EMBO/sd-character-level-ner huggingface dataset:
Figure 6A Diagram of the promoter structures of SAUR-AC and IAA19. Arrows indicate the primer positions for ChIP-qPCR. E-box, CACATG. G-box, CACGTG. B ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of BZR1/PIF4 target genes. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays. C ChIP-qPCR analysis showing that the association of BIC1 with the promoter regions of BZR1/PIF4 target genes is dependent on PIFs. The 6-d-old 35S:BIC1-Flag and 35S:BIC1-Flag/pifQ seedlings were used for ChIP assays. D ChIP-qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs. The 10-d-old 35S:BZR1-MYC and 35S:BZR1-MYC/bic1bic2 seedlings were treated with 1 μM epibrassinolide (eBL) for 3 h and then collected for ChIP assays. E ChIP-qPCR analysis showing that the enrichment of PIF4 in the promoter regions of its target genes is dependent on BICs. The 6-d-old 35S:PIF4-MYC and 35S:PIF4-MYC/bic1bic2 seedlings were used for ChIP assays.
Figure 1A. Schematic representation of RNAi screening strategy. B. Detection of ALDHbr cells by high content screening. The algorithm first detect nuclear region of interest (ROI) in the Hoechst channel (rainbow areas). Then, it computes the averaged ALDH signal in the ROI to identify ALDHbr cells (red nuclear ROIs) and calculate their proportion. C-D. Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death (cell viability>20%, B-score>│2.58│). Data represent mean ±SD (n=3). E. Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. ALDHbr cells are labelled in red. F. Volcano plot of the hits. Scatter points represent genes, the x-axis is the log2 fold change of the proportion of ALDHbr cells compared to the control, and the y-axis is the B-score. Genes targeted by a siRNA inducing a significant reduction (green dots; B-score>2.58, log2 FC<1) or increase (red dots; B-score>2.58, log2 FC<1) of the ALDHbr cell proportion are highlighted.
Upregulation of endosialin (EN) expression by activated hepatic stellate cells (HSC) in early liver fibrogenesis.Figure 1A EN qRT-PCR of murine liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), hepatocytes (Hep), and HSC (n = 3 per cell type). B EN qRT-PCR of human hepatocytes (n = 1) and HSC cell line LX-2 (n = 3). C EN immunofluorescence staining of LX-2 cells. Scale bar: 25 μm. D EN qRT-PCR of normal liver (NL) and fibrotic liver (FL,n = 8 each), and active or inactive cirrhotic liver (CL+/−, n = 10/ n = 2). E-H EN immunohistochemistry staining of human normal liver (E), fibrotic liver (F), and cirrhotic liver (G, H). Weak EN expression in portal tracts of normal liver (E). Abundant EN expression detected in activated HSC in fibrous septa of fibrotic (F), active cirrhotic (G), and inactive cirrhotic liver (H). Scale bars: 500 μm.I-N Higher magnification of EN-stained normal liver (I), fibrotic liver (K), and (active) cirrhotic liver (M) compared to alpha-smooth muscle actin (αSMA) immunohistochemistry on serial slides (J, L and N). Scale bars: 100 μm. O EN/αSMA mRNA ratio in normal liver, fibrotic liver, and active cirrhotic liver. P Collagen 1a (Col1a) expression in normal, fibrotic, and active cirrhotic liver determined by qRT-PCR.
Figure 5SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001. The mouse breast cancer cell lines 4T1 and 4T1-V5-SLC1A3 were orthotopically implanted into the mammary glands of pretreated NSG mice. Presented is the volume measurements of arising tumors at day 9. (n=13 mice for each group, except for 4T1+ASNase, n=12). Data are presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001. tumors were surgically removed once reached a volume of ~500mm3 and collected and subjected to LC-MS to determine the levels of asparagine, aspartate, glutamine and glutamate. Leucine level was used as a control. Results are based on five tumor samples and presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001. The human breast cancer cell lines MDA-MB-231 and MDA-MB-231-V5-SLC1A3 cells were intravenously injected into pretreated NSG mice. Once mice showed breathing problems, they were sacrificed, and lung and liver were collected and blindly scored for metastasis lesions. The p-value was calculated by one-tailed unpaired t test in Prism7. Data are presented as mean ± SEM (n=8). Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001. A schematic model depicting how SLC1A3-mediated aspartate and glutamate uptake promotes ASNase resistance. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.
Fig.Figure 6Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trehalose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations fromcounting of the three grids. ***P < 0.001 (Student's t-test). SD-N,nitrogen-starvation medium; WT, wild type YPD, complete medium.
Long-term loss of Miro1 in mature neurons leads to the initiation of the integrated stress response.Figure 6(A) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001. (C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test). (E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria. (F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).
In vitro uptake is independent of components involved in homotypic vacuolar fusion.Figure 6In vitro uptake is independent of components involved in homotypic vacuolar fusion. (A) Standard uptake reactions were performed (60 min, 27°C) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734-16 (Δpep4) and DBY5734-19 (Δpho8). Therefore, homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample. Four independent experiments were averaged. Error bars indicate SD. For averaging, uptake and fusion activities of the control sample without inhibitor were set at 100%. Inhibitors were used at the following concentrations: anti-Sec17p, 1 μM; anti-Sec18p, 1 μM; Gdi1p, 2.5 μM; anti-Vam3p, 1 μM; anti-Vam7p, 1 μM; and anti-Nyv1p, 1 μM. (B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.
CRY-independent circadian timekeeping occurs cell-autonomously(A) Representative double-plotted actograms showing wheel-running activity of wild type (WT) and CRY-deficient (CRY Knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness.Figure 1(A) Representative double-plotted actograms showing wheel-running activity of wild type (WT) and CRY-deficient (CRY Knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness. Note the 48 hour x-axis for WT versus 32 hour for CKO. (B) Mean period and amplitude (±SEM) of mouse behavioural data (n=4). (C) Longitudinal bioluminescence recordings of organotypic SCN slices from WT (black) and CKO (red) PER2::LUC mice (RLU; relative light units). (D) Mean period and amplitude (±SEM) of rhythmic SCN bioluminescence traces. (E) Circadian PER2::LUC expression in immortalised WT and CKO adult lung fibroblasts. Left panel shows two raw traces of a representative longitudinal bioluminescence recording, right panel shows same data detrended with a 24-hour moving average to remove differences in baseline expression. (F) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments (n≥3 per experiment, individual values ±SEM shown). P-values were calculated by an unpaired t test with Welch correction. Standard deviations differ significantly between WT and CKO (F test: p <0.0001).
Figure 4A Scheme of macroH2A and H2A variants constructs used in the study: full-length wild-type (WT), constructs with deleted macrodomain (ΔM), constructs with deleted linker and macrodomain (ΔLM), or hybrids consisting of the H2A histone fold fused to the linker of macroH2A1.1 (H2A + L) or the H2A fold fused to the linker and macrodomain of macroH2A1.1 (H2A + LM). Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant). Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).
Figure 4Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP-labeled WT-mDASCs (WT-Lung) or LL-37-mDASCs (LL-37-Lung) on indicated days. Scale bar, 1000 μm Engraftment ratio of transplanted cells in mouse lungs on indicated days. n=3. Error bars, S.E.M. *P=0.0474. Morphology of engrafted GFP-labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm. Anti-Ki67 immunofluorescence of engrafted GFP-labeled WT- and LL-37-mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.
Figure 5(A) An assembly of IgG (first row), DVL3 (second row) and KMT2D (third row) ChIP-Seq data in MCF7 for the HAUS5, WDR74, YWHAQ, CDNF/HSPA14 and PRDM10 genes, visualized by IGV. The bar below the DVL3 peak is the location of the statistically-qualified peak of KMT2D in MCF7. (B) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and KMT2D ChIP-Seq. (C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3). (D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468. (E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively. (F) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H3K27ac ChIP-Seq. (G) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H3K27ac ChIP-Seq. (H)Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H4K8ac ChIP-Seq (I) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H4K8ac ChIP-Seq. (J) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and super-enhancer targets in basal breast carcinoma (BRCA) (K) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and super-enhancer targets in luminal breast carcinoma.
Figure 2Pedigrees of the two families, indicating autosomal recessive inheritance. The genotype of the VPS16 c.2272-18 position is indicated. ?, not available for testing. (A) Family of patient A. The three sisters are healthy, but an older brother had previously died at 11 months from suspected heart failure after showing signs of developmental delay from an age of 3 months of age. (B) Family of patient B. The mother previously had two spontaneous miscarriages but the two sisters are healthy. Position of the intronic c.2272-18 variant in the VPS16 gene. cDNA analysis by Sanger sequencing of leukocyte RNA from patient A and a control sample. Reads beyond the alternatively spliced breakpoint yield double reads in patient samples, corresponding to the predicted mis-splicing and wildtype sequences.
Perturbing nuclear actin dynamics inhibits DNA replication in human somatic cells.Figure 1
Figure 3(a-c) Slices through Volta phase plate cryo-tomograms of the neck region in intact pig (a), horse (b), and mouse (c) sperm. Proximal centriole triplets are shown in green, distal centriole doublets in blue (A-tubule in light blue, (d-f) Three-dimensional architecture of the flagellar base, with the connecting piece in grey, the proximal centriole in green, distal centriole doublets in blue (A-tubule in light blue, B-tubule in dark blue) and singlets in pink, and electron-dense bars in yellow. The connecting piece was segmented semi-automatically with a neural network, while microtubules were traced manually.
Fig.Figure 2Allele frequencies (AF) of mutations detected in primary (PRI) patients' samples (x-axis) at initial diagnosis (INI) and relapse (REL) plotted against AF of corresponding mutations detected in matched PDX models (y-axis) for relapse type 1 (left) and relapse type 2 patients (right); black - mutations preserved between primary and PDX, blue - mutations, which were selected for/against in PDX models, green - relapse specific mutations selected for in INIPDX, absent or detected with a very low AF in primary patients' diagnosis sample, orange - subclonal mutations lost in PDX, red - clonal mutations lost in PDX, grey - mutations, which AF was affected by low blast content (BC) at relapse Allele frequencies of all mutations preserved between primary patients' samples (x-axis) and PDX samples (y-axis); PCC- Pearson's correlation coefficient
Figure 3(A) A principal component analysis (PCA) using the ATAC-seq data of HSCs in the steady state (untreated), early phase (5-FU 6d), or late phase (5-FU 10d). (B and C) A scatter plot comparing ATAC-seq peaks between untreated HSCs and those in the early phase (B) or between those in the early and late phases (C). (D) ATAC peaks overlapping with H3K27ac peaks detected in HSCs in each phase or CD48+ progenitor cells in the late phase after the administration of 5-FU were selected as cis-regulatory regions. (E) Scatter plots comparing peaks within cis-regulatory regions in L-ESLAM HSCs between the early and late phases after the administration of 5-FU. (F) The enrichment of genes related to "accessibility-increased regions at the late phase (Brown dot in Fig 3E)" (left) or "accessibility-decreased regions (Green dot in Fig 3E)" (right) within genes up-regulated in CD150+CD34- HSCs or CD150+CD34+ progenitor cells. (G) A scatter plot comparing the peaks of cis-regulatory regions between the steady state and early phase after the administration of 5-FU. (H) The enrichment of genes related to "accessibility-increased regions from the steady state to early phase (Purple dot in Fig 3G)" in the comparison between the steady state and early phase after the administration of 5-FU. (I) The enrichment of genes related to "accessibility-increased regions from the early to late phase (Brown dot in Fig 3E)" in the comparison between the early and late phases after the administration of 5-FU. (J) ATAC and H3K27ac peaks in the promoter and enhancer regions within the CD48 locus. ChIP-seq data for transcription factors obtained from the public database (GSE22178) were merged. (K) CD48 mRNA levels in HSCs after the administration of 5-FU. Data are shown as means ± S.D. (n=4, two independent experiments, N.S.; not significant by the t-test). (L) Average plot (top) and heatmap (bottom) of H3K27ac reads in HSCs in the early and late phases within accessibility-decreased (left: Black dot in Fig 3H), -unchanged (center: Blue dot in Fig 3H), and -increased cis-regulatory regions (right; Purple dot in Fig 3H) in the early phase. (M) Average plot (top) and heatmap (bottom) of H3K27ac reads in HSCs in the early and late phases within accessibility-decreased (left: Green dot in Fig 3E), -unchanged (center: Blue dot in Fig 3E), or -increased regions (right: Brown dot in Fig 3E) in the late phase.
Figure 3(A) Location of RAG1 M847 and M848 (surface mode, green) in the RAG STC. (B) Diagram of DNA sequence surrounding integration site with colors specifying relevant bases as in (C-F). (C,D) Local structures of top and front views of Paired RAG STC near the 12RSS integration site. (E,F) Local structures of top and front views of Dynamic RAG STC near the 12RSS integration site.
Increasing GABA concentration further increases Tor activity and inhibits autophagyWT cells were cultured under autophagy conditions with or without GABA for 6 h.Figure 5A S6 phosphorylation after 6 h in SD‐N was analyzed by immunoblotting with a loading control. B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.
Figure 1A) Schematic diagram of the functional regions of chicken and human CENP-C molecules. The canonical CENPC motifs for CENP-A binding (aa 655-675 in chicken CNEP-C, aa 738-758 in human CENP-C) are colored pink. Another CENP-A binding region (central domain) in human CENP-C is colored light pink. The C-terminal fragment (aa 601-864: CENPC-CT) and the CENPC motif-containing peptide (aa 643-683; CM peptide) derived from chicken CNEP-C, which were used for the in vitro CENP-A nucleosome binding assay, are diagrammed. B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA. C) Cryo-EM density map of the CENP-A nucleosome in complex with CENPC-CT (CA-CCCT complex) at a 4.5 Å resolution. The side views of the CA-CCCT complex along the two-fold axis are shown. The density corresponding to each molecule in the complex is color-coded, as indicated in the figure. D) Cryo-EM structure of the CENP-A nucleosome binding region of CENPC-CT. The left panel shows the cartoon representation of the structure of CENPC-CT bound to the CENP-A nucleosome with the cryo-EM density map. The densities derived from CENPC-CT and CENP-A molecules are color-coded in magenta and pale green, respectively. The functional elements in the CENP-A nucleosome binding region of CENPC-CT are depicted in the right panel. In addition to the CENPC motif, which recognizes the H2A/H2B acidic patch and the CENP-A C-terminal tail (C-tail), the CM upstream and CM downstream regions are identified in the CENP-A bound structure of CENPC-CT.
NOD2 accounts for intracellular pathogen sensing receptors in human alveolar macrophages.Figure 1(A) Intracellular detection of NOD2 (bold line in the histogram plot) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar cells (R1, dot plot). One representative experiment out of three experiments is presented. (B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported. (C) The upregulation of NOD2 gene expression was assessed by quantitative PCR. The results are depicted as the mean fold change in NOD2 gene expression relative to unstimulated cells (n = 11). (D) Subcellular localization of NOD2 was ascertained in unstimulated (left, ×80,000) and MDP‐stimulated cells (right, ×63,000) using an anti‐NOD2 antibody and detected with a secondary antibody coupled to 5‐nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.
Figure 6(A) AP illustrating the analysed parameters (B) Representative APs from control (CTRL), heterozygous (HET) and homozygous (HOM) CMs. (C) RMP, dV/dtmax , APAmax, APAplat, APD20, APD50 and APD90 of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs.
Figure 2Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from human neurons (A) Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from HEK293 cells expressing GFP-Aβ or GFP (B). C Effect of an RNase A inhibitor (RNasin) on RNase A mediated protein aggregation in neuronal cell lysate.