EMBO/sd-character-level-ner huggingface dataset:
Figure 7Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished. Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose. Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium. The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.
Figure 5(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture with the young CX3CR1+/GFP slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals no co-localization of young, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing young microglial cells.(B) Immunofluorescence analysis of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals co-localization of old, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bar: 10 µm (A and B). (C) 3D reconstruction of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (10 DIV) and immunostained using GFP (green) and M3.2 (red). GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3.2 positive immunoreactivity indicative of Aβ uptake. Overview at low magnification is shown in the left panel. Scale bar: 15 µm. A detailed plaque is shown at higher magnification in the right panels. Scale bar: 10 µm. (D) Immunofluorescence analysis of young CX3CR1+/GFP and old APPPS1/CX3CR1+/GFP slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV) and immunostained using GFP (green) shows alterations in microglial morphology of old cells upon culturing. Upper-left panels are enlargements of boxed regions. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bars: 50 µm.(E) Quantitative analysis of amoeboid microglial cells in young CX3CR1+/GFP (white bars) and old APPPS1/CX3CR1+/GFP (green bars) brain slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV). The values are expressed as percentages of amoeboid microglia from the total number of microglial cells. The values represent mean ± SEM from 3 independent experiments, each experiment including at least 250 cells per condition (*P < 0.05, ***P < 0.001).
Atg38 forms a homodimer.Figure 6.(A) Interactions between the indicated fusion proteins were analyzed as in Fig. 4 A. AH109cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d. (B) Analysis of Atg38 by gel filtration. Recombinant Atg38 protein was fractionated over a gel filtration column. Each fraction was subjected to SDS-PAGE and visualized by Coomassie staining. (C) Analytical ultracentrifugation of Atg38. The raw absorbance distributions and the best-fit model calculated by the SEDFIT program (top). Residuals of the fit (middle), sedimentation coefficients, c(S), and distributions (bottom) of Atg38 protein.
Figure 5A, pRIP3, a central mediator of necroptosis, was not expressed at the implantation sites of Rb1d/d mice but was expressed at those of Rb1f/f mice at 1500h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo; green dotted line, luminal epithelium; red dotted line, stroma. B, TNFα stimulated the expression of pMLKL, a critical mediator of necroptosis, in the primary mouse uterine epithelial cells. Scale bar = 50μm; blue signal, nuclei stained by DAPI; red signal, pMLKL. C, Uterine expression of Tnfα was elevated in the control mice (Rb1f/f mice) on days 5 and 6 compared to on day 4 (mean ± SEM, Student's t test; n=3 mice for each group). As for the uterine samples on days 5 and 6, those with implantation sites were examined. D, The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f/f and Rb1d/d mice. Immunostaining of TNFα at the implantation sites of Rb1f/f and Rb1d/d mice were performed. Scale bar = 100μm; arrowhead, embryo.
Elevated miR-21-3p and its inhibition are of clinical relevance to human skin inflammatory disorders.Figure 6(A) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and human squamous cell carcinomas (SCC). pri-miR-21 Healthy skin vs SCC P = 0.043; pre-miR-21 Healthy skin vs SCC P = 3E-05; miR-21-5p Healthy skin vs SCC P = 7E-05; miR-21-3p Healthy skin vs SCC P = 1.4E-06. ≥ 5 independent biopsies per condition. (B) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and humanpsoriasis lesions. pri-miR-21 Healthy skin vs Psoriasis P = 0.034; miR-21-5p Healthy skin vs Psoriasis P = 0.034; miR-21-3p Healthy skin vs Psoriasis P = 0.036; ns: non significant, ≥ 4 independent biopsies per condition. (C) RT-qPCR quantification of relative levels of IL6, IL1B, PTGS2 and TNFA levels in epidermis of abdominal healthy humanskin biopsies exposed to acute UV ex vivo, with or without topical treatment with miR-21-3p inhibitor (+) or mismatched control (-) as indicated. ns: not significant. IL6: no UV vs Ac-UV P = 0.036, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.019; IL1B: no UV vs Ac-UV P = 0.002, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.023; PTGS2: no UV vs Ac-UV P = 0.008, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.022; TNFA: no UV vs Ac-UV P = 0.032, ns = non significant. ≥ 7 replicates, a pool of three independent experiments performed with the skin of three different donors is shown.
Figure 10(A) Total GSH amount normalized to protein from kidney and (B) brain of wild-type (WT) and mutant (Mut) mice. Error bars represent SDs of at least 3 animals per group. (C) Thiosulfate levels in urine and (D) plasma normalized to protein amount. Error bars represent SDs of 9 animals per group (urine) and 6 animals per group (plasma). (E) H2S amount normalized to tissue from kidney and (F) brain. Error bars represent SDs of 7/8 animals per group. (G) Acylcarnitine C4, C5 and C6 levels from plasma, normalized to volume. Error bars represent SDs of 5 wild-type and 3 mutants.
Figure 3Ubiquitination reactions of either Nek2A or securin by the APC/CCdc20 and the effect of increasing concentrations of the MCC. Size exclusion chromatography peak fractions of APC/C complexes with either Nek2A Size exclusion chromatography peak fractions of APC/C complexes with Nek2A APC/CΔApc15 is the Apc15 deleted mutant APC/C, Size exclusion chromatography peak fractions of APC/C complexes with Nek2A APC/CΔApc15 is the Apc15 deleted mutant APC/C, Size exclusion chromatography peak fractions of APC/C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC. MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1 Size exclusion chromatography peak fractions of APC/C complexes securin MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1
Figure 1(A) Experimental design of translational profiling of mTOR-dependent mRNAs in vitro in NRCMs. Polysomal fractions were isolated on sucrose gradient and ribosomal footprints were recovered by ribonuclease digestion. NRCMs were treated with vehicle or Torin 1 (100nM for 3h) to block the mTOR pathway. (B) Scatter plot of ribosome occupancy in Ribo-seq of cultured cardiomyocytes in response to PE and Torin 1 treatment (blue dots - mRNAs upregulated after Torin 1, red dots - downregulated mRNA. RPKM - Reads per kilobase million. (C) Percentage of IRES (Internal Ribosomal Entry Site) and TOP or TOP-like containing mRNAs in total and Torin 1-sensitive mRNA pools. (D) Transcriptional (RNA-seq) vs. translational (Ribo-seq) control of mTOR-regulated mRNAs. (E) Network of enriched GO categories (biological process) among mTOR-sensitive genes into several functional groups represented by circles of different colors. Each node represents one enriched GO category with the size of the node is proportional to the number of transcripts. Edges indicate similarity (between the two connected GO categories). (F) Cumulative fraction of mRNAs relative to their fold change of Ribo-seq and RNA-seq between (G) all and mTOR-dependent transcripts two days after TAC surgery. Kolmogorov-Smirnov-Wallis test. (H) Immunoblot representing levels mTOR-dependent targets with defined 5'TOP motifs 3 hours and 2 days after TAC. n=3 biological replicates per condition. RT-qPCR representing levels mTOR-dependent targets with defined 5'TOP motifs 3 hours and 2 days after TAC. n=3 biological replicates per condition. (J) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1.* p≤ 0.05 vs sham surgery, # p≤ 0.05 vs TAC 2d surgery. t-test, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM (K) RT-qPCR for indicated mRNAs 2 days after TAC for indicated groups, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM
a) Schematic representation of the genome-wide systematic ORF suppressor screen in yeast.figf1(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.
Fig.Figure 9A, B Blocking autophagy by downregulating Atg5 suppresses the cellular defects caused by down-regulation of TOR signalling: (A) loss of lateral membranes, formation of actin bundles; (B) integrin mislocalisation (compare to Fig. 4D, E and Appendix Fig S3E and 4C). C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.
Figure 4A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points. C Immunoblot analyses of IRF3 dimerization in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) or poly (dA:dT) for 6 hrs. D Immunoblot analyses to detect the protein expression of ISGs in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with IFN-α or -β (50 U/ml) for 4 hrs. E, F Immunoblot analyses of total and phosphorylated (p-) STAT1 in shCtrl and shWTAP A549 cells, followed by stimulation with IFN-α (E) or -β (F) at different time points.
Miro ELM domains.Figure 2(A) Top, MiroS shown in the 'top view'. ELM1 and ELM2 are related by pseudo twofold symmetry, represented by the dyad between them. Bottom, overlay of the two ELM domains (rmsd of 1.8 Å over 84 Cα positions). ELM1 (blue), ELM2 (green) and identical or similar conserved residues in identical positions are shown as sticks. (B) Comparison of the ELM domain hydrophobic cores. The ELM1 core contains the UNL, modelled here as homoserine shown in spheres, occupying a ∼442 Å3 cavity highlighted with mesh. The ELM2 core includes three tryptophans in close proximity (W367, W391, W395 in orange). Hydrophobic residues in the LM/EF hand pair interfaces are also shown in sticks. LM, ligand mimic; UNL, unidentified ligand.
Figure 3Details of the EF hand loops. All loops are shown in approximately the same orientation, with entering and exiting helices ghosted for clarity. Residues of note are coloured in orange. cEF1 is shown in its apo state. cEF2 is shown in its apo, Ca2+‐bound and Mg2+‐bound states. In the cEF2 apo state, E354 is stabilized by nearby loop residue R344. In the Ca2+‐bound state, cEF2 loop residues coordinate Ca2+ in a pentagonal, bipyramidal arrangement, with E354 as a bidentate ligand. An ordered water molecule at the apex of one of the pyramids is represented as a small red sphere. In the Mg2+‐bound state, the coordination is octahedral with E354 as a monodentate ligand. The hEF1 and hEF2 loops adopt a conformation quite different from the cEF hands, and also lack the acidic residues that typically coordinate cations. Note the conserved hEF1 272VTL274 and hEF2 386VTL388 residues, which overlay identically in Fig 2A. cEF, canonical EF hand; hEF, hidden EF hand.
Figure 1 (A) Electron micrographs of immunogold-labeled Tau (arrows) in multivesicular bodies (yellow dashed lines) of rat hippocampus (scale bar: 250nm)  (B) Schematic diagram of proximity ligation assay (PLA), wherein primary antibodies against Hrs or Tau are detected by secondary antibodies conjugated to DNA. Ligation and amplification of DNA, resulting in green signal, only occurs when proteins are in close (20-50 nm) proximity  (C) PLA signal (green) for Hrs/Tau interaction (arrows) on an mCh-Hrs-labeled endosome (red) in N2a cells; scale bar: 5 μm (see also Fig. EV1). (D) Super-resolution images of N2a cells transfected with constitutively-active Rab5 (EGFP-Rab5-CA) and immunostained for Tau, revealing the presence of Tau in both membranes and lumen of early endosomes (scale bar: 5 μm)  (E) Super-resolution images of N2a cells transfected with mCh-Hrs and immunostained for Tau and EEA1. Higher-magnification insets (A and B) show Hrs- and EEA1-positive endosomes with juxtaposed Tau puncta present both in the membrane and lumen; scale bar: 5 μm
Figure 7(A) Flow cytometry analysis of single cell suspensions from the nasal epithelium. Cells were labelled with EpCAM and CD45 antibodies and gated accordingly. (B) ACE2, Syndecan 1 and Syndecan 4 cell surface expression on nasal epithelial cells, compared to polarized epithelial Calu-3 was confirmed by quantitative real-time PCR. (C, D) Nasal epithelial cells were exposed to SARS-CoV-2 isolate (hCoV-19/Italy, 100 TCID/mL) either directly or after pre-treatment with antibodies against ACE2 cell surface receptors (1 hour at 37°C ) or after pre-treatment with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Detection of viral binding after 4 hours at 4°C (C) and persistently-infected cells lysed after 24 hours at 37°C (D) was determined by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (C, D) ordinary on-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3), (D) (n=3 in duplicates).
Macrophages invading growing glioma are bone marrow derived.Figure 2A. F4/80 immunohistochemistry on a section of 5 weeks growth glioma implanted in ROSAmT/mG::Pdgfb-iCre mouse (50µm depth stack). B. Two-photon live imaging of LifeAct-GFP bone marrow transplantation in 5 weeks implanted glioma in ROSAmT/mG mice (100µm depth stack).B lower panels: 280µm; C. Two-photon live imaging of LifeAct-GFP bone marrow transplantation in 3 and 5 weeks implanted glioma in ROSAmT/mG. Macrophages relocate close to blood vessels during glioma growth (50µm depth stack) (n=4 mice). Statistical analysis: C: t-test. Error bars: meanSD. Scale bar:C: 100µm.
Figure 1(A) Schematic illustration of the two-step CENP-AOFF/ON assay using the Auxin (IAA) inducible degradation system. (B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells. (C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm. (D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments. (E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005. (F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images. (G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation.
Structural mapping of disease-related mutations on the C3b-regulator surfaces.Figure 4(A) Mapping of known disease-related mutations on C3b (Table 3). The high-resolution C3b three-dimensional structure is used as structural template, shown as white surface. The regulator contact surfaces (based on C3b-FH (CCP1-4) (Wu et al, 2009) and C3d-FH (CCP19-20) (Kajander et al, 2011; Morgan et al, 2011)) are shown as black contour. All mutations reported in Table 3 are shown in blue on the C3b surface. Disease-related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel. (B) Mapping of disease-related mutations on the FH (CCP1-4) surface using data from Table 4. Colors as in Fig. 4A. (C) Mapping of disease-related mutations on the MCP (CCP3-4) surface using data from Table 4. Colors as in Fig. 4A.
Pathological examination of Dnmt2-deficient mice.Figure 1Giemsa staining of a femoral section from 8-day-old mice showing endochondral ossification defects. Scale bar: 100 μm. Representative femur sections from 8-day-old wild-type and Dnmt2−/− mice. The staining of perivascular cells (GLS, green) is strongly reduced in Dnmt2−/− bone marrow. Nuclei are counterstained with DAPI. Scale bar: 100 μm. Quantitative analysis of capillary numbers. GLS-positive capillaries were counted in wild-type (wt) and Dnmt2−/− (D2-) mice, in three independent high-power fields (HPF) per mouse (n = 7). Quantification of cell numbers in haematopoietic tissues of 8-day-old mice (n = 8). Representative FACS plots of wild-type and Dnmt2−/− BM stained for Lin− Sca1+ and cKit+ cells. Quantification of bone marrow LSK cells in wild-type (three independent experiments, each with pooled BM samples from 4 to 6 mice) and Dnmt2−/−bone marrow (three independent experiments, each with pooled BM samples of 5-9 mice). Restoration of the LSK cell population in adult Dnmt2−/− mice. Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild-type littermates at the indicated age (2-3 independent experiments, each with pooled bone marrow samples from 3 to 9 mice). Increased LSK CD34− and reduced LSK CD34+cell populations in 8-day- and 1-year-old Dnmt2−/− mice compared to wild-type (two independent experiments, each with pooled bone marrow samples from 3-9 mice).
LncRNA CRNDE negatively regulated PTEN expression (A) RNA Immunoprecipitation (RIP) and (B) RNA pull-down assays were conducted to detect the association of LncRNA CRNDE and phosphatase and tensin homolog (PTEN)/ neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1).Figure 6(A) RNA Immunoprecipitation (RIP) were conducted to detect the association of LncRNA CRNDE and phosphatase and tensin homolog (PTEN)/ neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). (B) RNA pull-down assays were conducted to detect the association of LncRNA CRNDE and phosphatase and tensin homolog (PTEN)/ neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). (C) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1. (D) The expressions of PTEN and NEDD4-1 were measured using western blot in SGC7901 cells transfected with pcDNA-CRNDE or Mock. GAPDH was served as an internal control. (E) The expressions of PTEN and NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock. (F, The ubiquitylation of PTEN was measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock G) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors.