EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3). (B) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates. (C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody. (D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (D) Experimental protocol (D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5). (D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in spleniciNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.
Figure 3(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C. (B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm. (C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments. (D) Schematic representation of experiments (E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.
Figure 4This single cell example shows release dynamics of two synapses innervating the same IHC from either pillar or modiolar side. iGluSnFR signals (bandstop filtered at 33.3 Hz) in response to 50-ms-long step depolarizations to the given depolarization voltages are depicted (ruptured patch-clamp, 10 mM intracellular EGTA, 5 mM [Ca2+]e). The middle panel shows ΔF images of iGluSnFR, recorded from the mid-section of the IHC. * depict the first detected response in the given synapse. Note that pillar synapse 1 is already active at -49 mV, while modiolar synapse 2 starts responding only at -41 mV. (Scale bar 2 μm) A' The normalized iGluSnFR-AUC as a function of depolarization voltage. Dynamic range and V1/2 of the synapses are depicted in the lower panel. Left panel shows the linear regression analysis (solid lines) of V1/2 of synaptic Ca2+-influx (B), glutamate release (C), threshold (D) and dynamic range of release (E) as a function of position along the pillar-modiolar axis. Shaded areas depict the associated 95% confidence intervals. Significance of correlation coefficients is reported by a two-tailed p-value. Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. For comparison of pillar and modiolar synapses, either student's t-test (for normally distributed data) or Mann-Whitney-U test (for non-normally distributed data) was applied. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001
Figure 2Left: cylinder representation of the APC/C-Nek2A complex structure. Apc2 (light blue) and Apc4 (light brown) are highlighted, the rest of the APC/C is shown in transparency. (i) Two different views of cryo-EM density for the Apc2 and Apc4 subunits. Secondary structure elements of the Apc2WHB domain are highlighted. (ii) Cryo-EM density for the second MR tail of Nek2A.   Details of the second MR tail binding site formed by the Apc2 (including the repositioned Apc2WHB) and Apc4 subunits. Nek2AMR2 is shown in orange.   Nek2A ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants. APC/C2m is the N392A/E395A mutant of Apc2. APC/C2/4m is the N392A/E395A/R48A/H53A/S51A mutant of Apc2 and Apc4. Securin ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants.
a) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top).figf6(a) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top). (b) Luciferase activity in Jurkat cells transfected with the fusion in a (Slamf1-CD3ζ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ (ΔIgV-Slamf1-CD3ζ), and luciferase reporters as in a, then exposed to heat-killed E. coli F18. (c) Luciferase activity in Jurkat cells transfected as in a, then left uninoculated (−) or inoculated with 10 × 108 E. coli F18 (+), followed by the addition of monoclonal antibody (mAb; amount, under graph) 9D1 to SLAM (α-SLAM) or rat immunoglobulin G2b isotype-matched control antibody (Rat IgG2b). (d) Luciferase activity in Jurkat cells transfected as in a, then exposed to medium alone (Med), wild-type E. coli (JM101), or JM101 E. coli mutants lacking both Omp C and Omp F (HN705) or lacking either OmpC (ΔOmpC) or OmpF (ΔOmpF). Data are representative of at least three independent experiments (error bars (a), s.d.).
a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining.figf4(a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining. Values are means ± s.e.m. of three independent experiments. Bax transfection was used as a positive control. (b) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h. Quantitative analysis of these experiments is shown in a. Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining. Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis (nuclear condensation, cell shrinkage; approximately 80% of the cells). (c) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer. Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days. Morphological analysis (Diff-Quik) is also shown and revealed that the type of death was apoptosis (nuclear condensation, cell shrinkage). (d) CEM cells that either lacked Bcl-2 or expressed high levels of Bcl-2 received truncated Atg5 or GFP by lentiviral gene transfer. In the absence of Bcl-2, induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days. Apoptosis was largely blocked in cells expressing high levels of Bcl-2. GFP transfection did not affect viability of the cells in this system. (e) Staurosporine-induced Atg5 cleavage (5 h treatment) in CEM cells independent of Bcl-2 expression. Blots were stripped and reprobed with anti-GAPDH antibody. (f) Detection of autophagic activity by staining of acidic vesicular organelles (VAOs, red) followed by flow cytometry analysis in genetically modified HeLa cells. That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy (data not shown). (g) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities. Values are means ± s.e.m. of three independent experiments (lentiviral gene transfer for 24 h). The asterisks indicate P 0.05. n.s., no significant difference. The scale bars represent 10 μM.
Figure 1GSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled "miR-10b-i") or non-targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock). Statistical significance of the difference was determined by Student's t test, with p-values < 0.001 indicated by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.A. Cell viability was monitored at day 5 after transfection as described in Materials and Methods. B. The number and size of GSC colonies were monitored at day 5 after transfection. C. MiR-10b inhibition induces cleavage of caspases 3 and 7 in GSC, as determined by Western blot analysis at day 5 after transfection with the inhibitor. The signals were quantified using ImageJ and normalized to beta-actin. D. Flow Cytometry analysis of Annexin V and 7-AAD staining of GSC GBM8 at day 5 after miR-10b inhibition.
Figure 9A, B. miR-10b inhibition decreases GL261 cellviability. Cellviability was measured at days 3-7 after transfection with miR-10b inhibitor, non-targeting control, or Lipofectamine 2000 alone (mock).A. Phase contrast photographs of GL261 cultures at day 6 post-transfection.B. Growth curves of cultured GL261 cells, based on the viability assay. C. InVivo-jet PEI - formulated 2'-O-MOE-PS/PO miR-10b inhibitor or non-targeting control were infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS-containing oligonucleotides (green). GL261 tumor cells expressing M-Cherry are red. D. Osmotic delivery of miR-10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice. Micephotographs show tumor imaging in representative animals at day 3 after pump implantation, and average signals in photons per second are indicated. Tumorgrowth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment. E. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b. F. qRT-PCR analysis demonstrates that miR-10b target p21 was de-repressed in GL261 tumors upon miR-10b inhibition. mRNA expression levels were normalized to GAPDH.
Figure 2 (A) Schematic presentation of intracranial tumor implantation (B) Overexpression of human EGFL7 (hE7) or murine EGFL7 (mE7) increased tumor growth (n = 9; one-way ANOVA) (C) Immunocompetent mice bearing GL261 tumors. Representative MRI images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 34 d (control) vs. 29.5 d (hE7) vs. 31.5 d (mE7); n = 8; log-rank test). (D) Immunodeficient mice bearing U87 tumors. Representative magnetic resonance images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 70 d (control) vs. 42.5 d (hE7) vs. 40 d (mE7); n = 8; log-rank test) (E) miR-126 KO did not affect overall survival of GL261 tumor-bearing mice (n = 8) (F) EGFL7 KO prolonged overall survival of GL261 tumor-bearing mice as compared to wild-type (WT) littermates (44 d in KO vs. 35.5 d in WT; n = 8 each; log-rank test) (G) Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus-based approach. Knock-down of EGFL7 (shE7_1 or shE7_2) prolonged the median survival time of glioma-bearing mice (68 d or 69 d) as compared to scrambled control (shScr; 56 d; n = 6; log-rank test) (H) The blood vessel-specific and miR126-independent KO of EGFL7 in EGFL7fl/fl;Cdh5-CreERT2 mice prolonged the overall survival of GL261 tumor-bearing mice as compared to WT littermates (38 d in KO vs. 33 d in WT; n = 7 for KO; n = 5 for WT; log-rank test). All data are presented as mean ± SEM. Scale bars represent 5 mm
Figure 2(A) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming. The boundaries of the early-iPSCs formations are indicated by a blue dashed line. Scale bars: 100 μm. (B) Illustration of the experimental set-up for the single-cell Taqman-based qPCR assay. (C) Bar graph showing the percentage of cells isolated from early-iPSCs colonies or outside of the colonies (rest of population) expressing each of the 9 TRs. Data are shown as mean ± SEM of at least two independent experiments. 0.05 < (*) p < 0.1, *p < 0.05, **p < 0.01 by unpaired Student's t-test or Welch's t-test, when appropriate. (D) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs. Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments. Data are shown as mean ± SEM of at least two independent experiments. Anova test p<0.0001. (E) Schematic representation of the statistical analysis estimating the probability of co-expression of the nine TRs per cell within the early-iPSCs colonies. Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs (shown with different color) in 282 cells (right side legend). The co-expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure. The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells, while the right part depicts the co-expression of combinations of the nine TRs based on double and triple single-cell experiments. (F) Venn diagram model depicting the co-expression of the nine TRs in cells lying within the early-iPSCs colonies on day 6 of reprogramming. The area of each circle represents the percentage of cells expressing each TR, as determined from the single single-cell experiments and the overlapping areas between circles represents the percentage of co-expression of two or three TRs, as revealed from our double and triple TR single-cell RNA expression experiments. The shaded blue area highlights the percentage of cells co-expressing all the nine TRs. co-expression of the nine TRs in cells lying outside the early-iPSCs colonies.
Promoting macrophage recruitment in glioma drives early dysmorphia of the tumor blood vessel network.Figure 6A. MRC1 immunohistochemistry on sections of 3 weeks growth glioma in ROSAmT/mG mice treated with recombinant CSF1 or carrier. Recombinant CSF1 treatment induces blood vessel enlargement in early tumors (50µm depth stack). B. Quantification of Tomato positive macrophages. Recombinant CSF1 treatment induces increased macrophage recruitment compared to control (n=6 mice per group). Statistical analysis: B.D. one-way ANOVA followed by multiple comparisons Tukey's test; Error bars: B-C: meanSD; C. Vessel diameter quantification. Recombinant CSF1 treatment drives blood vessel diameter increase compared to control (n=6 mice per group). Statistical analysis: C. t-test. Error bars: B-C: meanSD; D. Tumor volume quantification in 3 weeks glioma implanted mice treated with recombinant CSF1, anti-CSF1 antibody or control antibody. Recombinant CSF1 treatment reduces tumor growth compared to control (n=6 mice per group). Statistical analysis: B.D. one-way ANOVA followed by multiple comparisons Tukey's test; Error bars: D: median interquartile. Scale bar: 100µm.
TBK1 expression leads to suppression of cytotoxic effects: (A, B) Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead.Figure 4(A Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (A) The percentage of cell death at DIV14, after co-transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 (n=3), B) Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (B) The percentage of cell death at DIV7, after co-transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 (n=8). (C) Quantification of cell death by a TUNEL assay in rat primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed at DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3). (D) The percentage of cell death., quantification by a nuclear condensation assay in mouse primary cortical neurons co-transfected with the indicated HTT N586 and kinases at DIV5, cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7. Cells were fixed, and the nuclei were stained with Hoechst dye (n=3). (E) Quantification of cell death by a TUNEL assay in mouse primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed on DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).
Figure 1MSCs were exposed to increasing LPS concentrations, thereafter washed and co-cultured with PMA activated neutrophils. Incubation of neutrophils with PMA alone served as positive control and DMSO treated neutrophils as negative control. Scale bars: 50μm. Quantification of NET bound elastase indicative of NET formation. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates. Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA-histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non-primed MSC (middle panels) or PBS injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10μm (upper row) and 50μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets.
Figure 1Figure 1. Cytoplasmic conical structures in Thermococcus kodakaraensis. (A) A tomographic slice shows a side view of a conical structure (c) in the cytoplasm, rotated and enlarged in (B). (C) A tomographic slice shows a side view of the cone in another cell, highlighting the subunit texture along the edge of the cone (arrowheads). (D, E) Top views of a cone at different heights show the inner ring (r; enlarged in inset to highlight 19-subunit structure) and outer cone. (F-H) Sequential slices through a side view of a cone in a lysed cell show the relative location of the ring in the cone. (I, J) Different views of a 3D segmentation of the cone shown in (A), embedded in a tomographic slice. s, S-layer; m, membrane; a, archaella; rib, ribosomes; rez, ribosome-excluding zone. Scale bars 100 nm; scale bar in (D) applies to (D, E); scale bar in (F) applies to (F-H); segmentation not to scale.
a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis.f4(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies. (b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin. (c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test. (e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm. (f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.
Figure 9A-C. The single or double-cysteine Bax proteins with or without the indicated mutations were synthesized in vitro, activated by tBid protein and targeted to the Bax-/-/Bak-/- mitochondria. Cytochrome c release from the mitochondria was measured using ELISA. The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B, and the lines are the means. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.
Atg38 is a novel component of complex I.Figure 1.(A) Proteins detected in Vps34-TAP immunoprecipitations by MS analysis are listed. The number of identified peptides of each protein and the peptide coverage are also shown. (B) Cells expressing TAP-tagged Atg38 and the indicated HA-tagged proteins were grown in YPD. The extracts were prepared and were immunoprecipitated by IgG-Dynabeads as described in Materials and methods. The whole-cell extract (left, "input") and the precipitated proteins (right) were analyzed by immunoblotting with the indicated antibodies. (C) Relative abundances of proteins identified by LC-MS/MS of Atg14-TAP and Atg38-TAP purifications from YPD-grown cells. Relative abundances are defined as emPAIprey/emPAIbait and are shown as mean ± SD of two independent experiments. (D) Cell extracts were prepared from wild-type and atg14Δ cells expressing Atg38-TAP as well as HA-tagged proteins of complex I components. The IgG Dynabead-precipitated proteins (bottom), together with the whole-cell extracts (top), were immunoblotted with the indicated antibodies. (E) ATG38-2GFP ATG17-2mCherry cells in wild-type and atg14Δ were cultured at 30°C with or without rapamycin. After 180 min, cells were analyzed by fluorescence microscopy. Bar, 5 µm.