EMBO/sd-character-level-ner huggingface dataset:
ECM triggers fibroblast state switching (A) Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days.Figure 2Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days. Cell number was measured by CellTiterGlo assay at indicated time points (n= 3 technical replicates of one representative experiment repeated 3 times). Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days (triangle). Controls were kept on TCP for 3 days before plating (circle). Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown (n= 3 technical replicates). Experimental design of de-epithelialized dermal (DED) organotypic cultures for reconstitution (left panel) and ECM degradation (right panel). Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding. Quantification of Ki67 positive fibroblasts at indicated time point after seeding (n= 8 section per condition of one representative experiment repeated two times). ECM degradation in DED organotypic cultures by collagenase treatment. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures at indicated time points after collagenase treatment. Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment (n= 8 section per condition of one representative experiment repeated two times). Note that fibroblasts around the injection sides start to proliferate after 48 hours. Nuclei were labelled with DAPI (blue) (D,F). Scale bars, 100µm (D,F). Data shown are means ± s.d. Fb, fibroblasts; PD, post digestion; TCP, tissue culture plastic; h, hours.
Figure 6A. The growth of CT26 tumor volume when treated with vehicle, TP-16 (75 mg/kg, p.o., daily), anti-PD-1 antibody (50 μg, i.p., twice weekly) or their combination (left) and percent change in tumor volumes between days 0 and 20 (right) (n=12). B. BALB/c mice with subcutaneous CT26 tumors were treated with vehicle, TP-16 (75 mg/kg, p.o., daily), PD-1 antibody (50 μg, i.p., twice weekly) or combined therapies for 2 weeks. Animal survival (time to tumor burden reaching 2000 mm3) was analyzed by the Kaplan-Meier method using GraphPad Prism (n=10). P values were calculated using the Log-rank test. C. Representative immunofluorescence staining images of tumor sections from CT26 tumor-bearing mice stained for CD8 and DAPI. Scale bars, 50 μm. D. Representative immunostaining images of tumor sections from CT26 tumor-bearing mice stained for p-STAT3, p-AKT and PD-L1. Scale bars, 100 μm. E. Transcriptome profile changes mediated by the synergy of TP-16 and anti-PD-1 analyzed using Agilent SurePrint G3 Mouse GE V2.0 Microarray. Venn diagrams depicting the number of changed genes (FC ≥ 2 or FC ≤ −2, P ≤ 0.05) compared with vehicle control following TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined therapy. F. The heatmap of T cell activation biomarkers for RNA-seq analysis. The unit of heatmap scale is expression value after centering and scaling by genes.
a, Immunoprecipitation of Flag–beclin 1 constructs with Nef–HA in HeLa cells 24 h post-transfection.f1a, Immunoprecipitation of Flag-beclin 1 constructs with Nef-HA in HeLa cells 24 h post-transfection. b, GFP-LC3-positive dots (autophagosomes) in MCF7 cells expressing GFP-LC3 and Flag-beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h. c, Sequences of beclin 1 amino acids 267-284, Tat-beclin 1 (T-B) and Tat-scrambled (T-S) control peptide. Red letters indicate amino acid substitutions to enhance hydrophilicity. d, Biochemical assessment of autophagy (p62 and LC3 immunoblots) in peptide-treated HeLa cells (3 h). e, f, Representative images (e) and quantification of GFP-LC3-positive dots (f) in peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h). Scale bars, 20 µm. g, GFP-LC3-positive dots in siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h). h, Model of Tat-beclin 1 peptide (left) based on corresponding elements of the beclin 1 evolutionarily conserved domain (ECD) structure (centre). Essential phenylalanine side chains, magenta; positions of solubility mutations, pink; lipid interaction site, yellow. ECD surface representation (right) illustrates exposure of corresponding peptide (cyan). i, p62 and LC3 immunoblots in peptide-treated HeLa cells (3 h). In b, f, g, bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. *P 0.05, **P 0.01; t-test.
Figure 5ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm. B, Normalized fluorescence intensity profiles across E. coli JM83 cell widths for CCCP and/or trypsin treated cells. Fluorescence was measured in ImageJ for 15 cells per condition and normalised to more clearly show distribution profiles for each condition. CCCP-treated cells showed peripheral ColE9* K469CAF647 fluorescence consistent with the colicin being bound at the OM. Trypsin treatment of cells in the absence of CCCP results in loss of this peripheral fluorescence but the presence of mid-cell signal consistent with internalisation. Treatment with CCCP and trypsin removed all ColE9* K469CAF647 fluorescence from cells, as in a. Error bars represent % SEM. C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.
Figure 2(A) L. pneumophila subpopulation proteomics. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry-positive/GFP-negative and mCherry-positive/GFP-positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP-producing subpopulation have a positive log2 ratio (q-value). (B, C) The ratio (GFP/mCherry) of selected proteins is indicated for (B) proteins relevant for flagellum formation and (C) cell division proteins and a T4SS substrate. The ratio of the mean intensities from 3 independent proteomics experiments is shown. (D, E) Expression of select L. pneumophila genes in amoebae. A. castellanii was infected (MOI 5; 6 h and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) PfleQ-mCherry-PflaA-gfp or (E) PlegC8-mCherry-PflaA-gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).
Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.f4Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a-d) Livers were subjected to solubility fractionation. 1% Triton X-100-soluble (a) and -insoluble (b) fractions were analysed by immunoblotting (a,b) and quantified (c,d). (e,f) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining (e). p62-positive areas were quantified (f). Scale bar, 200 μm (e). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
Rescue of Rb-deficient myotube degeneration by Bcl-2 and autophagy inhibitors.Figure 3.(A) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad.GFP or Ad.Bcl-2. Each point represents the mean ± SD of six fields from six independent experiments. Bcl-2-rescued, twitching Rb−/− myotubes are shown in Video 2. (inset) Western blot for Bcl-2 in Ad.Bcl2-transduced versus uninfected primary myoblasts. (B) Immunostaining for MCK (green) in Ad.Bcl-2-transduced control (ctrl) and Rb−/− myotubes at DM-6. (C) Western blot for MCK, myogenin, MHC, and troponin T in indicated cultures at DM-6 after transduction with Ad.Bcl-2. Tubulin was used as a loading control. Black lines indicate that intervening lanes have been spliced out. (D) Mean number of Rb−/− myotubes after 3-MA treatment, bezafibrate, or DMSO/vehicle as indicated. Counts are mean ± SD of six fields (n = 4). (E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA-rescued Rb−/− myotubes at DM-8. 3-MA-rescued, twitching Rb−/− myotubes are shown in Video 4. (F) Immunostaining for BrdU (green) and MHC (red) of 3-MA-treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10. (G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.
Figure 1(A) Model of Q cell position and migration direction in C. elegans L1 Larvae. Green arrow indicates that Q cells migrate along the AP body axis, whereas the dotted red arrows indicate that Q cells are potent to move towards the DV direction. (B) Schematics of the cst-1 and cst-2 gene models and DNA sequences of the cst-1 and/or cst-2 mutants that were generated via CRISPR-Cas9. Purple bar, Serine/Threonine kinase domain; blue bar, SARAH (Salvador-Rassf-Hippo homolog) domain. Arrow, sgRNA target site; green, target sequence; red, PAM sequence; blue, inserted nucleotide; dashed lines, deleted nucleotides. (C) Quantification of AQR position in adult WT, cst-1 and/or cst-2 mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. ***, P < 0.001 by Fisher's exact test. N = 50 - 109. (D) Quantification scheme of the angle (α) between the longitudinal axis of a migrating cell and the AP body axis. (E - F) Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (E) or cst-1; cst-2 double mutant (F) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. (G - J) Quantification of the QR.ap migration angle (G H), distance (I) and speed (J). Each line in (G I) represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student's t-tests, **, P 0.01; ***, P  0.001.
HIF-bound enhancers are in a poised state in normoxia.Figure 1Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:A B) HIF-1βChIP-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions. Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:C D) total RNA-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions. Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:E F) DNase-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions. Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:G H) H3K4me1 ChIP-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions. Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:I J), H3K4me3 ChIP-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions. Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:K L) H3K27ac ChIP-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions. Averaged signals at promoter-distal (>10 kb) HIF-binding sites and at the promoters of hypoxia-regulated genes for comparison are shown for:M N) RNApol2ChIP-seqMedian RPKM (reads per kilobase per million reads, scaled to background signal) from normoxic (red) and hypoxic (blue) MCF-7 cells are plotted for ±10 kb flanking regions.
Figure 6A Crystal structure of the Re9F06⋅RBD⋅Re5D06 complex (at 1.75 Å resolution; Appendix Table S1) as surface (left) or ribbon representation (right). The intramolecular disulfide bonds (shown as yellow sticks) are labeled. B Ribbon representation of the Re9F06⋅RBD⋅Re5D06 complex as in A, but with RBD-bound ACE2 shown as a semitransparent brown surface. Docking of ACE2 is based on the alignment of the RBD with the SARS-CoV-2 RBD⋅ACE2 complex (PDB ID 7KMS (Zhou et al., 2020b); RMSD=0.986 Å). Note the clash of ACE2 with Re5D06 and Re9F06. C Cryo-EM reconstructions of the Spike⋅Re5D06 complex (Appendix Table S2). SARS-CoV-2 Spike (Hsieh et al., 2020) was incubated with an excess of Re5D06, purified by size exclusion chromatography and vitrified immediately for structural characterization by cryo-EM (Appendix Fig S8). The two conformational classes detected were refined to high resolution (2.8 Å global). The sharpened maps are shown at low contour levels. The protomers of the homotrimeric Spike are shown in white, tan or gray, respectively, and are further color-coded as indicated. See Appendix Fig S8-12 for details and more information. D The SARS-CoV-2 RBD from A is shown as a ribbon, colored according to the indicated color gradient, with its N-terminus in blue and C-terminus in red. Disulfide bridges are depicted as yellow sticks. Re9F06 has been omitted for clarity. RBD side chains that interact with the fold-promoting VHH Re9F06 are shown as green "ball-and-sticks". See Appendix Fig S5 for a comprehensive analysis of the Re9F06⋅RBD interface. E Molecular details of the RBD⋅Re5D06 interaction. Left: overview of the RBD⋅Re5D06 complex, with the RBD shown as a green ribbon overlayed with its semitransparent surface. Re5D06 is shown as a ribbon (in magenta) with orange CDR loops. Right: Details of the RBD⋅Re5D06 interaction interface. RBD and Re5D06 are shown as transparent ribbons colored as on the left, with selected interface side chains depicted in green (RBD) or magenta (Re5D06). Blue marks nitrogen, oxygen is shown in red. A water molecule is shown as a yellow sphere. Dashed lines link interacting atoms (distance ≤ 4 Å). Lines pointing onto backbones indicate contacts to carbonyl-carbons or amide groups. F Crystal structure of free Re5D06, solved to 1.25 Å resolution (Appendix Table S1). Top: free Re5D06 is shown in magenta, with CDR3 (featuring three solvent-exposed tyrosine residues) colored in orange. For comparison, Re5D06 in its RBD-bound conformation is overlayed (RMSD=0.986 Å) in gray. Bottom: intramolecular interactions that stabilize CDR3. CDR3 side chains are depicted in orange; all other side chains are displayed in magenta. See E for further details. Extensive interactions in free Re5D06 stabilize a CDR3 conformation (with solvent-exposed Tyr 104, 109, 112) that requires only relatively minor structural changes for strong RBD binding.
Figure 2(A) Left: Immunoblot analysis of smooth muscle marker gene expression in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of SM -actin, SM22, and SM-calponin were normalized to -tubulin and expressed as a fraction of a control value. Results are expressed as means ± SD (**p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test). (B) qRT-PCR analysis of SMC transcription factor gene expression in control and FRS2 knockdown HASMCs. (*p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test. N=3). -actin was used for sample loading normalization. (C) Collagen gel contraction assays were used to determine the contractile ability of control or FRS2 knockdown HASMCs (*p<0.05 compared to control; unpaired two-tailed Student's t test. N=3). (D-F) Upper panels: Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 (10 m), TGFR2 or Smad2 shRNA lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to -tubulin, HSP90, or Smad2 and expressed as a fraction of a control value.
C state is a distinct pluripotency state (A-E) On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM).Figure 5On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM). After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of different pluripotent states (B) After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of Xist (C) After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells) their abilities to form chimeras (D) were determined. The characteristics of 5C-Oct4GFP+ cells were summarized and compared to naïve and primed PSCs in (E). (F) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. The obtained cells were maintained for additional 20 passages. The 5C-Oct4GFP+ cells cultured in primed medium lost GFP fluorescence. R1 ESCs-primed were primed PSCs generated from R1 ESCs. Scale bar, 100 μM. The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. (G) PSCs mentioned in (F) were used for RNA-Seq analysis. Similarity among gene expression profiles was determined. The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. The expression of Xist (H) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. the abilities to form chimeras (I-J) were determined.
Figure 6A, TOC1 protein expression pattern in 3-d-old seedlings in native promoter TOC1/5X/T135A/S175A lines grown in SD. White and black regions indicate light and dark period. Data from 3 independent trials were averaged; error bars indicate SEM. Statistical analyses were performed by Students's t test. Asterisks indicate significant differences between TOC1 wild type and its phosphosite mutants (*, p<0.05). Data information: TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ. B, Measurement of TOC1-GFP protein turn-over in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Data information: TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ. C, Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with student's t test, different letters indicate significant differences (p<0.05). Data from 3 independent trials were averaged.