EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Inhibition of cell-cell fusion by mAbs. Cell fusion was quantified by measuring the luciferase expression induced by full cell membrane fusion and mixing of cytoplasm, and the rate of inhibition by indicated antibodies compared to the condition without antibody is reported. Data depict mean ± s.d. (n = 4 biological replicates). (B) Inhibition of a post-binding step by FD20, but not by other mAbs. Antibodies were added to VeroE6-hACE2 cells during (filled) or after (open), the binding step of SARS-CoV-2 pp. Mean ± s.d. are plotted (n = 3 biological replicates). As control, cells were incubated at each step with an equal volume of PBS or Y112R. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). (C) FD20 pre-incubation with SARS-CoV-2 pp in the absence of cells strongly impairs entry. To test direct action on particles, SARS-CoV-2 pp and FD20 were co-incubated for 1 h, then the premixes were used to infect VeroE6-hACE2 cells for 6 h at 37 °C. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate the gained neutralization (%) by pre-incubation. As control, cells were incubated at each step with an equal volume of PBS or Y112R. Percentages of primary infection were calculated according to viral titers of PBS control conditions. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). ns, not significant. (D) Inhibition of cell surface binding of SARS-CoV-2 VLP by FD20. VLP-GFP were preincubated, or not, with FD20 and RBM-targeting mAbs (CB6, REGN10933, REGN10987) for 30 min at 37 °C. Then the VLP premixes were incubated with VeroE6-hACE2 for 1 h at 37 °C and the binding detected by flow cytometry. Control binding assays were performed using soluble ACE2 as a competitor. Data are representatives from three experiments.
Fig.Figure 5(A) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids. (B) Spearman's rho for circadian clock and clock-associated genes. (C) 8 conserved rhythmic signaling pathways identified by phase set enrichment analysis (PSEA).
Figure 7Average fractions of disordered residues in the skipped exons in each RBP-depleted RNA-seq dataset. SRSF3-depleted datasets are indicated in red. The q-value between SRSF3-depleted datasets and other datasets was calculated according to the gene set enrichment analysis (GSEA). Average fractions of disordered residues in S3-LCEs were included in the indicated categories. The genes containing S3-LCEs are classified into 3 groups according to the AnimalTFDB (Hu et al., 2019). The fraction in other RBP-dependent exons (gray box) is shown for comparison. ****p < 0.0001 by Steel-Dwass test. The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 6,005 for "Other RBP-dependent exons", n = 1,883 for "Others", n = 238 for "Transcription factor", and n = 251 for "Transcriptional coactivator"). Frequencies of all possible dipeptide sequences in S3-LCEs. Fold changes and P-values are shown as in Fig 1E. The color code indicates the number of P or S in a dipeptide. Droplet formation of mCherry-fused recombinant proteins of the S3-LCEs. Representative images are shown below the schematic of the mCherry fusion protein. Detailed information about the indicated S3-LCEs is shown in Fig EV4B. The recombinant proteins (20 nM) were incubated with 75 mM NaCl in droplet formation buffer. Temporal profiles of the MTS assay representing the number of C2C12 cells after transfection with the indicated siRNA. The targeted siRNA sites were different between siRNA sets A and B (Fig EV2O). Relative cell number was normalized to that of control cells (siCont) at 3 h. Means and SD are indicated (n = 3 wells each). Data were analyzed with two-way ANOVA and Tukey's post-hoc test (**p < 0.01, ****p < 0.0001 compared to siSrsf3).
Figure 5A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above. D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.
Figure 2(A) 2.6 Å cryo-EM map of RAG STC. Protein tetramer and DNA chains are segmented and color-coded as indicated. DNA in red, 12RSS with its covalently linked target DNA; DNA in yellow, 23RSS with its covalently linked target DNA (B) Orthogonal views of the STC DNA density map with RAG protein rendered transparent. Red and yellow, 12RSS and 23RSS and covalently-linked target site and flank DNA, respectively. (C) Diagram of DNA sequence surrounding sites of integration. (D,E) Front view (defined based on view in (B)) of local maps of target site DNA in Paired STC (left) and Dynamic STC (right) derived from sub-classification of the 2.6 Å cryo-EM map (top panel), and top views of the atomic models built from the two maps (bottom panel). (F) Atomic model of Paired RAG STC. One RAG1-RAG2 dimer omitted to allow visualization of target site DNA. Cyan arrow, integration site. (G) Superimposition of Paired RAG STC with the HFC (hairpin-forming complex) (left) and cartoon model of DNA from RAG HFC (left) and Paired STC (right). Orange, target site DNA.
Figure 6WB analysis performed in TA and VM muscles of mdx mice treated with anti-IGF2R using antibodies against CD20, pSer+pThr and vinculin. Densitometry analysis of data expressed as the pSer+pThr/CD20 and CD20/vinculin ratio in arbitrary units. One-way ANOVA *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group)(a). Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti-IGF2R using anti-IGF1R, anti-pospho-IGF1R and GAPDH. Densitometry analysis of data expressed as the pIGF1R/IGF1R ratio normalized to GAPDH content in arbitrary units. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group) (b). All values are expressed as the mean±SEM.
a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S.f2a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection. c, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets. d, Quantification of GBP-positive bacteria, LDH release and IL-1β secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means. e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium. f, g, Quantification of GBP-positive Salmonella in anti-IFN-γ-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 µm (c), 1 µm (e). Graphs show mean and 5-95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).
Aged lungs exhibit enhanced local adaptive immune responses despite circulatory deficiencies in the memory phase.Figure 2.phase.Young and aged mice were infected with PR8. A. Bulk RNAseq was performed on young or aged lungs at 60 d.p.i., data is displayed as a heatmap (purple = relatively low expression, orange = relatively high expression) of 658 differentially expressed genes (549 up-regulated in aged relative to young, 109 up-regulated in young relative to aged) with p < 0.001. B. GSEA for adaptive immune response genes with normalized enrichment score (NES) and false discovery rate (FDR). C. Ranked normalized geneset enrichment scores (ES) from the C5 and Hallmark (HM) databases and associated false-discovery rates (FDR (q)). D. Representative flow cytometry plot showing lung CD8+ T cells separated by CD69 and CD45 i.v. labeling, showing parenchymal (Res) and circulating (Cir) populations as indicated. E. Quantification of CD4+ (top panel) and CD8+ T cells (bottom panel) in lung circulation (Cir) or parenchyma (Par) 60 d.p.i. F. CD69+ parenchymal CD8+ T cells that either express CD103 (CD103+) or not (CD103-) were enumerated at 60 days post-infection. G. T (CD4+ & CD8+) and B lymphocytes (B220+) were quantitated in spleens at 60 d.p.i. (Spleen Ly #). H. Representative flow cytometry plots, and (I.) number of Db-NP (top) or Db-PA tetramer positive (bottom) circulating (Circ) or parenchymal (Res) memory CD8+ T cells.
Figure 5Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line. Proliferation as quantified by number of BrdU+ cells per unit in WT and Atf3−/−vehicle±HDT. Each data point = mean of counts from 40-50 gastric glands from an individual mouse, n=3-4 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation. Plot and statistical analysis as for Panel B but quantifying BrdU+ cells only in the bottom 1/3 of the unit, which are derived mostly from paligenosis and not from isthmal and neck stem and progenitor cells. Death of ZCs after injury plotted by quantifying the base dropout rate, which is the fraction of glands that do not reach the muscularis mucosa from the lumen. Each data point is the mean of 80-100 glands from an individual mouse, n=4 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation. Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation. Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.
Figure 1Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (A) ZIKV titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA. Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (B) IFNα is not highly induced in response to ZIKV infection in trophoblast cells. IFNα mRNA expression was inhibited in the first 24 h.p.i. and maintained at low level during ZIKV infection. Difference not significant (n.s.) by One-way ANOVA. Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA. Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. (D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.
Smooth muscle cells are the main source of VEGF-C in adult intestine.Figure 2Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm. Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm. Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm. Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm. Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.
Figure 6A For the image-based drug screening, Calu-1 cells were infected with SARS-CoV-2 in the presence of drugs with indicated doses for 48 h, immunostained, imaged and analyzed. The example images of mock-infected cells and virus-infected control cells (no drugs). The viral yield in cells was detected with an antibody for the viral N protein (virus-infected cells; upper image), and nuclear staining (mock-infected cells; lower image) (Scale bar: 200 μm). B The dose-response curves for six drugs with potential antiviral effects. The drug-induced inhibition of virus infection (count of N protein positive cells) was quantified using the area under the curve (AUC) values. C The effect of drugs on cell viability in the presence of the virus, e.g., cytotoxicity was evaluated by the count of nuclei. D Inhibition of interactions formed with DDX39B in the presence of MTX using co-IP assay. DDX39B is fused to the C terminus of a V5 tag, and the interaction partner is fused with a Strep-HA tag. The diagram (upper) shows the interaction pairs formed with DDX39B used for co-IP assay. Dot-blot results (lower) show the interaction pairs (arrangement as upper panel) in presence of different concentration of MTX as indicated on the top of the image. The image displayed is representative of 3 replicates (n = 3). E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.
ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Figure 5. ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell. Infected samples were collected as indicated above. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA391962; GEO: GSE100504).
Figure 3WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).   WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA). Schematics for the requirement of Stx16 and Stx17 in autophagic clearance of damaged mitochondria, surplus peroxisomes and invaded bacterial pathogens.
Diencephalic astrocyte subtypes share gene expression between ependyma and tanycytesA Violin plot showing scaled log normalized read counts of S100a6 expression for each cluster.Figure 3A Violin plot showing scaled log normalized read counts of S100a6 expression for each cluster. Color-coding corresponds to clusters In vivo validation of S100a6 expressing DIE astrocytes in Aldh1l1-eGFP mouse (3 mo) with immunolabelling for S100a6 (B: in the SEZ, LV: lateral ventricle, CC: corpus callosum. Scale bars: 200 µm , C In vivo validation of S100a6 expressing DIE astrocytes in Aldh1l1-eGFP mouse (3 mo) with immunolabelling for S100a6 C: in the DIE). Inset in left panel of C is depicted in middle and right panels. S100a6/GFP double positive cells are indicated by arrowheads. LV: lateral ventricle, CC: corpus callosum. Scale bars: 200 µm left panel C), 50 µm (middle and right panels C).
TAG .Figure 5(A, B) 25HC inhibits SARS-CoV-2 USA-WA1/2020 strain infection in Calu-3 cells. Cells were pretreated with ethanol or 25HC followed by infection. At 48h post-infection, RNA from supernatant (A) and infected cells (B) were extracted and quantified by qPCR. Data are mean ± SD of n = 3, representative of 3 repeats. (C) Progeny virus titer in supernatant was quantified by plaque assays. (D) Two-month-old differentiated lung organoids were pre-treated with 25HC (5µM) for 16h and infected with luciferase expressing SARS-CoV-2 pseudovirus at MOI =0.1 for 2 h and treated with fresh medium containing 5µM 25HC. Luciferase activity was measured at 24h post-infection.
Figure 3(A) THP1 cells were infected with HSV-1 KOS or an ICP27-deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity. (B) MDMs were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (C) THP1-derived cells deficient for cGAS or STING were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (D) HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2x104 cells per well). Reporter gene activity was measured in lysates isolated 24 h post transfection. (E) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity
Rescue of JAK‐STAT signaling leads to partial rescue of differentiation defect in GPCsWestern blot assaying level of pSMAD 5–6 h following BMP‐4 addition in control and Dgcr8Δ/Δ cells (n = 2).Figure 2Western blot assaying level of pSMAD 5-6 h following BMP‐4 addition in control and Dgcr8Δ/Δ cells (n = 2). Western blots and quantification (right panels, relative to actin) showing levels of pSTAT3 Y705 and total STAT3. Upper panel: Data for Dgcr8flox/Δ (control) and Dgcr8Δ/Δ cells 5-6 h following serum differentiation (n = 2). Light grey and dark grey bars represent the data for 2 experiments. Lower panel: Data for BakΔ/Δ;Baxflox/Δ;Dgcr8flox/Δ GPCs (control) and BakΔ/Δ;BaxΔ/Δ;Dgcr8Δ/Δ (TKO) cells during serum differentiation (n = 3). Quantification is shown on the right; error bars represent standard deviation. Representative images of GFAP immunofluorescence of Dgcr8Δ/Δ and BakΔ/Δ;BaxΔ/Δ;Dgcr8Δ/Δ cells and respective controls following 48 h of differentiation in the presence or absence of LIF (n = 3). Scale bars as shown.