EMBO/sd-character-level-ner huggingface dataset:
Figure 3A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6). A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) (data shown is representative of two independent experiments, n=3-6). C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).
Mpc1 or Mpc3 alone are not functional.Figure 4Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4). Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4). Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3). Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).
Figure 2(A) Western blot analysis and (B) Relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL), (n = 3). (C and D) Relative expression levels normalized to WT of (C) established UPRmt markers in 3 - 4 week old mice (n = 3 - 4). (C and D) Relative expression levels normalized to WT of (D) mitochondrial chaperones, proteases and TFAM in 3 - 4 week old mice (n = 3 - 4). (E) Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL), (n = 3) (F) Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Figure 6Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture. Measurement of HMGB1 released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture. Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm. Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody. qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.
Figure 2Schematic overview of McjD labelling schemes (A) and diffusion-based experiments (B) using confocal microscopy. Stochastic labeling is performed on the NBD (native cysteine C547) and TMD mutants (Y64C; L67C) Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicated Apparent ligand binding affinity values of McjD for AMPPNP (top), ATP (middle), and ADP-vanadate (bottom). Data points were obtained from the areas of fits from the E* histograms considering the ratio ligand-free/(ligand-free+ligand-bound) at the indicated substrate concentrations. Extraction of Kd values from a fit to the data is described in the Material and Methods section. Data for detergent-solubilized McjD for additional biochemical conditions is shown in Figure EV2
Figure 4A. Schematic diagram representing the domains and constructs of human (h) and zebrafish (z) Nup358 used in this study. TPR, tetratricopeptide repeat; LRR, leucine rich region; R1-R4, RanGTP binding domain; ZnF, zinc finger domains; IR, internal repeats; CHD, cyclophilin-homology domain. Dotted line indicates the deleted region in the indicated construct. FG and FxFG sequence positions are represented as short and long black lines, respectively. Amino acid positions are indicated in numbers. FL, full-length; N, amino-terminal region; M, middle region; C, carboxy-terminal region. B. HEK293T cells were co-transfected with GFP-maltose binding protein (MBP)-control or GFP-tagged version of indicated Nup358 fragments along with HA-AGO2. Immunoprecipitation (IP) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting (WB) using HA antibodies. C. Cells were transfected with indicated constructs and IP and WB were performed as described for B. D. HEK293T cells transfected with GFP-MBP (control), GFP-Nup358-C or GFP-Nup358-CΔIR (a mutant devoid of IR region) and HA-AGO2. IP and WB analyses were performed as indicated. E. Cells were transfected with HA-tagged version of indicated AGO subfamily member and GFP-Nup358-IR. IP and WB analyses were performed using indicated antibodies. F. Lysates from cells expressing GFP-control, GFP-human (h) Nup358-IR or GFP-zebrafish (z) Nup358-IR and HA-AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA-AGO2 by western blotting.
iNOS knockout mice are resistant to LPS-driven muscle wasting.Figure 1Quadriceps from saline or LPS treated, WT or iNOS KO mice, were isolated and used for western blot analysis with anti-iNOS (n=4). Ponceau S was used for total protein staining. (left) Quadriceps from saline or LPS treated, WT or iNOS KO mice, were isolated and used for western blot with anti-3NT (n=6). Total protein levels are also shown (right) Quantification of the 3NT to total protein ratio. Ratios are expressed relative to the saline treated controls. Representative images of tibialis anterior muscles. Scale bars represent 0.5 cm. Weight of tibialis anterior (D), quadricep (E) and soleus (F) muscle normalized . to initial body weight (n=15). (left) representative photomicrographs of H&E stained tibialis anterior muscle sections from control and iNOS KO mice injected with or without LPS. Scale bars = 100 μm. (right) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the tibialis anterior muscles from saline or LPS treated (top) WT control and (bottom) iNOS KO mice (n = 4). A total of 300 fibers per muscle were used for the analysis. Statistical comparisons, mean, and standard deviation of the mean are shown in the histogram legend. Change in grip strength between day of injection and endpoint of experiment. (WT saline n=12, WT LPS n=11, iNOS KO saline n=9, and iNOS KO LPS n=10).
Figure 3The antibody response of vaccinated naïve (plasma: n=64; saliva n=66) or SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects at 7-10 days after the second dose (T2) was compared to that obtained in COVID-19 patients (plasma n=28; saliva n=26). As a control, SARS-CoV-2 specific antibody response was analyzed in non-vaccinated subjects (n=19, Control). Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike IgG)=12, LoD (RBD IgG)=13.8, LoD (spike IgA)=54.22, LoD (RBD IgA)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA or total IgG concentrations of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU.
(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP.Figure 1(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times. (B) Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. Data information: Unless otherwise indicated experiments were performed three times. (C) ATG16L1 was phosphorylated in an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Phosphorylation of S278 and S287 in human (S278 marked in green, S287 marked in grey) was identified with high and low confidence, respectively. Conservation of amino acids 254-294 are shown using the Shapely colour scheme. Mass spectrometry was performed on a single experiment. (D) Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. Data information: Unless otherwise indicated experiments were performed three times. (E) HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated experiments were performed three times.
Figure 7(A, B) The homeostasis of PUFAs is determined by PUFA de novo synthesis and the balance of neuronal lipolysis and lipogenesis. The incorporation of PUFAs into phospholipids regulates normal neuronal functions (such as touch sensation) and affects neurodegeneration of mec-4(d).
Figure 5A. The spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ. B. The calculated mean and error of γ/κ for the sampled parameter fittings shown in Figure S2. The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $\frac{\gamma}{\kappa}\ \approx k_{1}\frac{{2P}_{\text{tot}}}{D_{2}}$.
Figure 5A, D. Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker (Centrin2-GFP; green), the microtubule marker (α-tubulin-mRFP; red) and no extra protein (A) or mouse JAM-A (D), 48 h after transfection of miR-449a mimic, JAM-A siRNA, or non-targeting negative control siRNA. Scale bar, 10 μm.B, E. Quantification of spindle rotation for cells as shown in A and D. Each dot represents a single cell. Red bar indicates median. Significance was tested by Pairwise t-test with Bonferroni correction. p-value = 2e-11 (Neg. control versus miR449 mimic), p-value = 0.01928 (Neg. control versus JAM-A siRNA), p-value = 0.17492 (Neg. control versus miR449 mimic, under JAM-A overexpression). * indicates p ≤ 0.05; ** p ≤ 0.01; p*** p ≤ 0.001 for all panels.C, F. Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in A and D; n ≥ 20 cells for all conditions, 2 independent replicates.
Figure 2Schematic overview of marker-tagged CNFY deletion variants. 3xFlag-tagged CNFY deletion variants were expressed in Y. pseudotuberculosis YP147 (ΔcnfY) from plasmids under control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody. To test secretion of the CNFY variants, full-length CNFY and different N- and C-terminally deleted variants fused to beta-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (ΔcnfY). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate. The data represent the mean ± SD of three independent experiments, carried out in triplicates. HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNF1, CNFY or the N- or C-terminally deleted toxin variants at 37°C for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody.
Figure 5(A) Venn diagram of 14 transcription factors (as recovered from the YEASTRACT database (Teixeira et al., 2006, 2018)) with at least one phosphorylation site changing two-fold in setup SR (grey) or SR igo1∆igo2∆ (blue). For the six overlapping transcription factors the log2 SILAC ratios of phosphorylation sites are shown (y-axis) in the corresponding setups (color code as above). The first two box plots illustrate the distribution of log2 SILAC ratios of all phosphorylation sites quantified in each setup ('all'). Gis1 and Rph1 were found to be significantly different in their distributions (p-value < 0.01, t-test relative to "all"). (B) M-Track proximity assay of Cdc55 fused to the protA-H3-HA prey-tag and Gis1, respectively Rph1, tagged with the HKMT bait-tag expressed in a rim15∆ strain background. Background signal intensity was defined using a strain expressing only Cdc55-protA-H3-HA and no tagged bait protein (noHKMT). Positive control: Nup2-HKMT, CDC55-protA-H3HA. me3: antibody recognizing me3K9H3; HA: 12CA5 antibody (C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side). (D) Mobility shift assay of Gis1 and Rph1 across several time points after Cdc55 depletion (without exposure to hyperosmotic stress). Cdc28: loading control (shown in Figure EV5C). (E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain. (F) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress across indicated strains expressing either wild type or point-mutated forms of Gis1 and Rph1 in a gis1∆rph1∆ deletion background, with the empty vector as control.
a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy.figf3(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death. (b) At 12-16 h post-transfection with GFP-LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P<0.01. (c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.05. (d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001. (e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b-e).
Figure 2(A) Example of raw pA-DamID data tracks from 2 million HAP-1 cells for Lamin B2, H3K27me3 and the Dam control from a single experiment. Sequenced reads are counted in 20kb bins and normalized for library size. (B) Same pA-DamID tracks of Lamin B2 and H3K27me3 as in (A) but after normalization to the Dam-only control (to correct for accessibility and amplification biases) and log2-transformation. (C) Correlation between the median peripheral enrichment of m6A-Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP-1 cells. The LAD definition is based on conventional Lamin B1 DamID data. Every point represents a single pA-DamID experiment for which both microscopy and sequencing data were generated, colored by the antibody used. The blue line represents a linear model with a standard error confidence interval in grey. The red dashed line represents the diagonal. The Pearson correlation coefficient was converted to a t-statistic and a one-sided t-test with n-2 degrees of freedom was used to determine statistical significance. (D) Comparisons of pA-DamID genome-wide data (bin size 20 kb) for different lamin antibodies in hTERT-RPE cells: Lamin B1 vs. Lamin B2 (left panel) and Lamin B2 vs. Lamin A/C (right panel). n denotes the number of independent biological experiments that were averaged for each data track. The blue line represents a linear model. The red dashed line represents the diagonal.
Figure 7Quantification of total length of vessels (A) Six images per tumor were analysed. Scale bar: 50µm. CD31+ tumor vessel area (B) and of αSMA+pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm. (D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm. (E, F) FACS quantification of CD8+ cytotoxic T cells (E) and CD69+ CD8+ T cells (F) over the total number of CD45+ in vehicle or glufosinate (20mg/kg) treated mice (n=6).
a) Body weight curves of male mice fed with the high-fat diet (HFD; 40% calories from fat) for 10 weeks starting from 5 weeks of age (0 on the.figf7(a) Body weight curves of male mice fed with the high-fat diet (HFD; 40% calories from fat) for 10 weeks starting from 5 weeks of age (0 on the x axis). Mice were injected with HDAd-TFEB either 1 week before (early inj), or 4 weeks after, (late inj) being placed on the high-fat diet, as indicated by the arrows. Values are represented as percentages of weight increase. Ctrl, control. (b) Whole-body composition analysis (EchoMRI) of the same mice as in a after 10 weeks of the high-fat diet. In a,b n = 10 mice per group; bars represent mean ± s.d. *P≤0.05;**P≤0.01; ***P≤0.001 compared with control high-fat diet group. (c-f) Total serum insulin, leptin, triglyceride and cholesterol levels in control and HDAd-TFEB mice kept on the high-fat diet for 10 weeks. Value are mean ± s.d. n = 10 add per group. *P≤0.05; ****P≤0.0001 compared with control. (g-i) Glucose and insulin tolerance tests in control and HDAd-TFEB mice challenged with the high-fat diet for 10 weeks. (g,h) Glucose (g) and serum insulin (h) levels at the indicated time points after glucose challenge. (i) Glucose levels at the indicated time points after insulin challenge. In g-i, values are mean ± s.d. n = 7 mice per group; *P≤0.05;**P≤0.01 compared with control. Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.
Fig.Figure 5(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats. (B) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates. (C) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7. (D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.
Figure 2A Mouse brain tissue was processed for immunohistochemistry by combining anti-lamin B1 (red) and anti-NeuN (green) antibodies. Representative images (maximal Z-projections) showing the distribution of lamin B1 in the striatum of 30-week-old wild-type (WT) and R6/1 mice. Yellow arrowheads show co-localization between lamin B1 and NeuN, and white arrowheads show NeuN negative lamin B1 positive nuclei. Scale bar 50 μm and 25 μm for low and high magnification, respectively. B Lamin B1 distribution in human putamen was analysed by immunohistochemistry. Antibody against lamin B1 (red) was combined with DAPI-Fluoromount G (blue) to label nuclei. Representative images show the distribution of lamin B1 in the putamen of non-affected individuals (CTL) and HD patients at different stages of the disease (VS II-IV: Vonsattel grades). Yellow and white arrowheads indicate MSNs and glial cells, respectively. Scale bar 50 μm and 20 μm for low and high magnification, respectively. C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry. Anti-lamin B1 antibody (red) was combined with anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue). Representative images show the distribution of lamin B1 at Vonsattel grade III. Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP-positive cells. Scale bar 10 μm.