EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Ketone body concentrations were measured in serum of adult C57Bl/6J mice that were fed a ketogenic diet for the indicated time periods. Data are presented as mean ± SD. n≥4; ***, p<0.001; unpaired Student's t-test. (B) RNA sequencing was performed on isolated cardiac endothelial cells of male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks. Volcano plot showing differentially expressed genes as genes with a log2 fold change ≤ -0.5 or ≥ 0.5 (vertical dashed lines) and p≤0.01 (horizontal dashed line). Data are presented as mean ± SD. n≥4; ***, p<0.001; unpaired Student's t-test. (C, D) Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks. (E) Gene set enrichment analysis of hallmark (GSEA gene set h, blue) and gene ontology (GO)-derived (GSEA gene set c5, green) gene sets enriched in cardiac endothelial cells of C57Bl/6J mice kept on a ketogenic diet compared to littermate animals kept on control diet. (F, G) Enrichment plots of the hallmark gene sets fatty acid metabolism and G2M checkpoint comparing expression pattern of cardiac endothelial cells isolated from C57Bl/6J mice kept on a ketogenic diet compared to littermate animals kept on control diet.
Figure 1(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype upon inhibition of protein synthesis with cycloheximide (chx). A plasmid-borne 3HA-Pgc1 expressed from the endogenous promoter was used. 3HA-Pgc1 was detected with anti-HA antibodies. Kar2 was used as a loading control and detected with anti-Kar2 antibodies. The graph on the right shows the average of four independent experiments; error bars represent the standard deviation. (B) The ubiquitination of 3HA-Pgc1 was analyzed in cells with the indicated genotype and expressing myc-tagged ubiquitin. 3HA-Pgc1 was immunoprecipitated using anti-HA and polyubiquitin-conjugated 3HA-Pgc1 was detected using anti-myc antibody. Input fraction corresponds to 2% of the total amount used for IP. (C) The degradation of 3HA-Pgc1 was analyzed as in (A) in cells bearing the CDC48 temperature-sensitive allele cdc48-6. Cells were grown 2,5 hours at 37C prior addition of cycloheximide. Inactivation of Cdc48 mutant protein was confirmed by stabilization of the ERAD substrate Erg1 in the same cells. (D) Membrane association of ubiquitinated 3HA-Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation. Cells of the indicated genotype and expressing myc-tagged ubiquitin were grown as in (C). Lysates were subjected to centrifugation on an optiprep density gradient. Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti-HA antibodies. Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively. Input fraction corresponds to 10% of the total amount used for IP. "Top", "mid", "bottom" indicate the fractions from the gradient. Asterisk marks a faster migrating unspecific band in the input bottom fraction.
Figure 1Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (A) CTGF interacts with LRP4 but not with MuSK. Full-length CTGF (CTGF-full-myc) was immunoprecipitated with the ectodomain of LRP4 (LRP4ect-Flag, lane 6) but not with the ectodomain of MuSK (MuSKect-Flag, lane 3). However, when CTGF-full-myc, LRP4ect, and MuSKect-Flag were expressed together, CTGF-full-myc was immunoprecipitated with MuSKect-Flag (lane 5). The CT domain of CTGF (CTGF-CT-myc) was sufficient to immunoprecipitate LRP4ect-Flag (lane 7). Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks. Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (B) Deletion of the CT domain of human CTGF (CTGF-ΔCT_ΔTSP1 and CTGF-ΔCT) impairs the interaction between CTGF and LRP4ect-Flag. Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks. Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (C) Anti-CTGF antibody immunoprecipitated human LRP4 deleted with the 1st, 2nd, and 4th β-propeller domains (Δ1, Δ2, and Δ4) but not with the 3rd β-propeller domain (∆3). Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (D) LRP4ect-Flag, but not MuSKect-Flag, bound to overlaid CTGF-full-mycAP and CTGF-CT-mycAP. In contrast, LRP4ect-Flag did not bind to CTGF-ΔCT-mycAP. Rspo2-mycAP were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (D) p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group. Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (E, upper panel) LRP4ect-Flag bound to overlaid MuSKect-mycAP in a dose-dependent manner of CTGF-full-myc, but not of CTGF-ΔCT-myc. (E, lower panel) LRP4ect-Flag bound to overlaid agrin-mycAP independent of CTGF-full-myc. Rspo2-mycAP and Rspo2-myc were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (E). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.
The role of SCOC and FEZ1 during amino‐acid starvation‐induced autophagy.Figure 9The role of SCOC and FEZ1 during amino‐acid starvation‐induced autophagy. We show that SCOC bound to FEZ1 is in a complex with ULK1 or UVRAG. Our results suggest that the inhibition of autophagy induction by FEZ1, possibly achieved through inhibition of ULK1 kinase activity or membrane targeting, is relieved by association of SCOC with the ULK1-FEZ1 complex. Also, amino‐acid starvation causes the dissociation of UVRAG from the FEZ1-SCOC complex and this may allow UVRAG to associate with the Vps34 complex and promote autophagy, most likely at later stages compared with the Vps34 complex containing Atg14 (not shown here). We hypothesise that SCOC or the SCOC-FEZ1 complex may coordinate the regulation of both the ULK1 and the Vps34 kinase complexes.
Pannexin-1 restrains the Production of Cytokines associated with Necroptosis.Figure 4A HT-29 cells were transfected with a siRNA for PANX1 (#3), or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T) for 6 hours. Shown is a normalized densitometric analysis for the presence of 120 cytokines with an antibody array. The color scale (0-10) represents the means of normalized densitometry values (n=2 technical replicates). B, C HT-29 transfected as indicated (B) or PANX1 knockout HT-29 cells (C) were treated with TQS for 6 hours. The mRNA levels of Cxcl8 relative to untreated NS or NC samples were measured by qPCR (means ± SEM, n=3 biological replicates, ****P<0.0001, ANOVA). D Lysates from cells treated as indicated were prepared and subjected to Western blotting analysis as indicated. Molecular weight markers (Mr) are shown. Data information Cleaved MLKL is indicated with an arrowhead. E Abundance of IL-8, measured by ELISA, in the supernatants of cells treated as indicated after 6 hours of stimulation (means ± SEM, n=4 biological replicates, ***P<0.001, ANOVA). HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (F). Data are means ± SEM of three (F) independent experiments (***P<0.001, ****P<0.0001, ANOVA). HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (G). Data information: Cleaved MLKL is indicated with an arrowhead. HT-29 cells were treated as indicated. The release of IL-8 in cell culture media was analyzed by ELISA (H). Data are means ± SEM of four (H) independent experiments (***P<0.001, ****P<0.0001, ANOVA). HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (I). Data are means ± SEM of three (I) independent experiments (**P<0.01, ****P<0.0001, ANOVA). HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (J). Data information: Cleaved MLKL is indicated with an arrowhead. HT-29 cells were treated as indicated. IL-8 release was analyzed in cell culture media by ELISA (K). Data are means ± SEM of four (K) independent experiments (**P<0.01, ****P<0.0001, ANOVA).
EDNA detects intracellular neutralization activity in SARS-CoV-2 convalescent sera.Figure 3(A) Capillary-based protein detection (Jess) of antibodies against SARS-CoV-2 antigens in convalescent sera. (B) Quantification of antigen-specific antibodies in convalescent sera. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. (C) 293T cells OE ACE2 infected with SARS-CoV-2 in the presence of electroporated convalescent sera. There are statistically significant differences in the ability of serum from different individuals to inhibit viral replication intracellularly (** = p< 0.005, * = p< 0.05). (D) 293T ACE2 OE/TRIM21 KOs reconstituted with either empty vector (EV) or TRIM21-expressing vector (TRIM21) infected with SARS-CoV-2 in the presence of electroporated convalescent sera. Intracellular neutralization of viral replication is only observed in cells reconstituted with TRIM21 (** = p< 0.005). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way (C) or two-way (D) ANOVA.
Loss of mtm‐3 causes increased autophagic association of ATG‐18 A–H Fluorescent images of embryos at the 4‐fold stage in the indicated strains expressing ATG‐18::GFP (A–D), ATG‐18(FKKG)::GFP (E, F), or ATG‐18(FTTG)::GFP (G, H).Figure 5A-H Fluorescent images of embryos at the 4‐fold stage in the indicated strains expressing ATG‐18::GFP (A-D), ATG‐18(FKKG)::GFP (E, F), or ATG‐18(FTTG)::GFP (G, H). J Number of wild‐type or mutated ATG‐18::GFP puncta in each strain. At least 15 embryos were scored per strain (mean ± SD). **P 0.0001; N.S: not statistically different. I Confocal fluorescent images of 4‐fold mtm‐3 embryos expressing ATG‐18::GFP (I′) and stained by DAPI (I) and anti‐LGG‐1 (I″) antibody. Insets show magnified views. Arrows indicate ATG‐18 puncta labeled by anti‐LGG‐1 antibody. Scale bars: 5 μm.
, Cell type-specific basal protein amounts in relation to pathway desensitization in cell lines and primary human hepatocytes The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were simulated by the mathematical model for untreated Huh7.Figure 6The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were simulated by the mathematical model for untreated Huh7.5 and HepG2-hNTCP cells as well as for cells stimulated with 1.2 pM and 608 pM Roferon for 24 hours. The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients. Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting. Detection was performed with antibodies specific to STAT1, STAT2, IRF9 or USP18 using chemiluminescense on a CCD camera-based device (ImageQuant). Average of at least N=3 are displayed with standard deviations. Data was used for calibration of a primary human hepatocyte-specific mathematical model and estimated values are shown with green squares. Experimental data and model fits of IFNα- and Roferon-induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor-depleted primary human hepatocytes prestimulated with 0, 2.8 or 1400 pM IFNα (patients 1-3) or 0, 1.2 or 608 pM Roferon (patients 4-6). Primary human hepatocytes from the same patients 1-6 as in (B) were used. Experimental data is represented by filled circles (N=1 per patient). Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα. Lines indicate model fits. Absolute and relative antiviral response is calculated by the mathematical model for each cell line and patient-derived primary human hepatocytes prestimulated with 0, 1.2 or 608 pM Roferon by simulating the amount of occupied GAS and ISRE binding sites upon stimulation with 608 pM Roferon. For the absolute antiviral response, the baseline-subtracted area under curve (ΔAUC) from 24 to 28 hours is shown. For the relative antiviral response, the ΔAUC from 24 to 28 hours is divided by the corresponding ΔAUC in cells without pre-treatment.
SPPL2c impacts on the integrity of subcellular compartments in HEK293 cells.Figure 5After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.
CRMP4 binds dynein via a specific 50 amino acid motif (A) Crystal structure (PDB code 4CNT) of a CRMP4 monomer (upper panel) and biological tetramer assembly (lower panel).Figure 4(A) Crystal structure (PDB code 4CNT) of a CRMP4 monomer (upper panel) and biological tetramer assembly (lower panel). The peptides that were selected to inhibit binding are highlighted and color coded as indicated. (B) Upper panel represent the binding site domain of dynein in CRMP4 and its deletion. These constructs were used in the IP that was performed in the middle panel. Middle panel - Immunoprecipitation of DIC followed by western blot analysis of CRMP4 in COS7 cells overexpressing either GFP-CRMP4, or GFP-CRMP4 with deletion of amino acid 100-150, or GFP-CRMP4 overexpressing cells that were pre-incubated with a 10 µm mixture of peptides 1-4 (size of ~91 KDa). Lower panel - Western blot analysis of total protein levels before the pull-down assay (DIC size: ~75 KDa). (C) Quantification of the Western blot in B from 3 independent repeats. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each technical repeat. One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.007, *p=0.0261. (D) Upper panel - immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - total protein input (size of ~150 KDa). (E) Quantification of the blot in D from 3 independent repeats. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, **p=0.01. (F) Upper panel - Immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - Total input (size of ~150 KDa). (G) Quantification of the blot in F. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0299. (H) Immunoprecipitation of DIC (size of ~75 KDa) followed by Western blot analysis of CRMP4 (size of ~64 KDa) in COS7 cells that were transfected with CRMP4 and AAV9-50aa or its control. IgG antibody was used as a control. (I) Quantification of the blot in H from 3 independent repeats. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0479.
Figure 2(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling. (B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm. (C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of "Nucleation & caging" with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlappin
Figure 4(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc, Log Rank, p < 0.001, n ≥ 16). (b) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc. Mann Whitney, n ≥ 10). (c) Kaplan-Meier survival plot of cohorts of induced AhCre-ERT Apc Pten and AhCre-ERT Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC led to a significant reduction in survival (Log Rank, p < 0.001, n = 14 vs 21). (d) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice (Mann Whitney, n = 9 vs 19). (e) cBioportal OncoPrint showing mutual exclusivity of HUWE1 mutations and increased MYC copy number and / or RNA expression in CRC samples (Log Odds Ratio: -1.144 [some tendency toward mutual exclusivity] , p = 0.042).
PERK knockdown normalizes mitochondrial Ca2+ overload and mitochondrial respiration.Figure 9(A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm. (B-E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group. (F, G) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK‐myc stained with MitoTracker Green. Scale bars: 10 μm. Quantitative analysis of mitochondrial morphology is shown in G. Data are mean±s.e.m. (n=3). *P0.05 versus the empty plasmid group.
Role of the ROD N-terminal region in filamentation (A) A predicted N-terminal extension (green) of the ROD β-propeller (red), slightly rotated from the view in B.Figure 5(A) A predicted N-terminal extension (green) of the ROD β-propeller (red), slightly rotated from the view in B. The propeller proper begins with strand β7d and ends with strand β7c, which leads into the helical domain. The N-terminal extension augments the sixth and seventh blades with external β-strands (β6e and β7e). The position of T13 and S15 on the extension is shown. (B) The AF2 model confidence score (pLDDT, displayed blue to red through green from highly to poorly reliable) highlights regions of the model predicted with high or poor confidence, respectively (C) MPS1-induced filamentation experiments demonstrate that the phosphomimetic T13E/S15E ROD mutant bypasses the filamentation blockade induced by the MPS1 inhibitor reversine. The T13A/S15A mutant prevents MPS1-induced filamentation altogether. Data information: Scale bars in panels C = 5 µm. Two technical replicates for panels C were performed. (D) N-terminal deletion mutant of ROD (∆15 and ∆26) removing the first 15 or 26 N-terminal residues that include the MPS1 phosphorylation sites Thr13 and Ser15 are unable to form filament in presence of MPS1 at 20ºC, but can form filaments upon mildly heating to 30ºC. Data information: Scale bars in panels , D = 5 µm. Three technical replicates for panel D were performed. (E) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS-PAGE separation of reactions. Data information: Two technical replicates for panels , E (F) Filamentation assays with the indicated combinations of 8 µM mChRZZSF, MPS1 (1 µM), and lambda phosphatase (0.4 mg/ml) in presence of 10 µM reversine. Dephosphorylation reactions were carried out on already formed filaments (see Methods). Samples were imaged by confocal microscopy. Dephosphorylation does not dissolve already formed filaments. Data information: Scale bars in panels , F, 5 µm. Two technical replicates for panels , F, were performed. (G) RZZ and RZZS complexes were reconstituted with the ZwilchE422A/D426A mutant and tested for filamentation at 20ºC in presence of MPS1 or upon mildly heating to 30ºC. Data information: Scale bars in panels G = 5 µm. Two technical replicates for panels G were performed. (H) Surface representation of the RZZ model depicting the position of ZwilchE422 and ZwilchD422 (in purple) and the positions of the ROD N-terminal region (green) and the highly conserved ZW10 N-terminus.