EMBO/sd-character-level-ner huggingface dataset:
Figure 4A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants. Complexes from mitochondrial membranes of Control, Subject 1 and Subject 3 fibroblasts were separated by BN-PAGE (Fig EV2) and analysed by complexome profiling. Sample preparation, mass spectrometry, data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier <PXD014936>. Assignment of complexes: III, complex III; IV, complex IV; III/IV, supercomplex containing dimer of complex III and 1-2 copies of complex IV; S, supercomplex containing complex I, dimer of complex III and 1-4 copies of complex IV. Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly. In subject complexomes, various stalled complex I intermediates are assembled: ND4 module (blue dashed box); Q-TIMMDC1-NDUFA13 complex I intermediate (orange dashed box); and Q-TIMMDC1-NDUFA13-MCIA (green dashed box). In control complexome, the Q module (orange dashed box) is assembled without TIMMDC1 or NDUFA13. Complex I modules are denoted according to Stroud and colleagues (Stroud et al., 2016). B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I. Heatmap (upper panel) and profile plot (lower panel) compare complex I between control (black), Subject 1 (red) and Subject 3 (blue). C Profile plot of complex III (average of all subunits). Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex (III/IV) in Subjects 1 and 3. D Profile plot of complex IV (average of all subunits) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3, but higher amounts of individual complex IV and small supercomplex (III/IV, black arrow). E Details of Q module-containing assembly intermediates of Subject 3. Intensity based absolute quantification (iBAQ) values of assembly factors, single subunit and averaged Q-module subunits were plotted to show abundance profiles. The cartoon (lower panel) indicate assembled intermediate complexes relative to the plot. F Details about ND4 module assembly intermediate of Subject 3. Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module, FOXRED1 and TMEM70.
Fig.Figure 2(A) NF-κB luciferase assay in HEK293T wildtype (WT) and NOD1/2 knockout (KO) NF-κB reporter cells upon various stimulations. (B) qRT-PCR analysis of IL8 expression upon indicated stimulations in HEK293T WT and NOD1/2 KO cells. (C) qRT-PCR analysis of Cxcl2 expression upon indicated stimulations in WT, Nod1/2 KO and Rip2 KO bone marrow derived macrophages (BMDMs). (D) Multiplex analysis of CXCL2 production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon indicated stimuli for 20 h.
Figure 4a) IMPDH2 was consistently destabilized from 4 hours post SARS-CoV-2 infection. IMPDH2 was previously shown to form a protein-protein interaction with nsp14 (green line) (Gordon et al, 2020b) and be a crucial factor in supporting SARS-CoV-2 replication (Gordon et al, 2020a). The SARS-CoV-1 nsp14 has a dual role in base pair mismatch repair and synthesis of the Guanine-rich 5' cap of viral RNA (Smith & Denison, 2013; Smith et al, 2015; Chen et al, 2009). Circular stability and abundance plots are from Caco-2 cells infected with SARS-CoV-2 as described in 1A (see also 1A for plot legend). Asterisk denotes significant regulation (see methods). b) Thermal destabilisation of UGDH and PAAF1 temporally coincide and both were previously shown to interact with the SARS-CoV-2 proteins nsp6 (UGDH), and M (UGDH and PAAF1) (Samavarchi-Tehrani et al, 2020). PAAF1 negatively regulates the proteasome by controlling its assembly/disassembly (Park et al, 2005) and has been previously linked to transcription of HIV-1 (Lassot et al, 2007; Nakamura et al, 2012). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3). Asterisk denotes significant regulation (see methods). c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods). D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).
(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.Figure 6(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc. (B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection. (C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody. (D) Immunoblot analysis of EphA2, ASC, NLRP3, caspase-1 and IL-1β in HEK293T cells were co-transfected with 200ng EphA2, 40ng ASC, 40ng caspase-1 and 200ng IL-1β, vector or wild NLPR3 or Y132F as indicated. The position of protein markers (shown in kDa) is indicated on the right-hand side. (E) Densitometry analysis *P<0.05 (unpaired t test). NS means no statistical difference. Shown is mean ± SD. Data represent three independent biological replicates.
TAG .Figure 1(A) IFNs and ISGs were induced by SARS-CoV-2 infection in lung epithelial cell lines: Calu-3 and A549-ACE2 were infected with SARS-CoV-2 at MOI=2 for 24h; A549 was challenged with IAV at MOI=5 for 9h; A549 was infected with HPIV3 and RSV at MOI=2 for 24h (Blanco-Melo et al., 2020). Color key represents log2(fold change) relative to uninfected control cells. CH25H was highlighted by red asterisk. (B) Expression of CH25H in heathy donors and COVID-19 patients. The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors, three moderate COVID-19 patients and six severe COVID-19 patients by scRNA-seq analysis (Liao et al., 2020). Characterization of SARS-CoV-2 pseudovirus. (C) SARS-CoV-2, SARS-CoV and VSV pseudovirus (VSVG) were concentrated by ultracentrifuge. Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS-CoV and SARS-CoV-2. VSV Matrix protein served as a loading control for viral particles. Black arrows indicate full length S and cleaved spike (S2 subunit), respectively. Characterization of SARS-CoV-2 pseudovirus. (D) SARS-CoV-2 pseudovirus infection in Calu-3 cells with or without treatment of coronavirus fusion peptide EK1. Luciferase activity was measured to quantify the infection. (E) SARS-CoV-2 pseudovirus infection in Calu-3 cells transduced with non-targeting control or ACE2 specific sgRNA. Luciferase activity was measured to quantify the infection. (F-G) Overexpression of CH25H restricts SARS-CoV-2 entry. Calu-3 cells transduced with lentivirus overexpressing CH25H or empty vector were infected with SARS-CoV-2 pseudovirus encoding Fluc or EGFP and pseudovirus infection was quantified by luciferase assay (F) or visualized by fluorescence microscopy (G).
Figure 2Localization of three binding sites is shown in the Munc18-1 tertiary structure (bottom right). Three potential binding sites, identified computationally by searching for pockets with favorable steric and electrostatic properties, were screened with the ZINC library. Chemical structures of top hits for each binding site are shown. Each compound was assigned a number for all following analyses.
Figure 3(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm. (B) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm. (C) MCF10A cells were grown to high density, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm. (D) Cells were grown as in (C), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test). (E) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and lysed while under tension. Anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test). (F) Cells were treated as in (E), fixed while under tension, and stained for TRIP6 and LATS1 as in (A). Scale bar=20µm. (G) MCF10A cells infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4), were grown at high density on PDMS membranes and were stretched or not (only stretched cells shown) at 17% elongation for 2 hours, fixed while under tension, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm. (H) Cells were treated as in (G) and YAP target gene (CTGF and Cyr61) and TRIP6 expression were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, T-test). (I) TRIP6 expression levels in (H) in control (shEGFP) and TRIP6 (shTRIP6) knockdown cells just prior to stretch were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, T-test).
Figure 6The knockdown efficiency of shRNA-GADD45A in human WI38 fibroblasts. The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A. Representative images of β-gal staining in human fibroblast cultures with control and shRNA-GADD45A treatments at passage 54 (C). Quantification of the percentage of β-gal positive cells in human fibroblasts with control and shRNA-GADD45A treatments (duplicates per group) (D). Representative images of GADD45A and γH2AX staining in colon sections from young (35-50 y/o) and old (75-85 y/o) people (E). Quantification of the percentage of GADD45A positive cells in young and old human colon tissues (n=8 per age group) (F). Quantification of the percentage of γH2AX positive cells in young and old human colon tissues (n=6 per age group) (G). Model for the role of Gadd45a in regulation of DNA damage response initiation. In G3Terc-/- mice, Gadd45a promotes the accessibility of telomeric heterochromatin and enhances the DNA damage response initiation (e.g. pATM activation) through a BER-dependent pathway. The initiated DNA damage response further causes upregulation of p53 and p21, which leads to impairment of intestine stem cell maintenance and function. In G3-dKO mice, the telomeric heterochromatin is condensed and damage response initiation is reduced upon Gadd45a deletion, which further leads to decreased expression of p53 and p21. Therefore, the maintenance and function of intestine stem cell is improved.
Fig4.Figure 4Overview of experiments for comparing wild type L. lactis MG1363 and CcpA mutant 445C1 in glucose-limited chemostats at D = 0.5 h-1. Samples were taken for dry weight measurements, external metabolite and amino acid analysis and proteome measurements. , Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S­0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S­0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names. C, Amino acid fluxes (mmol/gCDW/h) and D, Residual concentration (mM) of arginine. The arginine uptake flux is increased to such extent that the residual arginine concentration became undetectable in the CcpA mutant. Data points represent the individual chemostat fluxes and concentrations; bars are average values (n = 4). Statistically significant differences between amino acid fluxes were calculated with two-sided t-tests and marked with * (p < 0.05). Protein fractions. Protein fractions were obtained by normalizing iBAQ values by the total sum of all values. The obtained protein fractions were then averaged over the biological replicates (n = 4) for visualization. The fractions glucose transport, glycolysis, arginine pathway and remaining fraction are significantly different between the two strains (p < 0.001, calculated with two-sided t-tests).
Figure 3CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels). Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel). The relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one. Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively
Fig.Figure 7(A) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01 (B) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01). (C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).
Overview of the most pro­minent changes in the mitochondrial complexome in ρ0 cells.Figure 8Red dashed outlines, lost complexes containing mtDNA encoded components; red, decreased abundance; green, increased abundance; grey, no significant change.
Figure 2(A) Schematic of tRNA translocation. (B) mRNA-tRNA translocation monitored in a time-resolved Pmn assay upon mixing 80S 2C with Pmn in the absence (brown triangles) or presence of eEF2 and eEF3 (green, 0.13±0.01 min-1), eEF2 (red, 0.030±0.005 min-1) or eEF3 (blue, 0.10±0.01 min-1). Data are normalized to Met-Phe-Pmn formation in presence of eEF2 with the maximum value set to 1. Data presented as mean ± SEM of n = 3. (C) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction. (D) tRNAfMet(Flu) co-eluting with 80S 2C incubated without factors (orange), with eEF2 (red), with eEF2 and eEF3 (magenta), or with eEF2, eEF3 and TC-Val (green) for 15 min before loading on a BioSuite 450 size-exclusion column to separate ribosome-bound and -unbound tRNA. Data are normalized to tRNAfMet(Flu) bound to 80S 2C in the absence of eEF2/eEF3 with the maximum value set to 100%. Data presented as mean ± SEM of n = 3. (E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.
a) A schematic diagram of the ubiquitin-like lipidation reaction of autophagy.figf1. (b) Conjugation of LC3 and two other mammalian homologues (GL1 and GL2) with phosphatidylethanolamine (PE) was performed as described in ref. . Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8-12 μM LC3/GL1/GL2, 2 mM lipid (30 nm liposomes composed of 10 mol% bl-PI, 55 mol% DOPE, 0.15 mol% Rh-DOPE and 34.85 mol% POPC; see Methods for full definitions), 1 mM dithiothreitol and 1 mM ATP and were run at 30 °C for 90 min. Each reaction was run on a 12% SDS-PAGE gel and visualized by Coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7-GRL1 conjugate, the Atg3-GRL1 conjugate and a modified form of GRL1, GRL2 and LC3 that is labelled simply as GRL1/GRL2/LC3-intermediate and is probably the adenylated form of each LC3-related protein. (c) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes. Reactions are as in b, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mol% bl-PI, 0.15 mol% Rh-DOPE and POPC. (d) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material. Floated samples were run on SDS-PAGE and imaged by Coomassie. For c and d, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the vertical lines. Uncropped versions of all gels are included in Supplementary Fig. 7.
Transcriptional profile of bravo and wox5 QC cells.Figure 6Scheme of the methodology used for the QC-specific RNAseq. Plants expressing pWOX5:GFP in WT, bravo-2 and wox5-1 background were used for protoplasting followed by Fluorescent Activated Cell Sorting (FACS) to isolate QC for RNA sequencing. Area-proportional Venn diagram showing the overlap between BRAVO and WOX5 regulated genes in the QC (q-value< 0.05 and FC > 1). Expression of the 380 common BRAVO and WOX5 regulated genes from (B). Expression of the 53 genes from (C) showing similar expression in bravo and wox5 mutants. Expression of the 41 genes from (C) showing opposite expression levels in bravo and wox5 mutants.
Figure 6FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility (determined by ATAC-seq). Clusters 1-4 were HNF1β- positive while cluster 5 was HNF1β-negative. The heat-maps on the left show FOXA2, HNF1β, ATAC-seq and H3K27Ac profiles at FOXA2-bound genomic regions. The three heat-maps on the right show JunB ChIP-seq data and ATAC-seq data in wild type or Jun/JunB double knockout (DKO) CFPAC1 cells. Data are shown in +/- 3 kb regions centered on the summit of FOXA2 peaks. Characterization of clusters 1-3. Left: quantification of the indicated signals at FOXA2 bound regions in wild-type (WT) and HNF1β knock-out (KO) cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Middle: distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks. Right: over-represented motifs in clusters 1-3 and inferred binding models. Quantification of JunB ChIP-seq signals in clusters 1 to 5. (Right) Quantification of ATAC-seq signals in clusters 1 to 5 in wild type and Jun/JunB double KO (DKO) CFPAC1 cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Representative snapshot showing changes in FOXA2 occupancy, chromatin accessibility and histone acetylation in HNF1β knock-out CFPAC1 cells. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.
Figure 6 (A) Representative immunostaining of fibrinogen depositions in tumour-bearing mouse lungs received an injection of rhodamine-labelled B220+CD11c+NK1.1+HepELs derived from tumour-bearing- FX+/− or FX−/− mice (scale bar, 20 μm)  (B) Immunohistochemical quantifications of fibrinogen depositions in tumour-bearing mouse lung after an injection of B220+CD11c+NK1.1+cells derived from tumour-bearing- FX+/− or FX−/− mouse liver. Sorted B220+CD11c+NK1.1+cells were prepared from three mice per each genotype, and were i.v. injected. Shown are averages (N = 6/group, 4 sections/mouse) with SEM and Welch"s t-test
Figure 6a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white squares) and IL-12 either alone (white circles) or in the presence of LY294002 or MK2206 (black circles) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=8 independent experiments performed. b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle or IL-12 either alone or in the presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA and ionomycin. c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated as in c of n=8 experiments performed. d. Proliferation measured at day 4 of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that had been pre-incubated with either vehicle or IL-12 alone or with LY294002 or MK2206 at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression as in d at different Treg:Tresp ratios of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.