EMBO/sd-character-level-ner huggingface dataset:
A wide-range functionality of the inducible VpT6SS1-based platform in V.Figure 3A, B Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05. C-F Viability counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.
Effect of YAP on human cervical tumor growth in vivo.Figure 4A, B Representative images of tumorxenografts of ME180-MXIV (MX, right flank), ME180-YAP (A) (YAP, left flank), and ME180-YAPS127A (B) (YAPS127A, left flank) cells implanted in athymic nude mice. Red lines indicate the edge of tumors.C The growth curve of humantumorxenografts derived from control (ME-MX) and YAP-overexpressing ME180cervical cancer cell lines (ME-YAP, ME-YAPS127A) implanted in the athymic nude mice. Each point represents mean ± SEM of six tumors (control n = 10). D Representative images showing the relative size of tumors from control and YAP-overexpressing ME180 cervical cancer cells.E The average weight of tumor xenografts from the control and YAP-overexpressing ME180 cell lines. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents the mean ± SEM (n = 10 for control, n = 6 for YAP and YAPS127A groups). Bars with different letters are significantly different from each other (MXIV vs. YAP, P = 0.0008; MXIV vs. YAPS127A, P < 0.0001). F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells. G Expression of Ki67 in the tumor xenografts of ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells implanted in athymic nude mice. Ki67 was visualized using an Alexa-488 (green)-conjugated secondary antibody. Actin was visualized using an Alexa-594 (red)-conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 20 μm. Note the significant increase in the Ki67-positive cells in the ME180-YAP and ME180-YAPS127A tumor xenografts. H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector (shCtrl) or lentivirus-based YAP shRNAs (shYAP#1 or shYAP#2). I Representative images showing tumor xenografts derived from ME180-shCtrl cells (left flank) and ME180-shYAP#1 cells (right flank) (n = 6). J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA (left, shCtrl) or YAP shRNA#1 (right, shYAP#1) (n = 6).
Expression kinetics from a synthetic model system for ERK signal duration.Figure 1 Figure 1 - Expression kinetics from a synthetic model system for ERK signal duration. A: HEK293 with a stably transfected ∆RAF1:ER fusion protein were treated with ER antagonist 4-hydroxytamoxifen (4OHT, ON scenario in B). To generate pulses, ERK signalling was turned off using the MEK inhibitor U0126 (ON/OFF scenario in B). To distinguish between primary and secondary response genes, translation was blocked with cycloheximide (CYHX) in parallel to 4OHT stimulation (ON/CYHX scenario in B). In addition, we used actinomycinD (ActD) to determine mRNA half-lives via transcriptional shut-down and 4-thiouridine (4SU) to determine mRNA half-lives via metabolic labelling. B: Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two-hour pulse activation (ON/OFF scenario). Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes. Genes were ranked by their model-derived response time.
Figure 1(a) Schematic representation of the IgH locus of WT and of 8X-LexA binding element (LexA-BE) knocked-in CH12F3-2A cells. The a, b and c bars indicate the position of primers used for ChIP-qPCR in Fig. 1b. A protein in which the DNA-binding domain (DB) of LexA was tagged with 3X-FLAG and a nuclear localization signal (NLS) (FNLDD) was expressed in WT (FNLDD alone) and Sα-engineered (FNLDD- Sα-LexA) CH12F3-2A cells. (b) Chromatin immunoprecipitation using α-FLAG antibody was performed to confirm the pull-down of Sα chromatin from the Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The a, b, and c bars indicate the primers used for ChIP-qPCR as shown in Fig. 1a (n=3; mean + sd; two tailed unpaired Student's t-test). (c) Schematic representation of the iChIP coupled with SILAC based-mass spectrometry. (d) Functional classification of the Sα region binding proteins identified by iChIP. (e) Bar graph showing the proteins with ≥2-fold enrichment as calculated by the heavy to light SILAC ratio. (f) Effect of the siRNA-mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3-2A cells. Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=2; mean + sd).
A) The purity of the isolated lysosomes.Figure 5(A) The purity of the isolated lysosomes. Total cellular homogenates and lysosomal fraction (30 µg of proteins) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Actin was shown as a marker of cytoplasm. Hsp60 was shown as a marker of mitochondrial. LAMP-2A and Hsc70 were shown as markers of lysosomes. (B) The integrity of the isolated lysosomes. The release of β-hexosaminidase from lysosomes was indicated with incubation of lysosomes with para-nitrophenylphosphate (PNPP). The reaction was terminated with 0.25 mol/L NaOH. Release of all lysosomal β-hexosaminidase was induced by incubation with 0.1% Triton X-100. Release of all lysosomal β-hexosaminidase was induced by incubation with 0.1% Triton X-100. Values of β-hexosaminidase activity are expressed as percentage of total activity in the lysosomal fraction at time 0, 10, 20 and 30 min and are means±SE of three independent experiments. (C) Effects of Htt-552 on lysosomal stability. The release of β-hexosaminidase from intact lysosomes incubated with Htt-552-18Q or Htt-552-100Q. PC12 cells were transfected with Htt-552 for 48 h. Cell lysates were used as Htt-552-riched lysates. The lysates containing Htt-552 were incubated with isolated lysosomes and the lysosomal β-hexosaminidase activity at time 0, 10, 20 and 30 min was determined. Release of all lysosomal β-hexosaminidase from the same batches of lysosomes was induced by incubation with 0.1% Triton X-100. Release of all lysosomal β-hexosaminidase from the same batches of lysosomes was induced by incubation with 0.1% Triton X-100. The values are means±SE of three independent experiments. (D) Effects of lysosomes on the degradation of Htt-552. PC12 cells were trasfected with Htt-552 as described above and cell lysates were incubated with isolated lysosomes. The degradation of Htt-552-18Q and Htt-552-100Q after incubating with intact lysosomes was assessed with Western blot analysis. Values are the mean±SE of three independent experiments. **P<0.01 (compared with Htt-552-18Q without treatment); ##P<0.01 (compared with Htt-552-100Q without treatment). (E) Association of increasing Htt-552 with isolated lysosomes untreated (Binding) or pre-treated with protease inhibitors (Binding+Uptake) at 0 and 5 min. Values are the mean±SE of three independent experiments. (F) Association of increasing Htt-552 with isolated lysosomes untreated (Binding) or pre-treated with protease inhibitors (Binding+Uptake) at 5, 10 and 20 min. Values are the mean±SE of three independent experiments. **P<0.01 (compared with Htt-552-18Q without treatment); ##P<0.01 (compared with Htt-552-100Q without treatment). (G) Htt-552 in lysosomal membranes (MEMB) and matrices (MTX). Isolated lysosomes were incubated cell lysates containing expressed Htt-552 for 20 min, lysosomes were recovered by centrifugation. Lysosomal membranes and matrix were separated after hypotonic shock and centrifugation, and were processed for Western blot analysis.
The cysteine‐133 residue of Apg10p is essential for autophagy and the Cvt pathway.Figure 5(A) Autophagic activities were measured by an ALP assay ( Noda and Ohsumi, 1998). YTS3 cells (Δapg10PHO8::pho8Δ60) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600/ml in SC medium lacking tryptophan and then transferred to SD(−N) medium. Lysates from the cells after incubation for 0 and 4 h were used for assay. Error bars indicate the SD of three independent experiments. (B) Cell lysates from Δapg10 cells (TFD10‐L1) harboring empty vector, pRS316 (lane 1), wild‐type HAAPG10 (lane 2), HAAPG10C26S (lane 3), HAAPG10C133S (lane 4) or HAAPG10C137S (lane 5) on CEN plasmids were subjected to Western blotting analysis with anti‐API antiserum.
Figure 8A. Traces showing average changes (mean±sem) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of TG as indicated in the upper bar in SH-SY5Y S1-/- cells transfected with mcherry tagged constructs as indicated (n=46-105). B. Quantification of the Area Under Curve (AUC, mean±SD), calculated from traces shown in (A) with STIM1 (n=105), STIM1∆KK (n=56), STIM1A (n=75), STIM1A∆KK (n=46). ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test. Total n from three independent transfections with 2-3 technical replicates each. C C. Normalized endpoint NFAT translocation (mean±s.e.m) of control constructs and STIM variants with deleted PBD (∆KK) with or without addition of 1 µM PF-04957325, Kruskalis-Wallis Anova, n=18-19 for ∆KK constructs +/-PF, 56-120 for STIM1, STIM1A controls obtained from three independent transfections. **p<0.01, ***p<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. For clarity, not all differences are shown. D. Normalized endpoint NFAT translocation (mean±s.e.m) of cells transfected with indicated constructs that were treated ON with 1 µM ISA with or without addition of 1µM 6-Bnz-cAMP 30 min before measurement. * p< 0.05, **p<0.01, ***p<0.001, Kruskalis-Wallis Anova, n=26-50 from 3 biological replicates.
Ubiquitination of BAK on mitochondrial membranes and in cells inhibits its ability to mediate mitochondrial outer membrane permeabilisation and apoptosis.Figure 6(A) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK. (B) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function. Mitochondrially-enriched membranes from MEFs expressing BAK or BAK K113C were cross-linked (CuPhe) with UbG76C and treated with cBID for 30 min at 30°C. Supernatant (S) and pellet (P) fractions were then immunoblotted for BAK under non-reducing conditions, or cytochrome c or ubiquitin under reducing conditions. Representative of three independent experiments. Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS-PAGE. (C) Parkin-mediated ubiquitination limits BAK activation. HeLa+HA-Parkin cells were treated with antimycin A and oligomycin (AO) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT-737 and S63485 (BH3 mim) for 1 h. Intracellular flow cytometry was performed using the conformation-specific anti-BAK antibody G317-2. Representative histograms are shown and the fold increase in mean fluorescence intensity (MFI) is plotted from three independent experiments with error bars showing SD. Data are normalised to untreated control. (D) Parkin-mediated ubiquitination limits BAK apoptotic activity. BAK-/-BAX-/- HeLa cells expressing Parkin and BAK were treated with AO (2 h) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction. Error bars represent mean +/- SD. (E) Parkin activity limits cytochrome c release. HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments. Ubiquitinated BAK indicated (arrow). Graph shows densitometric analysis of three independent experiments showing cytochrome c release (supernatant/supernatant+membrane) relative to 100 nM cBID as 100% release. Error bars represent mean +/- SD.
The 2019-nCoV RBD/ACE2 binding interface compared with that of SARS-CoV RBD/ACE2.Fig. 3Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels. Sequence alignment of 2019-nCoV RBD and SARS-CoV RBD. Contacting residues in the 2019-nCoV RBD are indicated by black dots; contacting residues in the SARS-CoV RBD are indicated by red dots. Electrostatic potential map of 2019-nCoV RBD (left panel), and SARS-CoV RBD (right panel). The position of K417 in the 2019-nCoV RBD is indicated by black arrow. The N-terminal helix of ACE2 is shown as green ribbon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.
Figure 2The CVT path matches the polygon distribution of natural tissues. A) Polygon distribution Voronoi diagrams resulting from the iterative application of Lloyd´s algorithm. B-E) Density plot showing the frequency of a value for four, five, seven and eight sided cells respectively, depending of the percentage of hexagons (P6) in the CVT diagrams. The darker region reflects the higher probability of a determine value. Coloured circles represent the average values of each polygon class for the different sets of images analysed in this study. dWL, Drosophila larva wing disc, light green. dMWP, Drosophila mutant wing disc, violet. EYE, Drosophila eye disc, orange. BCA, biceps control adult, dark green. BNA, biceps neurogenic atrophy, dark blue.
Figure 1Colocalization of AcGFP-Siwi, AcGFP-BmAgo3 and mCherry-BmVasa in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant dissociates from BmVasa foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm. Colocalization of AcGFP-Siwi and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm. Colocalization of AcGFP-BmAgo3 and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm. Heatmap of colocalization ratio between fluorescence protein-tagged piRNA factors. Each colocalization ratio is quantified by using ImageJ plugin Comdet (see Materials and Methods) and averaged from n=3 independent z-stacks. Darker color represents more frequent colocalization. Known nuage proteins (BmVasa, BmAgo3, Siwi) form a cluster at the top-left corner. Siwi-D670A mutant, but not BmAgo3-D697A, clusters with BmSpnE, BmQin and BmDcp2 at the bottom-right corner, representing piP-bodies. See also Figures EV1 and EV2. Schematic diagram showing the proposed spatial compartmentalization of the BmN4 piRNA pathway. Nuage components: BmVasa, BmAgo3, Siwi; piP-body components: BmSpnE, BmQin, BmDcp2. Siwi-D670A mutation leads to the accumulation of the mutant itself in piP-bodies and colocalization with BmSpnE and BmQin. See also Figure EV1.
Figure 2A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate. B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT). C. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm. D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph. E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.
Apelin inactivation reduces sunitinib therapy-induced hypoxia and vessel permeability.Figure 5(A) Percentages of CA9+ cells adjacent to CD31+ tumor blood vessels in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=4); 100-200 peri-vascular intra-tumoral regions per group were counted. ***P<0.001; one-way ANOVA. Right panels show representative immunofluorescent images. Areas limited by dotted white lines indicate CA9+ areas. Scale bars = 50 μm. (B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of vessel permeability (K2) in NeuT+ mammary tumors followed over time. Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=5); ***P<0.001, compared to untreated control NeuT;Apln+/+ mice and §P<0.05 compared to sunitinib-treated NeuT;Apln+/+ mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analysed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus we did not perform any statistical analysis on the 6 weeks timepoints.
Mud does not determine planar spindle orientation in the FE.Figure 5A) The vertex long axis of cells does not correlate with the division angle in both the FE or ENE. The cell vertex distribution at interphase using both the vertex bipolarity and a modified method that takes into account the distance that vertices lie from cell barycentres (modified vertex bipolarity) does not predict the division angle. n=10 cells (FE), n=18 cells (ENE). Bars represent mean and standard deviation B, C) Pins and Mud are symmetric around the entire cell cortex in (B) mitotic follicle cells and (C) mitotic cells in the ENE. (B" and C") Confocal imaging of immunostained fixed tissue. Scale b D) Planar division orientation in the follicular epithelium is independent of Mud. Division angles were calculated relative to the elongating axis of the egg chamber using an unbiased analysis tool. Number of cells analyzed: w1118 n=52; mud3/mud4 n=43; Random number generator n=50; Mean division angle w1118= 31.6o, mud3/mud4= 30.9o, Random number generator = 46o; Wilcoxon Signed Rank Test, Theoretical Mean of 45, w1118 p < 0.0001, mud3/mud4 p = 0.0003, Random number generator p = 0.7779. Spindle angles in mud3/mud4 egg chambers are not significantly different from w1118 (p = 0.7859, Mann Whitney test). Spindle angles in w1118 and mud3/mud4 egg chambers are statistically different from the randomly generated list (p = 0.006 and p = 0.0044, Mann Whitney test) E) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0. Timepoints shown in minutes relative anaphase. Scale bars = 5 μm. F) The variance in mitotic spindle angle in the ENE reduces as cells approach metaphase. Graph shows root mean square of spindle angles approaching metaphase (defined at t = 0 secs). Number of cells analyzed= 22
Figure 6A. Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA (pulsed-field gel electrophoresis). B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L (pulsed-field gel electrophoresis). C. DRIP assay for DNA-RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9.6 antibody. Dot blots represent two replicates. Bars represent data from three independent experiments ± SD. The IgG signal was subtracted. Significance was determined using one-way ANOVA, p-values are indicated on the graph. D. C-circle (CC) assay for clone 35 upon POT1 removal. Bars represent data from three independent experiments ± SD. DNA from ALT positive cell line U2OS was used as positive control. Significance was determined using one-way ANOVA, p-values are indicated on the graph. In all experiments, POT1 deletions was induced with 0.5 μM 4-OHT for 4 and 7 days. E. C-overhang length analysis for clone 35 upon POT1 removal (pulsed-field gel electrophoresis, hybridization with Telo-G probe). F. Quantification and representative pictures for visualization of the PML protein (IF, green) and telomeres (FISH, red) in non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Arrows indicate co-localizations. Scale bar equals 10 µm. Bars represent data from three independent experiments ± SD. At least 150 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT. Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells, GRN163L - telomerase inhibitor Imitelstat, siNT -non-tagreting siRNA, negative control.
Transgenic flies (21D-GAL4>UAS-mCD8-GFP) with targeted GFP expression to L2 cell membranes.Fig 1(A)-The L2 cell bodies are located in the lamina cortex (*), axons with dendrites in the lamina synaptic neuropil (La) and terminals of axons in the second optic neuropil (Me-medulla); R-retina, arrows show the L2 cell terminals in the medulla. (B)-Cross-section of the lamina with GFP-labeled L2 cells with dendritic trees. (C)-A deconvolved image of L2 dendritic trees for which the perimeter was measured during analyses.