ± s.e.m.; n=3. (d,e) Pretreatment with Ros (d) or knockdown of Cdk5 (e) attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for d and n=8 for e. DMSO, dimethylsulphoxide. (f) Overexpression of EndoB1T145A significantly reduces the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP–LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=6. Uncropped images of blots are shown in Supplementary Fig. S8.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf3

(a) Left: lipid binding of EndoB1 is inhibited by mutating the five lysine/arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid (5E mutant). Right: purified GST protein was included as a negative control. S, supernatant fraction; P, pellet fraction that contains the lipid-bound proteins. (b,c) The 5E mutant of EndoB1 fails to dimerize (b) and interact with UVRAG (c) in 293T cells. (d) Overexpression of the 5E mutant of EndoB1 blocks starvation-induced autophagy in cortical neurons. Data are means ± s.e.m.; n=3. (e) Mutation of Thr 145 to alanine (T145A) or glutamic acid (T145E) has negligible effect on the lipid-binding property of EndoB1. Conversely, the 5E mutant is less able to bind lipid. The right panel shows quantified data as means ± s.e.m.; n=3. (f) Starvation increases co-localization of GFP–EndoB1 (green) with Atg5 (red) in neurons. The T145A mutation of GFP–EndoB1 does not affect its co-localization with Atg5-positive entities. Arrows denote GFP–EndoB1- and Atg5- positive vesicles. The right panel shows quantified data as means ± s.e.m.; n=9. Uncropped images of blots are shown in Supplementary Fig. S8.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf4

(a) Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells. Samples were run on the same gel and spliced together to create the image shown. (b) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells. (c) Diminished interaction between EndoB1 and UVRAG in Cdk5−/− mouse brain lysates. (d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) and Beclin 1 (e) in 293T cells. (f,g) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (f), or immunoprecipitation followed by immunoblotting (g). Uncropped images of blots are shown in Supplementary Fig. S8.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf5

(a) MPTP administration decreases the numbers of tyrosine hydroxylase (TH)- and cresyl violet (CV)-positive neurons in the substantia nigra. Representative images of tyrosine hydroxylase-stained brain sections are shown. Right panels show quantified data as means ± s.e.m.; n=9 for tyrosine hydroxylase and n=3 for cresyl violet. (b) MPTP administration upregulates the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the midbrain. Lower panels show quantified data as means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 inhibits MPP+-induced autophagy in cultured neurons. Data are means ± s.e.m.; n=5. (d) Cdk5 mediates the MPP+-induced EndoB1 phosphorylation at Thr 145 and the MPP+-induced increase in the LC3-II level in neurons. (e) Overexpression of EndoB1T145A attenuates MPP+-induced autophagy in neurons. Data are means ± s.e.m.; n=7. (f) MPTP fails to increase LC3-II and Thr 145-phosphorylated EndoB1 levels in the midbrain of p35−/− mice. The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated. Right panels show quantified data as means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf6

(a) Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice. Right panels show quantified data as means ± s.e.m.; n=3. (b) Overexpression of α-synucleinA53T mutant upregulates LC3-II level and Thr 145-phosphorylated EndoB1 in cultured neurons. Lower panel shows quantified data as means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 inhibits α-synucleinA53T mutant-induced autophagy induction in neurons. Data are means ± s.e.m.; n=7. (d) Cdk5 mediates α-synucleinA53T mutant-induced Thr 145 phosphorylation of EndoB1 and the increase of LC3-II in neurons. (e) Overexpression of EndoB1T145A attenuates α-synucleinA53T mutant-induced autophagy in neurons. Data are means ± s.e.m.; n=7. Uncropped images of blots are shown in Supplementary Fig. S8.
Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease (2011)
figf7

(a) Pretreatment with 3-MA, but not z-VAD-fmk, significantly reduces MPP+-induced neuronal death. Data are means ± s.e.m.; n=6. (b) Knockdown of Atg5 expression abrogates MPP+-induced neuronal loss, whereas it reduces neuronal survival in untreated cells. Data are means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 expression abolishes MPP+-induced neuronal death. Data are means ± s.e.m.; n=8. (d) Overexpression of EndoB1T145A inhibits MPP+-induced neuronal death. Data are means ± s.e.m.; n=5. (e) Pretreatment with 3-MA, but not z-VAD-fmk, reduces α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6. (f) Knockdown of Atg5 expression abolishes α-synucleinA53T mutant-induced neuronal death, whereas it reduces neuronal survival in vector-transfected cells. Data are means ± s.e.m.; n=3. (g) Knockdown of EndoB1 or Cdk5 expression abolishes α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=4. (h) Expression of EndoB1T145A inhibits α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.
Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from Cancers of Unknown Primary (2023)
Figure 4

Figure 4. G842C mutation leads to spontaneous activation of PlxnB2 signaling

(A) Representative images of immunofluorescence analysis (with anti-PlxnB2 antibodies) of the phenotype of COS7 cells transfected with wild-type or mutated PlxnB2, compared to the collapsed phenotype induced by the cognate ligand Sema4C (1 μg/ml) in cells expressing the WT receptor. Scale bar: 50 μm. A wide series of representative microscopic images is further shown in Appendix Figure S2. (B) The stacked bar graph shows the fraction of collapsed PlxnB2-expressing cells, identified as having a surface minor of 2000 μm2 (measured by ImageJ software), out of 50-100 cells counted per each condition, from different fields and n>3 experiments). The statistical analysis was performed comparing WT untreated cells with all other groups, by Fisher's Exact Test, which indicated a *****p value < 0.00001 for Sema4C-treated and for G842C-PlxnB2-expressing cells (confirmed by ANOVA analysis on individual cell values, shown in Appendix Fig. S2). By Fisher's test, the only other mutants significantly different from WT were R820H (p< 0.001) and R843Q (p<0.01), although ANOVA analysis on individual cell values shown in Appendix Fig. S2 didn't confirm such significance for other PlxnB2 variants.
Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from Cancers of Unknown Primary (2023)
Figure 5

Figure 5. Knocking-down G842C-mutated, but not wild-type PlxnB2, hampers CUP cell viability, clonogenic capacity, and tumorigenic growth in mice.

(A) The graphs show the analysis of cellular viability in shPlxnB2-tranduced agnospheres, compared to their respective scramble-transfected controls, after 24, 48, 72, and 96 hours of growth in culture. Values are mean ± SD of n≥3 independent experiments (6 technical replicates for each). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons (Graph Pad Prism 8.0.0): **p = 0.0053; ****p < 0.0001. (B) Limiting dilution assays (LDA, see details in Methods) were used to assess the clonogenic capacity of dissociated CUP cells derived from same shPlxnB2-tranduced agnospheres analyzed above, compared to their respective controls. Analyses generated by the ELDA software are shown below, reporting the estimated stem cell frequency (percentage of clonogenic cells) with confidence intervals (C.I.) and statistical analysis by chi-square test. (C) After transplantation in immunodeficient mice of CUP cells described above, the volume of ADX43- and (D) ADX901-shPlxnB2 tumors was assessed, in comparison with the respective controls. Plotted values indicate the mean ± SD (n = 8 each group). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons: **p = 0.0060; ****p < 0.0001.