EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Schematic illustration of the tumor model in which YUMM1.7 melanoma cells were injected subcutaneously into syngeneic wildtype C57BL/6 mice. 5-FU (25 mg/kg) or DTIC (10mg/kg) were administered intraperitoneally from day 6 post cancer cell injection, with one dose every two days. Tumor and spleen were harvested at day 16. B. Western blot for Ctrl and STING KO YUMM1.7 cells, assayed with STING and Gapdh antibodies. C. STING-KO or sgRNA control (Ctrl) YUMM1.7 cells were treated in vitro with the indicated concentrations of DTIC. Cell viability were determined using the CellTiter-Glo assay after two days, with relative cell viability shown. N=3.   D. For cells in (C), proliferation assays were performed with cell viability assayed on indicated days after plating. Relative luminescence in arbitrary units (AU) is shown. N=3. YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (E) Representative images of tumors and spleens. Image panels were cropped from the same picture. YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (F) Mean tumor volumes of Ctrl or STING KO YUMM1.7 with DMSO, DTIC or 5-FU treatment were followed during the experiment. N=3 to N=5, as shown in (E). YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (G) Tumor (left panel) and spleen (right panel) weights were determined at the endpoint for the experiment followed in (F). Each dot represents data from a mouse.
Figure 5Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).
a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG).figf2(a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R2 = 0.8984). Values were pooled from five independent experiments. (c) Potential confounders in half-life analyses. Top, total protein ideally decreases over time in a predictable manner (top right panel). Middle, toxicity reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Bottom, protein aggregation might also shorten half-life estimations if the protein cannot be immunoprecipitated (black arrows, bottom right panel) or lengthen it if the protein is more stable and successfully immunoprecipitated (gray arrows, bottom right panel). (d) OPL. Primary cortical neurons were transfected with EGFP and TDP43(WT)-Dendra2, photoconverted then imaged by AFM. Dead cells (arrows) were excluded at the time of death. Pre, before photoconversion. (e) The half-life of TDP43(WT)-Dendra2 was determined by fitting a first-order exponential curve to the data (R2 = 0.9558). (f) Including cells with aggregates prolonged TDP43(WT)-Dendra2 half-life (pink line, R2 = 0.9003; *P 0.0001, F 15.34, extra sum-of-squares F-test). Excluding cells that died over the 36-h experiment also prolonged half-life (cyan line, R2 = 0.7171; **P 0.0001, extra sum-of-squares F-test). Error bars represent s.e.m. Values were pooled from eight wells per condition, with experiments performed in quadruplicate.
Figure 2A Schematic of the functional domains of EDC3 and the fragments used to construct GFP fusion proteins. The phosphorylation site on EDC3 serine 161 is shown. B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3). C 2D [1H,15N] HSQC spectrum of EDC3104-197 (100 µM EDC3104-197; 20 mM HEPES pH 6.2, 500 mM NaCl, 0.5 mM TCEP; 298 K; 14.1 T). HSQC spectrum shows a single cross peak for each N-HN pair, i.e. one per amino acid (other than prolines). The lack of chemical shift dispersion in the 1H chemical shift dimension is typical for intrinsically disordered protein/region (IDR), as they lack the hydrogen bonding network in secondary structure elements of folded proteins. D 2D [1H,15N] HSQC spectra of EDC3104-197 with (red; 1:2 molar ratio) and without (blue) Pim1. We titrated Pim1 into 15N-labeled EDC3, and the 2D [1H,15N] HSQC spectrum of EDC3 (residues 104-197) when bound to Pim1 was measured. Direct comparison with the unbound 2D [1H,15N] HSQC spectrum of EDC3 showed that while the majority of EDC3 peaks are unchanged, ~30 peaks become either broadened beyond detectability demonstrating they directly bind to Pim1 or show small chemical shift perturbations. Upon completion of the sequence-specific backbone assignment of the EDC3 IDR, we identified that EDC3 residues 137 to 167 are necessary for the interaction with Pim1.
Figure 3Schematic of HSATIII-ChIRP-MS at various time points during and after thermal stress exposure. Bar graph of relative ChIRP-MS enrichment score. ChIRP-MS enrichment score for each protein was calculated as (the average number of identified peptides at each time point + 0.00001) / (the average number of identified peptides at 37°C + 0.00001) and normalized to the maximum value of each protein. Western blot-validation of HSATIII-ChIRP. nSB proteins (YTHDC1, WTAP and SRSF9) were detected by western blotting using specific antibodies (input 1%). HSATIII lncRNAs were detected by semi-quantitative RT-PCR (input 100%). In; input, PD; pulldown by ChIRP. Changes of nSB localization of m6A-related factors at various time points during and after thermal stress exposure. HeLa cells were exposed to thermal stress (42°C for 2 h) and cultured at 37°C for the indicated periods. m6A-related factors were visualized by HSATIII-FISH and IF using anti-WTAP antibody (D) or anti-YTHDC1 antibody (F). Nuclei were stained with DAPI. Scale bar: 10 µm. (E, G) Box plot of relative intensities of WTAP (D) and YTHDC1 (F) to HSATIII within nSB areas in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=30).
Figure 1A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z=0, two images below, z=-4 µm and z=-2 µm, and above, z=+3 µm and z=+5 µm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A.B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in A. Additional z-slices of this spindle are shown in Fig EV1B.C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits.D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1786 kinetochore-PRC1 pairs (n=29 cells) and vertical spindles 1133 kinetochore-PRC1 pairs (n=16 cells).E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles.G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle.
Figure 5A. In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry: endoderm (α-fetoprotein, AFP), mesoderm (α-smooth muscle actin, SMA), and ectoderm (α-tubulin, TUJ1). Nuclei (DNA) were stained by Hoechst (blue). Scale bars: 200 μm. B. F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice. C. Pictures demonstrating germline transmission; endogenous Oct4-GFP signal was detected in the gonads of F1 pups. Scale bars: 200 μm.
Figure 5A-C. Three-dimensional reconstruction (left) and Imaris-based morphometric analysis of CX3CR1+ brain microglia or F4/80+ liver and kidney MΦ in adult Irf8-/- or Irf-8+/+ mice, respectively. Quantification of cell density of Iba1+ microglia and F4/80+ liver and kidney MΦ in adult Irf8-/- or Irf-8+/+ mice, respectively (left). Scale bars = 10 µm. For morphological measurements each symbol represents one mouse with at least six examined cells per mouse. Data represent mean ± s.e.m. Significant differences were examined by an unpaired t-test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001); n.s.: not significant.
Figure 3(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. (B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91. (C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura). (D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase. (E) Model illustrating the role of Prx1 in coupling H2O2 to oxidation of the mitochondrial glutathione pool.
A and components of the cohesin complex on chromatin.Figure 8A. Scheme representing the strategy used to identify protein interacting with BAZ2A on chromatin (Chromatin-IP). B. BAZ2A-interacting proteins on chromatin. Mass spectrometry analysis of FLAG immunoprecipitates from wt and F/H-BAZ2A ESC+2i. Values of peptide number are shown as the difference between peptides obtained in FLAG-IP of F/H-BAZ2A and wt ESC chromatin extracts. Data are from three independent experiments. The proteins found enriched in FLAG-IP of F/H-BAZ2A ESCs in all three experiments are shown. Further BAZ2A-interacting proteins identified in only two or one of the immunoprecipitation (IP) experiments Error bars represent s.d. C. Anti-HA IP of wt- and F/H-BAZ2A-ESC+2i and ESC+serum. Western blot shows the interaction of BAZ2A with SNF2H, TOP2A and SMC1a. D. TOP2A and BAZ2A regulate gene expression in ESC+2i via a shared pathway. qRT-PCR of genes regulated by BAZ2A in ESC+2i (left panel) and in ESC+serum (right panel) upon TOP2A or BAZ2A knockdown by siRNA. ESCs were treated with the corresponding RNAs for 4 days. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001). E. Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A-regulated genes in ESC+2i observed upon TOP2A or BAZ2A knockdown. ESCs were treated with the TOP2 inhibitor ICF-193 for 24 hours. mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, ** < 0.01, *** < 0.001, **** < 0.0001). F. ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC+2i but not in ESC+serum. Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA-control. #1-#4 indicated sequences with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic region that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).
Figure 1(A) miRNA profiling identified miR-574-5p as the commonly upregulated miRNA in the hearts of aged mice, MI mice, and patients; and miR-574-3p is induced in exercised hearts and MI patients. (B) Sequence and location of miR-574 gene in the human genome. (C) miR-574-5p and miR-574-3p were highly expressed in the explanted failing hearts from chronic HF patients (n=17) versus donor non-failing hearts (n=6). (D) miR-574-5p and miR-574-3p were induced in the hearts of mice with isoproterenol (ISO) infusion (4 weeks; n=10-12). (E) miR-574-5p and miR-574-3p were induced in the hearts of mice under transverse aortic constriction (TAC) surgery (4 weeks; n=8-10).
Figure 7Several proteins coordinating the usage of two ends of a DSB suppress BIR: (1) Mre11-Rad50-Xrs2 complex (MRX) that promotes synchronous resection of two ends of a DSB, (2) D-loop unwinding helicase Mph1 that stimulates SDSA, and (3) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture. All of these proteins are particularly important to reduce BIR when recombination occurs between short repetitive sequences.
Retrograde CRMP4-dynein complex formation mediates MN loss in ALS (A) Representative images of CTX signal in healthy and C9orf72 human IPSC-derived MNs before and after Sema3A application.Figure 6(A) Representative images of CTX signal in healthy and C9orf72 human IPSC-derived MNs before and after Sema3A application. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 40 μm. (B-C) Quantification of CTX signal in healthy and C9orf72 IPSC-derived MNs before and 3 days after applying Sema3A to distal compartment, compared with untreated control. 3 independent chambers in each condition were analyzed. Average of ~150 neurons per condition monitored. Student's t-test, n=3, Data presented as mean ±SE, *p<0.05. (D) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A or untreated. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 30 μm. (E) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A. 3 independent chambers in each condition were analyzed. ~200 neurons were monitored per each condition. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p<0.05, **p<0.01. Dynein inhibitor and Dynasore treatments were used as a negative control. (F-G) Representative images and quantification of C9orf72 iPSC-derived MNs in the proximal compartment of an MFC before and after Sema3A treatment with and without 10 ug peptides 1-4. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. 3 independent chambers in each condition were analyzed. ~200 neurons per condition monitored. Scale bar: 40µm. One-way ANOVA Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, **p =0.004.
CoQ biosynthetic gene expression.Figure 4A-E mRNA expression levels of Coq9 (A), Coq7 (B), Adck3 (C), Coq5 (D) and Coq6 (E) on cerebrum of Coq9+/+, Coq9Q95X and Coq9Q95X mice at 3 months of age. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.F-J mRNA expression levels of Coq9 (F), Coq7 (G), Adck3 (H), Coq5 (I) and Coq6 (J) on kidney of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.K-O mRNA expression levels of Coq9 (K), Coq7 (L), Adck3 (M), Coq5 (N) and Coq6 (O) on triceps surae of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. *P < 0.05; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice.
Ubiquitination is required for Fpn‐mediated ferritin degradation.Figure 3(A) FM3A (black) and ts85 (grey) cells were transiently transfected with a plasmid containing CMV‐regulated Fpn‐GFP and incubated in the presence of FAC (10 μM Fe) and 0.5 μM hepcidin for 24 h. Hepcidin was either maintained (+) or removed (−) to allow Fpn‐GFP localization at the plasma membrane. Cells were maintained at the permissive temperature (33°C) or moved to the restrictive temperature (39°C) and incubated for 6 h. Cells were harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. (B) FM3A and ts85 cells were treated as in (A) but FAC was replaced with 1.0 × 10−7 M Tf(59Fe)2. Ferritin was immunoprecipitated and the specific activity of 59Fe‐ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate. (C) ts85 cells were treated as in (B) and cell extracts applied to size exclusion chromatography. Ferritin content in selected fractions was measured by ELISA. Black bars represent assembled ferritin (>400 kDa), gray bars represent monomeric (50 kDa). (D) FM3A and ts85 cells were treated as in (A) but cells that had been incubated at the restrictive temperature (39°C) were then returned to the permissive temperature (33°C) and incubated in the presence of cycloheximide (75 μg/ml) and 10 μM FAC for 1 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.
Figure 2A. Bar diagram shows the initial rate of GPX-like activity of Vs under different assay conditions. B. Dependence of the initial reaction rate for H2O2 reduction on varying concentration of the catalyst, Vs. C. Michaelis-Menten plot with the variation of H2O2 (0-400 µM) in the presence of Vs (20 ng/µL), GSH (2 mM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C. D. Michaelis-Menten plot with the variation of GSH (0-7 mM) in the presence of Vs (20 ng/μL), H2O2 (200 µM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C. E, F. Lineweaver-Burk plot with varying concentration of H2O2 (E) and GSH (F) in presence of Vs nanozyme, respectively. The concentration of NADPH was constant (0.2 mM) in all the assay conditions. G. Surface area measurement by N2 adsorption, desorption isotherm and distribution of pore size (Inset). The measured surface area of Vs was 13.3 m2/g. H. Enzyme kinetic parameters and BET surface area values of different forms of V2O5 nanomaterials # reported from our previous literature (Ghosh et al., 2018).
The C-terminus of Trs85 binds to the TRAPP core Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex.Figure 4Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.   As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.   As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.   Network map showing the relative positions of cross-links between the C-terminus of Trs85 and the core subunits Trs31 and Trs20. View of the cryo-EM structural model showing the interaction surface between Trs85, Trs31 and Trs20. Inset depicts several basic residues from Trs85 that are in close proximity to several acidic residues of Trs20. Yeast complementation assays performed in a sensitized strain (BET3-GFP::HIS3, trs85∆::KANMX) to assess the functionality of Trs85 mutants within the region shown in E. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector) Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector) Live cell fluorescence microscopy of Trs85-mNeonGreen and mRFPmars-Ypt1. Localization of Rab1/Ypt1 to punctate structures is indicative of its activation by TRAPPIII. Scale bar is 2 microns. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)