EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments. Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B) HMGB1 levels in the medium of CT26 cells treated with BoxA or 1μM MTX for 24 hours (n=3). Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in CT26 cells treated with increasing concentrations of BoxA or 1μM MTX for 24 hours. β-actin is shown as loading control (n=3). Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D) Surface CD47 on CT26 cells after incubation for 24 hours with the indicated concentrations of BoxA. The experiment shown is representative of 2 performed, in biological triplicate. Data information: , bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) Cell proliferation. CT26 cells (3x105) were seeded at t-16 hours, then exposed to increasing concentrations of BoxA or 1 μM MTX and counted after further 24 hours (n=3). Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (F) Western blot analysis of cleaved caspase-3 in CT26 cells exposed or not to BoxA or MTX for 24 hours. α-tubulin was used for normalization (n=2). Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (G) Scheme of the experiment. Twenty-two BALB/c mice were inoculated i.p. with 3.5x104 CT26-LUC cells and treated with either 800 μg BoxA or PBS 3 times a week, 10 times in total. Yellow arrows represent BLI imaging. (H) Tumor growth was detected by BLI. Lines that do not reach day 20 correspond to mice that were sacrificed for ethical reasons. One BoxA treated mouse survived without any BLI signal and was re-challenged with CT26-LUC cells at day 65; at day 79 it was sacrificed and no tumor masses were detected. (I) Kaplan-Meier survival curves of mice shown in panel H. Statistics: Gehan-Breslow-Wilcoxon test. (J) BLI was measured two hours after re-challenge (upper images) and one week later (lower images). The survivor was imaged also at much higher sensitivity (note the different scales for radiance).
Figure 1JNK signaling is activated during a distinct phase of EMT and becomes a critical determinant of mesenchymal identity. (A) Heatmap showing standardized expression (Z-scores) of genes differentially expressed upon TGF-β-induced EMT in NMuMG cells. (B) Venn diagram showing overlap of upregulated genes at individual time-points. (C) Same as in B for downregulated genes. (D) Representative immunoblots (n=3) showing phosphorylation state of Smad2 and JNK during TGF-β-induced EMT in NMuMG cells. Lamin B serves as loading control. (E) Quantification of one set of representative immunoblots (in figure D) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF-β-induced EMT in NMuMG. (F) Representative immunohistochemistry image for pSmad2 and pJNK in non-invasive (n=34) and invasive (n=14) breast cancer samples. Scale bars for 10X and 40X images are 200 µm and 50 µm respectively. (G) Quantification of Immunohistochemical staining for p-JNK and p-Smad2 as in (F) in invasive tumor samples (n=34). The pie chart shows the percentage of tumors with high p-Smad2 and low p-JNK or high p-JNK and low p-Smad2 or equal p-Smad2 and p-JNK. (H) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells (d0) and cells treated with TGF-β for seven days in the presence of JNK inhibitor (d7 JNKi) or control (d7 DMSO). Staining was performed with antibodies against Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. It is important to note that the untreated epithelial cells are highly proliferative and appear small in size, round in shape and adhere to surrounding cells whereas day 7 TGF- β-treated (mesenchymal) cells are much bigger in size, elongated in shape and detached from the nearby cells. Scale bar, 100 μm, 63X magnification. (I) mRNA levels for classical EMT markers in cells treated as in (F) were measured by qRT-PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. (J) Transwell migration assay for NMuMG cells treated as in G. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.
Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.Figure 2Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.S. sclerotiorum A2 (A) and wild type (B) strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were post-fixed in osmium tetroxide, and embedded in Spurr's epoxy resin. A microtome was used to cut 400 nm sections. Toluidine blue stain was used to reveal fungal hyphae. H = hyphae. The dotted line represents the leading edge of the visible lesion. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227.
Figure 5As demonstrated by our study and shown in the model, Bax core dimers form parts of the proteolipid wall that separates the nonpolar membrane from the aqueous pore. Other regions of the Bax oligomer and radially arranged lipids may form other parts of the wall. The diameter of the pore will be determined by the stoichiometry of the Bax oligomer and the number of the radially arranged lipids. In order to release a hydrated cytochrome c from the mitochondria, the diameter of the pore must be > 60 Å, the diameter of cytochrome c plus a water shell. The length of a core dimer along its longest axis (L) = 45 Å. Since the axis tilts 60° from the bilayer normal, a core dimer can cover 39 Å of the lipid bilayer around the pore (M). Assume the pore diameter = 60 Å, and the pore wall only consists of Bax core dimers, six Bax core dimers or 12 Bax proteins are required to form a hexagonal pore that can release cytochrome c. The structure of cytochrome C in the pore was generated from PDB file 3ZCF using PyMOL program.
LINC00941 is a suppressor of keratinocyte differentiation A Workflow of the performed RNA-sequencing experiment.Figure 2A Workflow of the performed RNA-sequencing experiment. B LINC00941 knockdown efficiency (siLINC00941) in epidermal tissue on day 3 of differentiation as obtained by qRT-PCR measurements (n = 4-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant). C Heatmap of differentially expressed genes (padj < 0.05 and -1 > log2FC > 1) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes (n = 4-5 epidermal tissue cultures/knockdown group). D GO term analysis of downregulated (padj < 0.05 and -1 > log2FC <1) genes in LINC00941 deficient organotypic epidermal tissue on day 3 (n = 4-5 epidermal tissue cultures/knockdown group).
∆Np63-driven SCC cells of various tissues are vulnerable to USP28 depletion Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours.Figure 6Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours. Cbioportal. Expression of TP63 (left) and USP28 (right) in human Lung (n=498), Cervix (n=254), Oesophagus (n=96) and HNSC (n=522) SCC tumours and normal non-transformed tissue (nLung=338 nCervix=3, nOesophagus=11 and nHNSC=44). In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test statistical analysis. Xena UCSC software. Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung (nADC=513 and nSCC=498), Cervix (nADC=47 and nSCC=254) and Oesophagus (nADC=89 and nSCC=96). Blue dots: ADC; Red dots: SCC; R= Spearmans correlation coefficient; m: Slope. Xena UCSC. Spearman's Correlation values of the gene list '' Squamous cancer markers'' and TP63, c-JUN, c-MYC or NOTCH1 in Lung (n=1011), Cervix (n=301), Oesophagus (n=185), HNSC (n=522) and PAAD (n=178) tumours. Intense blue: Low correlation; Intense red: Strong correlation. Gepia software. Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63, KRT14 and USP28 protein abundance in H1299, LUDLU-1adh, HELA, SiHa, Ca Ski, PANC-1 and BXPC-3 (ACTIN as loading control). Cells were seeded at equal cell density and counted after five days, Brigth field images of control or sh-USP28#2 infected H1299, LUDLU-1adh, Hela, CaSKI, PANC-1 and BXPC-3 cells before quantification. Scale bar = 30µm Relative number of H1299, LUDLU-1adh, Hela, CaSKI, SiHa, PANC-1 and BXPC-3 sh-USP28#2 cells compared with sh-NTC control cells. p-values were calculated using two-tailed t-test statistical analysis. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.
Structure similarity between SARS-CoV-2 RBD and SARS-CoV RBD.Figure 4. Structure similarity between SARS-CoV-2 RBD and SARS-CoV RBD.RBD is shown in a space-filled model with colored surface. ACE2 is shown as gray tube model. The three glycosylation sites in SARS-CoV are labeled. Note that N357ST in SARS-CoV is changed to N370SA in SARS-CoV-2, which is different from the NXS/T pattern required for glycosylation, and hence this site is more likely to be unglycosylated. The two possible cross-reactive regions are marked with yellow circles.
Figure 6Figure 6. Speculative model for TERRA's role in mediating telomerase-dependent elongation of short telomeres. See main text for details.
a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b).f3(a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b). c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel. (d) Co-IP experiments to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes in vivo. FLAG- or GFP-tagged Atg14L or UVRAG was transfected alone, or both were co-transfected into HEK 293T cells and their interaction with endogenous Beclin 1 was probed by co-IP using GFP antibody, followed by western blot with anti-Beclin 1 antibody.
Figure 7HME cells were treated with 750 μM of H2O2, with and without pre-treatment with olaparib. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.01 in two-way ANOVA. The data represent the results of three experiments. HME control (vec) and KO1-3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min. PAR accumulation was assessed by the Western method, with anti-PAR. β-actin is shown as a loading control. HME vec, KO1 and OAS1hi cells were incubated with olaparib for 30 min, then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h. Bars represent means of triplicates ± SD from three experiments. * p<0.05; ** p<0.01 (relative to H2O2-treated vec cells) in Student's t-test. KO1 cells transfected with vector (KO1-vec), wild-type OAS1 (KO1-OAS1 wt), or mutant OAS1 (D75A/D77A, KO1-OAS1 mut) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method. Loading controls: β-actin and overall protein levels were visualized by stain-free technology. The experiment was repeated three times; a representative image is shown. Quantification of the relative abundance of PAR in (D). Images obtained in three independent experiments were analyzed; the data are plotted as mean + SD *, p<0.05 by Student's t-test. KO1 cells transfected with vector, wild-type OAS1 (OAS1 wt), or mutant OAS1 (D75A/D77A, OAS1 mut) were treated with 500 μM of H2O2. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ****, p<0.001 in two-way ANOVA. The data represent the results of three experiments.
Figure 6A. Schematic representation of the experimental paradigm. Concentrations used: 200ng/mL Sema3A and Slit2. Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Sema3A (B) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=3 (B) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant. Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Slit2 (C) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=4 (C) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant. D. Schematic representation of the experimental paradigm. Abbreviations: co-MO, control morpholino. Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino. Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.
Fig.Figure 3A) FM4-64 and GFP excitation/emission spectra. B) FRET-based in vivo strategy to distinguish localization of FM4-64 in the inner or outer leaflets of vacuolar membranes. EGFP tags were added to the cytosolic C-terminus of Vph1 or to the lumenal C-terminus of Nyv1. C) Assay by microscopy. Half of two cultures of cells expressing Vph1-EGFP or Nyv1-EGFP were labeled with FM4-64, washed and mixed with the other, non-labeled half of the same culture. Cells were analyzed by spinning disc confocal microscopy. The intensities of GFP fluorescence were tracked along the dashed lines and compared between neighboring FM4-64-labeled and unlabeled cells. D) FACS analysis. The strains were labeled washed and analyzes by FACS. Shown are side (SSC-A) and forward scatter (FSC-A), as well as the fluorescence intensity distributions for FM4-64 and for GFP in the absence or presence of FM4-64. E) Comparison of the GFP mean fluorescence from FM4-64-stained cells and unstained cells (20'000 each). F) Possibilities for FM4-64 transfer between the inner leaflets of two tethered vacuoles. Flipping between the trans-leaflets of a hemifusion structure; reversible flipping into the outer leaflet, establishing a minor pool that then transfers through the buffer or through the outer leaflet; or passage through a fusion pore.
Figure 7(A) Picrosirius red staining visualized under polarized light and Elastica van Gieson (EvG) staining) of forepaw toes from 12-wk-old WT or RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Note the reduced signal for picrosirus red in RDEB mice treated with 0.1 mg/kg Ang-(1-7) indicating less parallel alignment of and/or thinner collagen fibrils, and additionally the more organized and less disrupted appearance of elastic fibers (black in the EvG staining). (B) Quantification of picrosirius red stained forepaws visualized as in A. A digital threshold was applied to the red channel before quantification. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant; n = 10-15. Individual data points, mean ± S.E.M are shown. ( Data information scale bar = 100 μm (C) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A. α-SMA, α-smooth muscle actin; Thbs-1, thrombospondin-1. Data information: β- actin or β-tubulin was used as loading control (D) Dermal β-arrestin-1/2 (red) and NFκB (red) staining in forepaws from WT and RDEB mice treated as in A. Nuclei counterstained with DAPI (blue). Data information scale bar = 20 μm. (E) Western blot for the indicated proteins on tissue lysates from eyes, esophagus or tongue from RDEB mice treated as in A. Data information: β- actin or β-tubulin was used as loading control
Figure 1Pa (Pseudomonas aurantiaca) and Aci2 (Acinetobacter sp.) were co-cultured with a 104 dilution every 24 hours and 0.1%w/v alanine supplied as the only carbon source. Two biological replicates were performed for each of four initial Pa fractions: 0.1, 0.25, 0.75, and 0.9. Population sizes were measured via colony counting after the first, third, fifth, and seventh days (Materials and Methods). With only alanine supplied, Aci2 competitively excluded Pa. An identical experiment was performed with 0.1%w/v glutamate supplied as the only carbon source. Aci2 again competitively excluded Pa. A third experiment was performed with both 0.05%w/v alanine and 0.05%w/v glutamate supplied. Pa coexisted with Aci2 and reached a population fraction between 0.34 and 0.42 independent of initial starting fraction. Two-resource coexistence was unexpected given the single-resource competitive exclusions.
Figure 2(A) LMP1 is localized to the inner mitochondrial membrane. Isolated mitochondria were incubated with proteinase K (40 μM) for 30 min before LMP1 expression was detected (left); the mitochondria were further subjected to submitochondrial fractionation. VDAC1, TIM23, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively (right). (B) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy，and use imageJ to calculate the co-location level of both. The nuclei were stained with DAPI (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± S.E.M. (n = 6, biological replicates per group). (C) IP/IB analysis was used to detect the interaction of LMP1 with endogenous ANT1 in CNE1-LMP1-expressing cells. (D) Detection of the LMP1-ANT1 interaction using a proximity ligation assay. Red fluorescence corresponds to the PLA positive signal and blue fluorescence corresponds to nuclei (DAPI staining; scale bar, 10 μm). (E) LMP1 interaction with ANT1 in vitro. GST-ANT1 expressed in bacteria was incubated with a Flag-LMP1 protein and the proteins were subjected to Western blot analysis. (F) ANT1 interaction with LMP1 is dependent on its domain 2 in 293T cells. ANT1 and LMP1 domain architecture and schematic representation of its respective deletion constructs used in this study in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 and Myc-tagged ANT1 mutants. IP of cell lysates using a Flag or Myc antibody. (G) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 mutants and Myc-tagged ANT1. IP of cell lysates using a Flag or Myc antibody.
SARS-CoV-2 S recognizes hACE2 with comparable affinity to SARS-CoV S.Figure 2.Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS-CoV-2 SB (B) or SARS-CoV SB Sequence alignment of SARS-CoV-2 SB and SARS-CoV SB Urbani (late phase of the 2002-2003 SARS-CoV epidemic). Identical and similar positions are respectively shown with white or red font. The single amino acid insertion at position 492 of the SARS-CoV-2 SB is indicated with a period at the corresponding SARS-CoV SB position. The 14 residues that are key for binding of SARS-CoV SB to hACE2 are labeled with a star.
Figure 5(A) Schematic representation of wild-type (wt), targeted (flx) and deleted (Δ) Cdca5 alleles (left) and Southern blot of mouse genomic DNA (right). Red triangles, loxP sites. (B) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ/+ mice (newborn mice, n = 136; E9.5, n = 54). (C) Gene deletion and G0 phase synchronization protocol for mouse embryonic fibroblasts isolated from Cdca5 flx/flx mice expressing ERT2-Cre. (D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins. (E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment. (F) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium. (G) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition. (H) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition. (I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts. (J) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment. Representative images from every category are shown; size bar, 10 µm; error bars denote s.e.m.; n > 500 per genotype.
Figure 3A Model calibration with mCFU-E wild-type cells, and mCFU-E cells overexpressing SHIP1 or PTEN, and BaF3-EpoR wild-type cells. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories. B Model prediction of pAKT dynamics in BaF3-EpoR cells overexpressing PTEN or SHIP1. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. C Model prediction for the basal expression level of the dual specific phosphatase (DUSP). DUSP abundance ratio between BaF3-EpoR and mCFU-E cells was identified by the mathematical model. The solid line indicates the profile likelihood. The dashed red line indicates the threshold to assess point-wise 95% confidence interval. The asterisk indicates the optimal parameter value. D Experimental validation of basal DUSP expression in mCFU-E and BaF3-EpoR. Quantitative RT-PCR was performed with all samples on the same plate, allowing a direct comparison of mRNA levels between mCFU-E and BaF3-EpoR cells. Data is normalized to the Rpl32 gene. Ratios of expression in BaF3-EpoR cells compared to mCFU-E cells are shown as box plots. N=10. For details, see Appendix K.