EMBO/sd-character-level-ner huggingface dataset:
Single cell quantification of p53 dependent transcription highlights distinct patterns of gene expression upon DNA damage.Figure 2Fluorescence microscopy images of smFISH probes CAL-Fluor 610 (grey) overlayed with Hoechst 33342 staining (blue) at 3 h after 10 Gy IR or the indicated target genes in A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization. We quantified RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). smFISH based single cell analysis of gene expression patterns highlights distinct RNA counts for p53 targets. RNA counts per cell are displayed as boxplots (see Data Visualization section); n: number of analyzed cells, fc: median fold of induction relative to time point basal (indicated by grey lines) Distributions of RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Despite a clear change in median levels (m: median), single cell analysis reveals a strong dispersion that overlaps for the different conditions, as shown by the strongly overlapping distributions of RNA counts per cells. For better visualization, probability density estimates (PDF) based on a normal kernel are shown
Figure 3A. Root mean square deviation (RMSD) calculations of both SPOP WT and Q165P mutant on CA atoms with reference to the crystal structure (3HQI.pdb). B. Overall architecture of SPOP dimer. Superposition of all Cα atoms of WT structure (a) and Q165P structure (b) from simulation at 80 ns over the crystal structure. The two SPOP molecules in the crystal structure are colored cyan and gray, respectively; the WT structure from simulation are red and warm pink, and Q165P are green and pale green, respectively. C, D. Close-up views of the MATH domain from top of (B). The Cα atoms of Q165 or P165 are represented by spheres. E. Several amino acids are predictively important for SPOP dimerization. Mutations on the following residues might destroy the dimer interface for MATH domain: any residue in blue color to Pro at the strand where Q165 locates or any residue in green color to Pro at the strand on the dimer interface. Residues mutated in PCa patients F102, F133 and Q165 are in red. Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F) Co-IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P (G).
Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells.Figure 1Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells. Cells were mock infected or infected and immunoblotted for actin, p62 (A) or LC3 (B). Cells were infected for 15 h and labeled using antibodies to p62 and capsid (C). Cells were transfected with GFP‐LC3‐B, infected for 15 h and labeled with anti‐capsid antibody (D). Mice were infected for 3 days, then whole‐cell lysates of muscle were immunoblotted for LC3 or actin (E) and muscle sections were labeled with p62 and capsid antibodies (F). Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I). Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.
Characterization of oligo#5 and its derivatives.Figure 2A. Titration of oligo#5. An increasing amount of oligo#5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene and the RNA was analyzed by RT-PCR. -RT: negative control without reverse transcriptase.B. Quantification of three independent experiments. The half-maximal effect was seen at 13.1 nM concentration. C. Influence of oligo#5 shortening on alternative splicing of the serotonin 2C receptor. Oligo#5: 5' AGU AUU GAG CAU AGC CGC 3' (18mer); Oligo#5-3: 5' GAG CAU AGC CGC 3' (12mer); Oligo#5-10: 5' G CAU AGC CGC 3' (10mer) were used in transfection assays analyzed by RT-PCR.D. Statistical analysis of the effect of oligonucleotide shortening. The effect of all oligos testes is significant, oligo#5: p= 0.0015; oligo#5-3: p=0.031; oligo#5-10: p= 0.048, n=6. E. Influence of cy3 label on the 5' and 3' end of oligo#5. Using a phosphate linker, Cy3 was added to the 5' or 3' phosphate, respectively and the oligonucleotides assayed by RT-PCR. Oligo#5 with cy3 at the 5' end showed 86% inclusion, with Cy3 at the 3' end 17% exon inclusion.F. Statistical analysis of the effect of oligonucleotide modification. The effect is significant, p=0.009, n=4.
The unique IgI3 structure is present in Gram-positive bacterial species and evidence for binding CEACAM1.Figure 7Amino acid based Maximum Likelihood tree of β-IgI3 homologs. Clades of related IgI3 domains are shown, with clade I representative of β-IgI3 domain sequences. Branches are coloured by bacterial species from which the sequence originates, except those with no species designated. Maximum likelihood values bootstrap values > 60 are shown as a circle, where larger circles indicate a higher bootstrap support value. Superposition of the IgI3 domain from β protein (blue) onto predicted IgI3 domain structure of clade II sequence of S. pyogenes R28 protein (red). Close-up of the truncated C strand, α-helix and D strand of β-IgI3 (blue) and R28-IgI3 (red), indicating the corresponding R28-IgI3 residues present at sites in β-IgI3 that bind CEACAM1 (CC1). Binding of recombinant (r)CC1-HIS to dynabeads (DB) coated with β-IgI3, R28-IgI3 or control. Mean and SD is shown for n = 6 independent replicates. Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates. Binding of recombinant rCC1-HIS (10 μg/mL) to R28-expressing GBS strain H1147 and R28-expressing S. pyogenes strain AL368. Data representative of n = 3 replicates. The per residue free energy contribution spectrums of β-IgI3/CC1-N interface and R28-IgI3/CC1-N interface. The residues with binding free energy contributions lower than -2.0 kcal/mol are identified as key (favourable) residues and coloured blue (for β-IgI3) or red (for R28-IgI3). Free energy contributions at -2.0 kcal/mol and 2.0 kcal/mol are indicated by dashed lines. The position of the truncated C strand, α-helix and D strand is shaded for each protein. The mean and standard deviation is representative of estimates from 50 docked models..
ORP5 and ORP8 localize at ER-PM and ER-mitochondria contact sites.Figure 1(A) Domain structures of human ORP5 and ORP8. PH, Pleckstrin homology domain; ORD, OSBP-related lipid binding domain; TM, transmembrane domain. (B) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 showing variable expression levels. Arrows indicate ORP5 and ORP8 localization to cortical ER (cER). Scale bar, 10 μm. (C-D) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]). (G) Representative electron microscopy of mitochondria and (H) quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells expressing EGFP-ORP5, EGFP-ORP8 together with HRP-KDEL, to visualize the ER. Control (Ctrl) consisted of HeLa cells overexpressing only HRP-KDEL. (I) Crude mitochondria, mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein (25g) from each fraction were loaded on a 4-20% gradient SDS-PAGE gel and immunoblotted using anti-ORP5, anti-ORP8 and anti-IP3R-3 (MAM protein), anti-PDI (ER protein) and anti- cytochrome c (mitochondrial protein). TC,Total cell; MAM, mitochondria-associated ER membrane .
Figure 1A Molecular structure of the HIV-PI Nelfinavir (NFR) and schematic representation of the protocol used to generate NFR resistant clones. HeLa cells were maintained with 10μM NFR. After 15 days, few clones proliferating in the constant presence of 10μM NFR in the culture medium were selected and expended for further analysis. B Dose response curves for viability of parental cells (in red) or selected clones (in black) upon treatment with NFR for 48h. The green dashed box highlight concentration in the physiologically relevant range. Curves are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone. Bar graph represents the EC50 (half maximal effective concentration) of the dose responses. Data are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 1way ANOVA analysis of variance (P value in red) followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01. C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real-time PCR relative to β-actin (mean and s.e.m. from 3 independent batches of cells are shown). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level. Tubulin is used as loading control. Histogram represents mean and s.e.m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates (see also source data). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *** P value ≤ 0.001.
Snai1 function is essential for in vitro organoid formation and survivalVillinCreERT2 Snai1fl/fl organoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls.Figure 4VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002. Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.
RecA pulsing is predictive of cell fate in M.Figure 4Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm. Representative time traces of individual lineages of cells fed with fresh medium (C) or spent medium (D). Different styles are used for lineages with different fates: death and lysis (L); SYTOX-positive (SX+); and survival (S). Line breaks represent division events in the same lineage, where a single random daughter cell is tracked for clarity. Vertical bars represent fresh (white) or spent (dark shading) 7H9 medium, CIP (yellow), and SX (blue). Comparison of single-cell growth rates prior to CIP exposure for "D" cells that die and "S" cells that survive and regrow after CIP washout (E), and lag time of regrowth (F). Cells were cultured in fresh (EXP) or spent (STAT) flow medium. Gray and black lines indicate mean ± SD. Significance by Kruskal-Wallis and Dunn's multiple comparison test (E) and unpaired Mann-Whitney test (F): ns, not significant; ****p < 0.0001; 6 < n < 84. The data shown are from 2 independent experiments. Single-cell fate is independent of the growth rate before exposure to the drug. However, cells cultured in spent medium (STAT) grow at significantly slower rates, survive at higher frequencies, and recover faster after CIP washout compared to cells cultured in fresh medium (EXP). Representative GFPdes (G) and mCherrywt (H) fluorescence time-traces for single-cell lineages of cells cultured in fresh medium (EXP) and cells cultured in spent medium (STAT), and having different fates. Vertical bars are color-coded as in C and D. Single-cell fate as a function of VMR of P2P1recA-GFPdes (I) and RecA-mCherrywt (J) fluorescence prior to CIP exposure. Significance by Chi-square test of independence for cells that die (n = 84) and cells that survive (n = 54). The data shown are from 2 independent experiments. Cells that do not experience DNA damage prior to drug exposure (RecA-non-pulsing cells) survive more than cells that do experience DNA damage (RecA-pulsing cells).
Figure 1(A-C) Representative images of Cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti TLR2 antibody following by staining with APC-labeled secondary antibody or (B) with TLR1 antibody following by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labelled with lipid dye, DiD (C). Then rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars 10µm. The mean Pearson correlation coefficient observed of TLR2-p or scrTLR2-p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S.EM (n=19-61).
Unipolar insertion and low mobility of LPS.Figure 6Localization of S-LPS (green) in the eFluor-labeled (magenta) inducible rough strain Δgmd plac-gmd possessing the old pole marker PdhS-mCherry (red). The strains Δgmd and Δgmd plac-gmd -IPTG were used as negative controls. Scale bars: 2 μm. Quantification of S-LPS insertion from (A). Numbers are indicating mean percentages. n = 468 bacteria (3 biological replicates). Demograph represents fluorescence profile of S-LPS labeling from (A). Cells were oriented with the old pole at the bottom of the graph by PdhS-mCherry and sorted by cell length. NFI: normalized fluorescence intensity. n = 468 bacteria (3 biological replicates). Short pulse labeling of LPS (2 h) by Kdo-N3 (green) on eFluor-labeled (magenta) bacteria expressing the old pole marker PdhS-mCherry (red). Scale bars: 2 μm. Quantification of LPS insertion sites from (D). Numbers are indicating mean percentages. n = 566 bacteria (3 biological replicates). Demographs represent fluorescence intensities of eFluor (OM, left) and Kdo-N3 (LPS, right) labeling. Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS-mCherry localization. NFI: normalized fluorescence intensity. n = 566 bacteria (3 biological replicates).
Figure 8Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A) Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test). (C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm. After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D). With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).
Figure 6(A) Immunocytochemistry of patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) and healthy kidney tissue-derived control tubuloids (CNTSWT-1 and CTNSWT-2) for PAX8, TP63 and F-actin. Scale bar = 100 µm. (B) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in control and cystinotic tubuloids (n = 3). (C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3). (E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3). (F) Representative images of control and cystinotic zebrafish. (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in control and cystinotic zebrafish (n = 3). (H) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in cystinotic zebrafish after treatment with cysteamine (1000 µM), bicalutamide (10 µM), and a combination of cysteamine and bicalutamide (1000 µM and 10 µM, respectively) (n=40 embryos per group). (I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group). (J) Deformity rates in ctns-/- zebrafish after treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group). (K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).
Figure 2(A) Actin co-sedimentation assay shows that CA7 binds to F-actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4) n = 3 independent replicates at each actin concentration, two-way ANOVA, P < 0.001. (B) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5 and 23 min). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. (C) Quantification of the mean increase in filament length (n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross-sections for individual filaments /bundles (n = 30 - 31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within-unit factor and the presence of CA7 as a between-unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non-significant. (D) Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 minutes. Scale bar 5 μm (full height is 20 μm). F-actin bundles can be detected as clear lines in kymographs. (E) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22 min -23 min 20 sec). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. This is a representative video from 8 similar experiments.
A common gene regulatory program governs naïve state maintenance, EpiSC resetting and somatic cell reprogramming.Figure 7(A) 0.717 cABN predictions compared with published data on gene combinations that do (dark green) or do not (white) enable MEF reprogramming (B) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in LIF containing medium n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Red dashed lines indicate empty vector + OSKM (C) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in 2i+LIF. n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Empty vector + OSKM reprogramming in LIF ('control no 2i') was included as a control for the effect of 2i addition. Red dashed lines indicate empty vector + OSKM control level. Box plots indicate min, median (D) Recapitulation of the gene activation kinetics during MEF reprogramming. Top, the number of regulation steps required for permanent activation of the indicated gene. Tfcp2l1 and Sall4 are found to activate earlier than Nanog and Esrrb. Bottom, gene expression measured from sorted populations of reprogramming intermediates from O"Malley et al. ( (E) Delineation of gene activation at single cell level. Top, experimental scheme used in Buganim et al. (2012) for the isolation of reprogramming intermediates profiled by single-cell RT-qPCR. Bottom, comparison of the predictions of the sequence of gene activation between gene pairs (left) along the reprogramming trajectory in OSKM+LIF, compared with experimental measurements from Buganim et al. (2012). Each table shows the percentage of single cells at the indicated stage of reprogramming (column) that have the indicated expression state of the gene pair considered (row) (F) Summary of the predictive accuracy of the three cABNs progressively refined against experimental results, with the 0.717 cABN having the highest predictive accuracy for each set of the investigation