EMBO/sd-character-level-ner huggingface dataset:
a) Confocal micrographs of HeLa cells 2 h after infection with S.figf3(a) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to Cocoa, NDP52 and ubiquitin (Ub). (b) Colocalization, at the indicated time points after infection, of ubiquitin-positive S. Typhimurium with NDP52 or Cocoa. (c) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to NDP52 and LAMP1. (d) Colocalization, at the indicated time points after infection, of NDP52-positive S. Typhimurium with LAMP1. DAPI, 4,6-diamidino-2-phenylindole. All data are representative of at least two independent experiments. Error bars, s.d.; three coverslips per experiment, 50 marker-positive cells evaluated per coverslip. Scale bars, 10 μm; arrowheads, bacteria shown in insets.
Figure 6In this model, curcumin inhibits adipogenesis by reducing the expression of ALKHB5, an m6A demethylase, which leads to higher m6A-modified TRAF4 mRNA. TRAF4 mRNA with higher m6A level is recognized and bound by YTHDF1, leading to enhanced translation of TRAF4. TRAF4, acting as an E3 Ring ubiquitin ligase, promotes degradation of adipocyte differentiation regulator PPARγ by a ubiquitin proteasome pathway thereby inhibiting adipogenesis.
E2HTH substitutions differentially impact substrate ubiquitination by the E3 APC/C.Figure 6A, B Schematic of UBE2S~Ub (A) and UBE2C~Ub interactions (B) with the APC/C. UBE2S~Ub uses its C-terminal extension to bind to a groove formed by APC2 and APC4. The UBE2SHTH interacts with APC2 and the APC11 RING domain positions the substrate-linked Ub for chain elongation. UBE2C~Ub is positioned by the APC11RING and APC2 winged-helix B (WHB) domains during substrate ubiquitination (Brown et al., 2015; Brown et al., 2016). C Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS-PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch's t-test (**** p ≤ 0.0001, n=5 independent, technical replicates). Error bars: SEM. D Similar to C, Ub-securin ubiquitination by APC/C and either UBE2D2 WT or charge-swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one-way ANOVA (**** p ≤ 0.0001, n=4 independent, technical replicates). Error bars: SEM. E Representative fluorescent scan of an SDS-PAGE gel of APC/C-dependent polyubiquitination of Ub-CyclinB* by a panel of UBE2SHTH charge-swap variants. Quantification in Fig EV5C. F The recombinant UBE2S ER/ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild-type (WT), monitored by immunoblot. G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA (siFF) or two UBE2S siRNAs (siUBE2S).
Figure 3(a) Correlation of uPA or PAI-1 protein expression and neutrophil infiltration in tumors as assessed by ELISA as well as immunohistochemistry and light microscopy in human breast cancer samples (histological grade: G1), representative images (scale bar: 100 μm) and quantitative data are shown. (b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort. (c) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test). Proliferation of (d) 4T1 breast cancer cells upon exposure to recombinant murine uPA-PAI-1, the uPA-PAI-1 inhibitor WX-340, or primary neutrophils isolated from the peritoneal cavity of WT mice undergoing 6 h of intra-peritoneal stimulation with uPA-PAI-1 with or without addition of a NE inhibitor as assessed by a MTT assay, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. neutrophils; *p<0.05 vs. neutrophils + vehicle / #p<0.05 vs. 2 % FCS medium; One-way ANOVA / t-test). (e) bEnd.3 microvascular endothelial cells upon exposure to recombinant murine uPA-PAI-1, the uPA-PAI-1 inhibitor WX-340, or primary neutrophils isolated from the peritoneal cavity of WT mice undergoing 6 h of intra-peritoneal stimulation with uPA-PAI-1 with or without addition of a NE inhibitor as assessed by a MTT assay, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. neutrophils; *p<0.05 vs. neutrophils + vehicle / #p<0.05 vs. 2 % FCS medium; One-way ANOVA / t-test). (f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).
Figure 6All genes passing hard-thresholding in each experiment are shown. K-means clustering was used to cluster responsive genes (K = 4 for Fmp48 and K = 2 for Stp4 and Hmx1) and GO slim gene-sets enriched in each cluster are shown.
Figure 2Evaluation of expression by RNA-Seq and number of peptides required to detect an annotated protein by MS in M. pneumoniae. The plot represents the relationship between expression levels (average expression from RNA-Seq data) and number of possible unique tryptic peptides (UTPs) for two sets of studied proteins: detected (blue dots) and not detected (orange dots) by MS. B) Evaluation of thresholds and artefactual signals in MS data. The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP, 1 UTP and 1NUTP, or ≥2 UTPs for three categories. Color code: annotated (blue bars), putative new (orange bars), and decoy set (gray bars). C) Number of SEPs detected by increasing the number of experiments. Color code is the same as in panels B. Each line represents the accumulated number of different SEPs detected (Y-axis) when combining 1 to 116 MS datasets (X-axis) from M. pneumoniae. Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets (e.g., x=80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples).
PIPKIγi5 recruits SNX5 to inhibit LAPTM4B association with Hrs A–C Co‐immunoprecipitation (co‐IP) of wild‐type (WT) or C‐terminal deletion mutants of HA‐PIPKIγi5 with SNX5 (A) or LAPTM4B (B and C) in HEK293 cells cotransfected with indicated proteins.Figure 7A-C Co‐immunoprecipitation (co‐IP) of wild‐type (WT) or C‐terminal deletion mutants of HA‐PIPKIγi5 with SNX5 (A) or LAPTM4B (B and C) in HEK293 cells cotransfected with indicated proteins. Δ675 indicates a deletion from amino acid 675 to the C‐terminus. D PIPKIγi5 promotes SNX5 association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐SNX5 and Flag‐LAPTM4B. E SNX5 inhibits Hrs association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐Hrs and Flag‐LAPTM4B. F A model for the PIPKIγi5-LAPTM4B pathway in EGFR trafficking. Hrs is an established key regulator of EGFR intraluminal sorting. LAPTM4B, by promoting Nedd4‐mediated Hrs ubiquitination, inhibits the recognition of ubiquitinatedEGFR by Hrs and therefore inhibits EGFR intraluminal sorting and lysosomal degradation. PIPKIγi5 directly binds to LAPTM4B and antagonizes the function of LAPTM4B in EGFR sorting by generating PtdIns(4,5) P2 signals and recruiting SNX5.
Paneth cell dysfunction in OGT-deficient mice.Figure 6 A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048)   B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm   C Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031)   D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice   E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells   F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm   G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462)   H Representative images of cultured organoid on Day 3   I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5)   J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049  J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049  J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049
Fig.Figure 5A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and a-tubulin as loading control were detected by immunoblotting. B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1. C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05 D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between "Ubc9 wt"and "Ubc9 DA" cells. The average size of "Ubc9 wt" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.
Figure 5Art2-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, GidSR4, GidAnt, or Gid10 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot. Mdh2-3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10, GidSR4, GidAnt, or Gid4 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot. Art2-3xFLAG or Art2P2S-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, or GidSR10∆C for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot. In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C-terminus (pep*).
MPC1 inhibition stimulates mitochondrial respiration coupled to ATP synthesis in brown adipocytes.Figure 2(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment. Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (C) Representative Western Blot analysis of MPC1, MPC2 and actin. (D) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in (C) from n=3 individual experiments. Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (E) mRNA levels of MPC1 and MPC2 from n=3 individual experiments. Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment.
Neocortex expansion and increased cortical neuron production in embryonic 11B mouse neocortex at E18.Figure 2A Representative DAPI staining (white) of E18.5 wildtype (WT) and 11B mouse neocortex illustrating the measurements of cortical perimeter (blue), cortical thickness (red) and ventricular length (green). Data information: Images are single optical sections Scale bar, 1 mm B-G Quantification across the rostral-caudal axis (sections 1-9, B-D) and only the rostral part of the brain (sections 1-3, E-G) of cortical perimeter (B, E), cortical thickness (C, F) and ventricular length (D, G) of E18.5 WT (white) and 11B (black) mouse neocortex. Data information: Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test ; two-tailed unpaired Student's t-test , * P < 0.05, ** P < 0.01, **** P < 0.0001. WT vs. 11B, P < 0.0001; section number 3, P = 0.0322; section number 8, P = 0.0411 WT vs. 11B, P < 0.0001; section number 1, 2, P = 0.0233; section number 3, P < 0.0001 (C). P = 0.0476 (E); P < 0.0001 (F) H Representative immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines). I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines). J Data information: Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0287 (I, IZ); P = 0.0039 J-L Quantification of Tbr1+ (J), Ctip2+ (K) and Satb2+ (L) neurons in the CP in a 100 µm-wide field of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines). Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0085 (J); P = 0.0093 (K); P = 0.0157 (L).
FLIPR assay results for interactors positively affecting CFTR Cl- channel activity.Figure 3Assays were performed in HEK293 cells for A) F508del-CFTR For the F508del-CFTR measurements since this mutant does not respond well to Fsk alone (Fsk + VX-770 potentiator is required to obtain a detectable signal) we compared the CFTR inh172 (inhibitor) response (F1/'F-CFTRinh172'; where F1 represents peak RFU immediately before inhibitor addition and 'F-CFTRinh172' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle). For each of F508del-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d. Assays were performed in HEK293 cells for B) wt-CFTR. For the wildtype measurements, we compared the maximum Fsk response (Maximum F /F0; where F0 represents baseline RFU right before the stimulation). For each of wt-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.
Figure 5(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of DB12s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the Guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of DB12s of ZIKV and WNV are well folded, but that of DB12 of DENV2 is partially folded. The Kratky plots for the two PK3 mutants of DB12s, the ZIKV- DB12-PK3∆ (green) and WNV- DB12-PK3∆ (black), respectively, are also included for comparison, suggesting reduced compactness upon PK3 mutations. (D) The ab initio shape envelopes of the DB12s of DENV2 (left), ZIKV (middle) and WNV (right) in two views. The atomic models from de novo structure modeling by Rosetta were superimposed onto the respective envelopes. The back-calculated scattering profiles of the de novo atomic models are fitted to the respective experimental scattering profiles in (A). (E) The ab initio shape envelopes of the PK3 mutants of ZIKV DB12s, the ZIKV-DB12-PK3∆ (left) and WNV-DB12-PK3∆ (right) in two views, suggesting open conformations upon PK3 mutations.
Proposed mechanism by which MPC inhibition induces lipid cycling and and the activation of the malate-aspartate shuttle.Figure 6Inhibition of mitochondrial pyruvate import in brown adipocytes results in the induction of lipolysis, a shift towards fatty acid utilization and an increase in energy expenditure. The increase in energy expenditure mediated by the addition of energy demand contributed by the activation of lipid cycling, involving a recurrent cycle of fatty acid release and re-esterification. To support fatty acid oxidation in the absence of pyruvate import the TCA cycle relies on carbons from glutamine and the activation of the malate-aspartate shuttle. Thus blocking mitochondrial pyruvate import can promote an increase in energy wasting even in the absence of adrenergic stimulation. LD, lipid droplet; MPC, mitochondrial pyruvate carrier; CPT1, Carnitine Palmitoyltransferase1; ATP, adenosine triphosphate; AMP, adenosine monophosphate; OGC1, oxoglutarate carrier 1; Aralar, mitochondrial aspartate-glutamate carrier.
Figure 1(A) Pedigrees of the studied family. A child patient (Patient: IV.4) with elevated serum ALT, AST and TBA levels, and her nuclear family members (III.4, III.5 and IV.3) were evaluated by WGS analysis. Mutated alleles are depicted as "a" for SEMA7A. Squares symbols: male; circles: female; solid: the patient. The reference alleles are depicted by a plus sign. The arrow points to the proband. (B) The homozygous p.R148W mutation in SEMA7A was further confirmed by Sanger sequencing. (C) The protein domain architecture of SEMA7A and conservation of the R148 position in Vertebrata. (D) Structures of WT SEMA7A and R148W mutant show local conformational changes, as a result of the replacement of an arginine (carbon atoms in yellow) with a tryptophan (pale) at position 148, which is highlighted in ball-and-stick models. (E, F) The surface electrostatic potential map of the WT and R148W mutant proteins, respectively, in which positively and negatively charged resides are expressed in blue and red, respectively and non-polar residues are denoted in white.
ADAP-deficient CD8+ CTLs reduce PD-1 expression and enhance cytotoxicity.Figure 2A OT-I CD8+ CTLs were conjugated with CFSE-labeled OVA257-264-pulsed or non-pulsed EL-4 cells for 30 min, fixed, and stained with anti-ADAP (red). B OT-I CD8+ CTLs were transfected with plasmids expressing ADAP or GFP, then stimulated with 10 nM OVA257-264-pulsed EL-4 cells to detect surface PD-1 expression. Representative of three independent experiments. C, D WT and ADAP−/− OT-I CD8+ CTLs were stimulated with 10 nM OVA257-264-pulsed or unpulsed EL-4 cells for 4 h to examine surface expression and the mRNA levels of PD-1 (C), in vitro cytotoxicity at different Effector:Target ratios (D; mean of triplicates ± SD). Graphs are representative of at least three independent experiments. E WT and ADAP−/− OT-I CD8+ CTLs were pretreated with 10 μg/ml anti-PD-1 antibody or IgG control, followed by incubation with 10 nM OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD). F Naïve CD8+ T cells were isolated from WT, SKAP55−/−, ADAP−/−, and SKAP55−/−ADAP−/− splenocytes, then stimulated with plate-bound anti-CD3/CD28 for 12 h to assess PD-1 mRNA levels, and for 48 h to check surface PD-1 expression (mean of triplicates ± SD).