EMBO/sd-character-level-ner huggingface dataset:
Figure 4Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10. De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D). H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic. ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H. Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR.
Figure 4A. Outline of experimental set up to monitor DNMT1 DPC levels in S phase and mitotic cells. B. HeLa cells released from double thymidine block in early S phase were pulse-labeled for 30 min or not with 5-azadC in the presence of SUMOi in late S phase. Following 5-azadC removal, cells were incubated with SUMOi and nocodazole, and mitotic cells were collected by shake-off, as outlined in (A). Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies. C. U2OS cells transfected with indicated siRNAs and then treated as in (B) were collected in late S phase or mitosis (M), subjected to stringent pre-extraction and immunostained with DNMT1 antibody. DNMT1 foci formation was quantified by QIBC analysis (red bars, mean; >7400 cells analyzed per condition). Data are representative of three independent experiments. D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated. E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated. F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies. G. HeLa cells transfected or not with indicated siRNAs were synchronized in early S phase by double thymidine block, released and pulse-labeled 5,5 h later with 5-azadC for 30 min in the presence or absence of SUMOi. Following 5-azadC removal, cells were subjected to live-cell imaging analysis, and the duration from late S phase to mitotic entry (nuclear envelope breakdown (NEBD)) was quantified (red bars, median; at least 83 cells, pooled from three independent experiments, were analyzed per condition; **p<0.01, ns: not significant, Mann-Whitney test). H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.
Figure 8Wet-spun yarn containing human myogenic precursors showed progressive cell differentiation over time (arrows in day 3); dashed box enlargement reveals still undifferentiated cells after 15 days of culture (Upper panel). Middle and lower panel showing MHC immunostaining (red) performed on microfiber yarns and bulk structure loaded with human myoblasts upon 15 days of culture, revealing a significant difference in terms of myotube organization between the two hydrogel systems, with the wet-spun constructs greatly outperforming the bulk gels. Such difference is quantified in the polar plots. Nuclei were counterstained by DAPI (Blue).
Figure 4(A) BRD4 occupancy (left panels) and H3K27ac levels (right panels) at regulatory regions associated with ERS UP or LIVER-ID genes (8 regions at ERS gene loci and 10 regions at LIVER-ID gene loci were assessed by ChIP-qPCR in MPH (10 independent experiments) or mouse liver (10 mice per group) to define changes induced by acute ERS. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. (B) Left panel, BRD4 mRNA (4 independent experiments) or protein expression levels (5 independent experiments; densitometric quantification in MPH subjected to acute ERS. The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance. Right panel, Total protein extracts from MPH pre-treated for 3h with 0.01µM MZ1 followed by addition of 1µM thapsigargin (ERS) for 4h were subjected to Western blot with an antibody against BRD4. LMNA was used as loading control. (C) Heatmaps showing Log2 FC (relative to the DMSO/Control condition) for 5 ERS UP and LIVER-ID genes issued from RT-qPCR analyses of MPH pre-treated with 500nM JQ1 (left) or 0.01µM MZ1 (right) followed by addition of 1µM thapsigargin (ERS) for 4h (3 to 6 independent experiments). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance. (D) Heatmaps showing average H3K27ac ChIP-seq signals in MPH at LIVER-ID domains overlapping (+) or not (-) with BRD4 SE. The arrow indicates the position of gene transcriptional start sites. (E) Box plots showing Log2 FC ERS/Control in MPH (3 independent experiments) (left panel) or mouse liver (3 mice per group) (right panel) for genes associated with LIVER-ID + BRD4 SE or LIVER-ID - BRD4 SE Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Student's t-test was used to assess statistical significance, *P < 0.05.
Figure 2A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK1; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control. C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes. D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures. E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).
Mec1-S1991 phosphorylation participates to RNAPII degradation on HU.Figure 5(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin. (E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.
Figure 7Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm. Quantitative assessment of clCasp3+ cells in the 3D cultures. N = 4. Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm. Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4. Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm. Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.
Irgm2 regulates GBP-independent caspase-11 targeting to Gram-negative bacteria.Figure 2(A) Measure of LDH and IL-1β release in WT, GBPChr3-/- and Casp11-/- BMDMs were Irgm2 was knocked down 16 hours after exposure to 2,5 µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences. (B) Cell death (LDH) and IL-1β release evaluation in Irgm2-silenced WT and GBPChr3-/- BMDMs infected for 16 hours with either S.Tm orgA- or F. novicida (MOI 25). Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences. (C) Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA--mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2-/-BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n=3 independent experiments; scale bars 5 µm. For quantifications, the percentage of GBP-associated bacteria was quantified and 'n=' refers to the number of intracellular bacteria counted in each experiment; quantifications from n=3 independent experiments were then plotted and expressed as mean ± SEM. (D) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.
Figure 7A Sections of 1205Lu xenografts from Fig. 6B were stained with picrosirius red and imaged under transmission light (upper panels) or polarized light (middle panels) (scale bar, 500 µm) or imaged by second harmonic generation (SHG) microscopy (lower panels) (scale bar, 50 µm) to examine collagen fiber network upon the mono or combined regimens. B Quantification of collagen maturity and fiber thickness in 1205Lu xenografts stained with picrosirius red using polarized light microscopy. Birefringence hue and amount of collagen fibers were quantified as a percent of total tissue area (2 to 4 fields per tumor section, n = 4 tumors per condition). C Quantification of collagen fibers using SHG microscopy in tumor sections from (A). Error bars represent mean ± s.d. of 4 independent fields per section, n=2 tumors per condition. ***P=0.0002, Mann-Whitney test.
Fpn‐mediated decrease in ferritin is not prevented by chloroquine or leupeptin.Figure 1(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments. (B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.
RIPK3 amyloid fibril structure A Structure of the RIPK1:RIPK3 RHIM core, prepared using PyMol Graphics System from PDB 5V7Z.Figure 7Structure of the RIPK1:RIPK3 RHIM core, prepared using PyMol Graphics System from PDB 5V7Z. The RIPK3 sequence is coloured in blue and RIPK1 is coloured in green. Tetrad residue side chains are shown as sticks; Tyr, Asn/Gln, Ser and Cys side chains are shown in wheat, red, grey and yellow, respectively. Core tetrad interactions across the opposing β‑sheets are indicated by the dotted box Model of M45:RIPK3 RHIM core, prepared by appropriate mutation of RIPK1. The M45 sequence is coloured in cyan and RIPK3 in blue. Tetrad residue side chains are shown as sticks; Tyr, Asn/Gln, Val and Cys side chains are shown in wheat, red, grey and yellow, respectively. Core tetrad interactions across the opposing sheets are indicated by the dotted box Schematic representation of RHIM fibrils formed by full-length proteins with partner domains (e.g. kinase or RNR, coloured green) flanking either side of the amyloid core formed by RHIMs (coloured grey) Confocal microscopy image of a single fibril composed of YPet-M451-90
Figure 6A, Representative time-lapse sequence of GFP-Fbl1 in P19 cells expressing the plasmids indicated defining t=0 when the chromatids separate (anaphase). B-C, Analysis of the number (B) and the total area (C) of the GFP-Fbl1 foci in P19 cells expressing the constructs indicated for a period of 360 minutes. Band width represents SEM and grey areas represent time points with significant differences between the control and the Trnp1 expressing conditions. The ∆1-16Trnp1 expression does not show any statistical difference compared to the control condition. D, Representative time-lapse sequence including one mitosis of H2B-Venus in P19 cell expressing the indicated constructs. E, Volcano Plot illustrating the length of M-phase from condensation to de-condensation of chromosomes in minutes. Each dot represents an individual cell from 3 different biological replicates. GFP=61 cells, Trnp1=62 cells and ∆1-16Trnp1: 40 cells. F, Bar graph showing the percentage of cells expressing Trnp1-IRES-GFP or GFP as control in each phase of the mitosis indicated in different color as indicated at different times after release from synchronization in G2. 100 cells per condition and time were analyzed. G, Bar and dot plots depicting the proportion of GFP+ NSCs (Pax6+) in mitosis (pHH3+) showing a significant decrease 24h after Trnp1 compared to control IUE. N= 4 brains from 2-3 different litters.
Figure 4A, B Intersection of the top 200 differentially expressed genes (sorted by statistical significance) in tumourspheres and tumours (Nfib high-expression vs. the respective controls) and putative transcriptional targets of Nfib determined by ChIP-seq in mammary gland (Shin et al, 2016) (A) and epithelial-melanocyte stem cells (Chang et al, 2013) (B) (association rule: Basal+extension: 5000 bp upstream, 1000 bp downstream, 10000 bp max extension, curated regulatory domains included). D Kaplan-Meier survival analysis of NSG mice inoculated i.v. with SUM159PT gNFIB shCTRL (n = 9), SUM159PT gNFIB sh1 ERO1A (n = 7), or SUM159PT gNFIB sh2 ERO1A (n = 7) cells. Two-tailed log-rank test. E Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 8 (SUM159PT gNFIB shCTRL) and 20 (SUM159PT gNFIB sh1 ERO1A or SUM159PT gNFIB sh2 ERO1A) days after i.v. injection of the respective cells. n = 4, means ± s.d., two-tailed Student's t-test. F Kaplan-Meier survival analysis of overall survival of mice injected i.v. with SUM159PT gNFIB shCTRL (n = 10), SUM159PT gNFIB sh1 ERO1A (n = 12), or SUM159PT gNFIB sh2 ERO1A (n = 13) cells, two-tailed log-rank test.
Figure 5A, B Western blot of whole-cell (WCL) and P-body (PB) purified from PC3-LN4 cells stably expressing GFP-DCP1a used as a P-body tag. The P-bodies (PB) were isolated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. C Immunofluorescence (IF) of PC3LN4 cells under normal culture condition with anti-phopsho-EDC3, anti-EDC3 Abs, and DAPI. Images captured under 40x magnification; scale bar: 10µm. D Immunofluorescence of PC3-LN4-EDC3 S161A (SA) cells under normal culture condition with anti-DCP1a and anti-EDC3 Abs as well as DAPI. Images captured under 40x magnification; scale bar: 10µm.
Figure 4(A) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment. Scale bar 5 µm. (B) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP, RPA1 and RAD51. H2B is shown as a loading control. CHD1 depletion leads to decreased (C) CtIP and (D) RPA1 recruitment to I-SceI-induced DSB in U2OS19 ptight13 GFP-LacR cells. CHD1 was depleted in U2OS19 ptight13 GFP-LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co-immunostained for γH2AX and CtIP or RPA1. The number of cells with CtIP or RPA1 foci from (C) and (D) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD (n=3), more than 50 cells were counted. Scale bar 5 µm. (E) PC3 cells were transfected either with mock or siCHD1 followed by empty vector, wt mChd1 (Wt) or ATPase-mutant (Mt) mChd1. After 48 h of transfection cells were treated with of 4-OH tamoxifen (4-OHT) for 24 h and processed for PLA with γH2AX and CtIP antibodies. Cells demonstrating positive focal interactions indicative of DNA repair hubs (punctate staining) are present only in control and (Wt) CHD1-rescued cells but not following knockdown or reconstitution of an ATPase-mutated CHD1 (Mt). Scale bar 20 µm. (F) PC3 cells which stably express HA-mChd1-ERT2, were transfected with either mock or siCHD1. After 24 h of transfection cells were treated with 4-OHT for 24 h to induce HA-mChd1-ERT2 nuclear translocation. Western blot analysis of chromatin fractions for CHD1, ERT2 (mChd1), CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ. The relative quantification for the indicated proteins is shown in the graph. See also Figure EV4A-EV4P.
Figure 2(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue. (B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores. (C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.
Figure 1(A) Molecular structure of CNP520 (B) X-ray structure of CNP520 bound to the BACE-1 active site (resolution: 1.35 Å), with binding pockets S1 (filled by P1) and S3 (filled by P3) and catalytic residues Asp32 and Asp228. (C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point. (D) Structure of the aminopyridine generated by metabolic cleavage of the amide bond in CNP520.
Figure 7Working model summarizing the results obtained in this work. The blue arrows indicate wild type situation, red arrows/text indicate the changes in cystinotic cells and green arrows indicate intervention with medication. DMKG; dimethyl α-ketoglutarate, ROS; Reactive oxygen species.
Figure 5(A) Inhibition of SARS-CoV-2 infection by ACE2-Fc in the plaque assay. Mixtures of ACE2-Fc and SARS-CoV-2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37°C. The ACE2-Fc and SARS-CoV-2 premixtures were removed, and the cells were washed once with PBS and overlaid with methylcellulose with 2% FBS for 5-7 days before being stained with crystal violet. Those results showed increase of plaque formation was regarded as no inhibition of plaque formation. TPCK-treated trypsin: N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin. Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on virus titers The culture medium and cell extracts were harvested 24 hours post-infection for real-time PCR (B) Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on protein expression. The culture medium and cell extracts were harvested 24 hours post-infection for western blot (C). The NP/PCNA represents the relative NP expression as compared to that of PCNA, which served as a loading control. The NP/PCNA numbers below the panel are the ratios of NP/PCNA normalized to that of the human IgG control group. PCNA: Proliferating cell nuclear antigen. (D) Schematic illustration of the ACE2-Fc pretreatment and full-time experiment procedure, delineating the stages where the ACE2-Fc was present during the experiment. (E) The cell lysates from the pretreatment and full-time experiments were harvested and immunoblotted with the indicated antibodies. (F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.