EMBO/sd-character-level-ner huggingface dataset:
Figure 2A) Quantification of micronuclei containing 0, 1 or 2 CENP-A signals in HeLa YFP:CENP-A cells transfected with control and H2A.Z.2 siRNA for 72 h. 160 micronuclei were analysed. Error bars indicate SD of three biological replicates. The image represents an example of a micronucleus containing a single CENP-A signal. The insets are enlargements of the MN. Scale bar 5 μm. B) Representative images of metaphases from YFP:CENP-A (green) HeLa cells treated with Control (top) or H2A.Z.2 (bottom) siRNA. The white arrowheads indicate the mis-aligned chromosomes. Scale bar: 10 μm. C) Representative images of metaphase spreads from Control (top) or H2A.Z.2 (bottom) siRNA-transfected HeLa cells after FISH with a Chr17 centromeric probe (green). The arrowheads indicate the separated sister chromatids. Scale bar: 10 μm. D) Representative images of HeLa cells transfected with Control (top) or H2A.Z.2 (bottom) siRNA, fixed, and stained for Sgo1 (green). Scale bar: 10 μm. E) Quantification of Sgo1 localisation in pro-metaphase cells from the experiment in (D). Error bar represents SD of two biological replicates. 35 pro-metaphase cells were analysed. F) Representative images of HeLa cells treated as in (D) and stained for Aurora B (green) and alpha tubulin (grey). Scale bar: 10 μm. G) Mitotic index of HeLa cells transfected with Control (grey), H2A.Z.1 (blue) or H2A.Z.2 (red) siRNA and treated with nocodazole for 0, 2, 4 or 6 h. At least 800 cells were analysed for each category. Error bar represents SD of three biological replicates. **=p<0.01; ***=p<0.001 ns=not significant (Chi square test). H) Left panels: Representative images of HeLa cells treated as in (D) and stained for HP1α (green). Scale bar: 10 μm. Right panels: Representative images of HeLa cells treated as in (D) and stained for h3T3ph (green). Scale bar 5 μm I) Violin plot of centromeric H3T3ph intensity of pro-metaphase/metaphase cells from the experiment in (H). The median is shown as bar. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01. J) Representative images of HeLa YFP:CENP-A (green) mitotic cells after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. K) Violin plot of centromeric CENP-A intensity of pro-metaphase/metaphase cells from the experiments in J. (Control si N=197 H2A.Z.2 si N=260). The bar represents the median. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01. L) Representative images of HeLa mitotic cells stained for CENP-C after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. M) Violin plot of centromeric CENP-C intensity of pro-metaphase/metaphase cells from the experiments in L. (Control si N=1083 H2A.Z.2 si N=686, from 3 biological replicas). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.0001.
Determination of accumulation of autophagosomes using GFP-ATG8a.Figure 3(A) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
Figure 1A) Correlation matrix of Pearson correlation coefficients for RNA-seq data obtained from multiple PDAC cell lines (two biological replicates for each of the nine cell lines were used). The matrix was hierarchically clustered. The relationship with grade is shown. Source data were deposited in GEO. B) Heatmap showing TFs differentially expressed in Lo-G and Hi-G PDAC cell lines. Differentially expressed genes were identified based on a log2(fold change)> + 1 and an FDR < 0.1. Hierarchical clustering was based on Pearson correlation coefficients. C) Representative RNA-seq snapshots. D) Western blots for selected TFs. One experiment out of three independent biological replicas is shown. E) IHC on selected showing TF staining in G1 and G3 areas from human PDACs. The areas in the insets are shown at 4x higher magnification in the panels below. Data from one representative patient out of nine tested are shown.
Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice.Figure 3.Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B). PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D) Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain (E and F) by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD (F).
Figure 1a) Caco-2 monolayers consisting of 1.5e6 cells per sample were infected with SARS-CoV-2 at an MOI of 0.5 (see materials and methods). Three biologically independent replicates were collected per time point. Samples were subjected to vacuum manifold-based 2D-TPP. After aggregate removal, the remaining soluble fractions were labelled with isobaric mass tags (TMTpro) to enable protein quantification. Samples were combined in a 2D-TPP layout in order to compare protein stability and abundance throughout the infection time course (Becher et al, 2018). The uninfected (t-1) time point was used as a reference to calculate fold changes (FC). Significant protein changes (abundance and thermal stability) are represented as circle plots for individual proteins, as previously described (Becher et al, 2018). Inner circle corresponds to protein abundance and the outer circle corresponds to stability changes (n=3). b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM. c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).
The PC1/PC2_AA complex channel has greater Ca2+ permeability than the homomeric PC2_AA channel.Figure 3A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test). C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA. D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test). E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA. F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used. G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test). H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.
Figure 8A, B. Jurkat cells were treated for 10 and 30 min with PMA (200 ng/ml) and ionomycin (iono) (300 ng/ml). After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or DAF-FM-DA (B) to evaluate the production of H2O2 or NO, respectively. Values are expressed as units of DCF or DAF-FM fluorescence relative to untreated cells (arbitrarily set as 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 (A) and n = 6 (B) independent experiments. *p<0.01; **p<0.001; ***p<0.001. C. (left) Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml). Basal and phospho-active form of ATM, and GSNOR were assessed by Western blot. Vinculin was used as loading control. (right) Phospho:basal level ratios of ATM along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05. D. Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml), in the presence or absence of ATM inhibitor (KU55933) (5 μM) or GSNOR inhibitor (N6022) (25 μM). Cell death was assessed cytofluorimetrically upon staining with propidium iodide (PI). Values are expressed as % of sub-G1 population of PI-stained cells and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 5 independent experiments. *p<0.05; **p<0.01. E, F. CD4+ T cells were incubated for 30 min with anti-CD3, anti-CD28 and anti-CD49d (stimulation). After stimulation, cells were incubated with 5 μM 2',7'-H2DCF-DA (E) or DAF-FM-DA (F) to fluorometrically evaluate the production of H2O2 or NO, respectively. Values are shown as units of DCF or DAF-FM fluorescence relative to non-stimulated cells (arbitrarily set as 1). Values are shown as fold change and represent the median plus range with all the experimental points of n = 3 independent experiments. **p < 0.01. G. CD4+ T cells were stimulated for 96 h with anti-CD3, anti-CD28 and anti-CD49d in the presence or absence of ATM inhibitor (KU, upper panel) or GSNOR inhibitor (N6022, bottom panel). Cell death was assessed cytofluorometrically upon staining with Annexin V (AnV) and PI. Values are expressed as fold change of AnV+/PI+ cells relative to control (CD4+ without inhibitor, vehicle, arbitrarily set to 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 independent experiments. *p<0.05; ***p<0.001. H. In the same experimental settings, populations of stimulated (proliferating) CD4+ T cells were identified cytofluorometrically and included in rectangles. Values (as % of total population) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments. *p<0.05; ***p<0.001.
Contribution of GSDMD in the effect of VHHASC on the release of IL-1β and cell death induced by PFO or nigericin.Figure 4THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-). (A) Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA-differentiation, where indicated. Data is from one representative of two independent experiments. THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-). (B) IL-1β concentration or percentage of LDH released into cell-free supernatants of PMA-differentiated THP-1 cells that were treated with VHHASC (200 µg ml-1) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml-1, right panels) for 2h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-). (C) Confocal microscopy imaging of live PMA-differentiated and nigericin-treated (10 µM) THP-1 cells expressing human ASC-GFP (green) in the presence of AlexaFluor647-labeled VHHASC (VHHASC-AF647, 10 µg ml-1, cyan) in the medium. Cells were either left untreated or incubated with VX-765 (50 µM) for 1h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm.
Figure 4Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. cellular DNA was sequenced to perform an Inference of CRISPR Edits (ICE) analysis C-D: MyD88KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (C) and CXCL10 (D). Done with n=4 Data information: Bars represent mean values and equal symbols represent equal donors. Statistical significance was determined using Mann-Whitney one-tailed T test in (C) as MyD88-KO data had no variance and values of 0. For (D) a Mann-Whitney two-tailed T test was used for comparison between conditions of mock and MyD88 KO donors. E-F: Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIFKO or TRIG+MyD88KO (E) and RIG-IKO or RIG-I+MyD88KO (F) pDCs. Done with n=2
Figure 1(A) Sphere formation in MGC-803 cells incubated with fresh medium or conditioned medium from MGC-803 cells for 7 days. The number of spheres was quantified and indicated on the right. Scale bar = 100 µm (n = 3 biological replicates; mean ± standard error of mean (SEM); *P = 0.0146; two-tailed unpaired Student's t-test). (B) Sphere formation in MGC-803 cells with indicated treatment. The number of spheres was quantified and indicated on the right. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; no significant differences; two-tailed unpaired Student's t-test). (C) Sphere formation in MGC-803 cells incubated with an equal volume of phosphate-buffered saline, supernatant after differential centrifugation, or sEVs. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; ns = no significant difference; *P = 0.0390; two-tailed unpaired Student's t-test). (D) Sphere formation in MGC-803 cells treated with sEVs (20 μg/mL) from five gastric cancer cell lines as indicated (n = 3 biological replicates; mean ± SEM; ***P = 0.0007 (MGC-803), **P = 0.0061 (HGC-27), and **P = 0.0018 (BGC-823); two-tailed unpaired Student's t-test). (E) Expression levels of LSD1 in MGC-803, MKN-45, HGC-27, BGC-823, and NCI-N87 cell lines and their corresponding sEVs. The samples with equal amounts of proteins were loaded (n = 3 biological replicates; mean ± SEM; compared with the NCI-N87; *P = 0.0432 (cell/MGC-803), *P = 0.0300 (cell/HGC-27), **P = 0.0306 (cell/BGC-823), **P = 0.0018 (sEVs/MGC-803), **P = 0.0029 (sEVs/HGC-27), and ***P = 0.0003 (sEVs/BGC-823); two-tailed unpaired Student's t-test; GAPDH was used as a loading control for cell lysis; CD9 was used as a loading control for sEV lysis). (F) Establishment of LSD1 knockout (KO) MGC-803 cell line. Con indicates MGC-803 cells, while KO indicates LSD1 KO MGC-803 cells. Transmission electron microscopy images (G) of sEVs from MGC-803 and LSD1 KO MGC-803 cells. Scale bar = 100 nm. size distribution (H) of sEVs from MGC-803 and LSD1 KO MGC-803 cells. Scale bar = 100 nm. (I) Expression levels of LSD1, CD63, CD9, TSG101, and calnexin in sEVs from MGC-803 and LSD1 KO MGC-803 cells. sEVs were extracted using two different extraction methods (ultracentrifugation (UC) and commercial kit). Calnexin is an sEV negative marker. UC, sEVs isolated using the ultracentrifugation method; Kit, sEVs isolated using the commercial kit. (J) Expression levels of LSD1, CD63, and CD9 in sEVs with indicated treatment.
Astrocyte-specific Stat3 deletion reduces Aβ plaque burden and Aβ levels without altering APP metabolism (A-B) Aβ plaque burden, as assessed by plaque load and size using an anti-Aβ antibody, was strongly reduced in APP/PS1-Stat3KO vs.Figure 3Aβ plaque burden, as assessed by plaque load and size using an anti-Aβ antibody, was strongly reduced in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 300 µm). Plaque density, assessed by staining brain sections with thioflavin (yellow), remained unchanged in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 250 µm). Electrochemiluminescence ELISA after sequential extraction from whole-brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1-42 and Aβ1-40 were significantly reduced (Mann-Whitney test), whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1-42 and Aβ1-40 (Mann-Whitney test). Western Blot quantification of full-length amyloid precursor protein (APP) and its C-terminal fragments (CTF) showed no differences between both groups (Mann-Whitney test; for all datasets, n = 9 male mice for both groups (age, 11 months)).
Time course and temperature dependence of uptake.Figure 3time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed. (B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.
Figure 1A Experimental workflow for MPRA: oligonucleotide library is cloned into an empty vector and a reporter gene and promoter are inserted using restriction sites within the oligonucleotide. The assembled plasmid is transfected into primary CD4 T cells and RNA is extracted after 24 hours. RNA barcode counts are normalised to their respective counts in the input plasmid library (DNA), which is sequenced separately. B Adapted MPRA plasmid incorporating RSV promoter. C Principal component analysis of scaled element counts (sum of barcodes tagging same genomic construct in mRNA) in resting and stimulated CD4 T cells from 12 donors. Dotted lines indicate samples from the same donor. D Heatmaps showing pairwise comparison of MPRA activity for all constructs (mRNA/DNA) between donors - left panel: resting CD4 T cells; right panel: stimulated CD4 T cells.
HH signaling and Smaug control each other in vivo (A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells.Figure 5(A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells. Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E. Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference (B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype. (C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.
Figure 1(A) The MET p.Y1234C mutation segregated with disease phenotype in the arthrogryposis family. Filled symbols denote affected individuals, open symbols indicate unaffected individuals, and symbols with slashes represent decreased individuals. Asterisks indicate a mutation is present, # means wild type. (B) Phenotypes of affected individuals. Camptodactyly, absent flexion crease, and limited forearm supination were observed. (C-E) T1 weighted MRI scan on upper limbs of subject IV:7. (C) The pronator quadratus absence of affected side was indicated by a red arrow. (D) No difference was found in palmar muscles. (E) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow. (F-H) T1 weighted MRI scan on upper limbs of subject IV:8. (F) Increased epimysial fat was indicated by red arrow. (G) Completely loss of thenar eminences of both hands were indicated by red arrows. (H) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows. (I) The MET variants by Sanger sequencing was indicated by red arrow. (J) 293T cells were transfected with FLAG-tagged MET/METMut/Vector plasmids, and 48 h post transfection, cells were treated with 10 ng/mL recombinant human HGF for 1 h. Then MET/METMut protein purification and tyrosine kinase assay were conducted. Western Blot pictures representative of n = 3 experiments. METMut means p.Y1234C mutant MET. EGFR means epidermal growth factor receptor and serves as a positive control. NC means negative control.