EMBO/sd-character-level-ner huggingface dataset:
Apical actomyosin shows a dynamic relocalization during early egg chamber development.Figure 6Live confocal imaging of Myosin-II shown by Zipper::YFP (A at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6 Live confocal imaging of Myosin-II shown b Spaghetti squash-mCherry (B) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6. (B) The localization Sqh-mCherry is shown relative to adherens junction marked by Shg::GFP
Figure 3A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm. B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm. C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control. D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.
Cell lines derived from ALKAL2-driven NB respond to ALK TKI treatment.Figure 6Murine NB cell lines were generated from tumours arising in Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) mice. A. Alkal2 expression in cells derived from Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) NB. Immunoblotting analysis for ALKAL2 and tubulin in the indicated mouse NB cell lines. Whole cell lysates (30μg) were analysed in each lane. B. The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) cell lines. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test. C-D. Brigatinib suppressed tumour spheroid formation and spheroid viability. Cells (#3456 or #3540) were treated with brigatinib (0, 150nM) for 4d in ultra-low attachment plates. The spheroid number was analyzed by IncuCyte® Live Cell Analysis Systems. Data are presented as means ± SEM from three independent experiments. *p<0.05, two-tailed unpaired student t-test. Scale bar (C) is 200μm. E. Tumour spheroids formed from either Rosa26_Alkal2;Th-MYCN (#3540) or Alk-F1178S;Th-MYCN (#3456) were formed at indicated cell number in ultra-low attachment plates for 3d and followed by brigatinib (0, 150nM) for 10d. Inhibitor was re-fed every other day. Cell viability was determined by CellTiter-Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments. *p<0.05, ****p<0.0001, two-tailed unpaired student t-test. F. Cell viability. Mouse tumour derived cell lines #3540 (Rosa26_Alkal2;Th-MYCN) and #3456 (Alk-F1178S;Th-MYCN) were treated with brigatinib (125nM and 500nM) and viability was evaluated by using a resazurin-based assay. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test. G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.
Figure 8SCFFBW7α together with GSK3 regulate SOX9 protein levels through the ubiquitin-proteasome system. FBW7α mutations, such as those detected in SHH-medulloblastoma, lead to stabilization of SOX9 protein and subsequently promote SOX9-driven EMT-like reprogramming in medulloblastoma. Small molecule inhibitors of the PI3K/ mTOR pathway can be used to promote GSK3 / FBW7α-mediated SOX9 turnover and may provide a novel strategy to target SOX9-driven medulloblastoma.
Figure 2Schematic diagram (A) of IFN-γ release assay IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C). Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test). D-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).
Meningeal macrophages display subtle changes through the absence of the microbiota under steady-state conditions.Figure 5(A) UMAP plot illustrating the color-coded aggregated transcript counts of published core signature genes for CAMs. The scale bar represents color coded sequencing depth-corrected log transformed transcript counts. (B) UMAP representation of 545 individual cells of SPF (green dots) and GF (yellow dots) mice. (C) UMAP representation of the meningeal macrophages of SPF and GF mice color-coded for the clustering results of the Seurat algorithm. The mappings between colors and clusters are shown on the right. The cell type annotations are based on published gene expression profiles. (D) Stacked bar plot (Marimekko chart) color-coded for relative contribution of meningeal macrophages derived from SPF (green) and GF (yellow) mice. P-value ranges are provided for significantly enriched regions per cluster, based on hypergeometric tests. Benjamini-Hochberg correction was applied to correct for multiple testing (*P < 0.05; ***P < 0.001). (E) Volcano plot of the differential gene expression analysis of of n=144 SPF and 87 GF mMΦs based on Wilcoxon Rank Sum tests. The shown genes are filtered based on an average log2 fold change (avg. log2FC) > 0.2 and adjusted p-value < 0.05. (F) Heatmap of the top 10 differentially expressed genes per cluster (adjusted p-value of <0.05 based on Wilcoxon Rank Sum tests). The scale bar represents color-coded Pearson residuals. (G) UMAP maps color-coded for transcript counts of genes enriched in subsets of the captured cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts. (H) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm. (I-O) Quantification thereof. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments.
Gene expression profile of P240 PR-SET7ΔHepA livers and comparison with transcriptomes of ductal progenitor cells.Figure 8Gene expression profile of P240 PR-SET7ΔHepA livers and comparison with transcriptomes of ductal progenitor cellsA Overexpression of stem cell-specific, oncofetal hepatic and proliferation-dependent genes in P240 PR-SET7ΔHepA livers. Volcano plot of normalized RNAseq data obtained with P240 wild-type and PR-SET7ΔHepA livers. Significance cutoff value was set at P = 0.05 in -log10 scale (horizontal dashed line) and fold change cutoffs at −0.6 and 1 in log2 scale (vertical dashed lines).B Volcano plots showing dots corresponding to representative stem cell marker genes (red), oncofetal hepatic genes (blue) and proliferation-dependent genes (black) are indicated.C, D Comparison of differentially expressed genes in P240 PR-SET7ΔHepA livers with those reported in Lgr5-LacZ+ sorted cells or MIC1-1C3+ sorted ductal cells (Huch et al, 2013).
Oligomerisation of BAK restricts its ubiquitination and induces Parkin activity.Figure 4(A) BN-PAGE and SDS-PAGE of HeLa+HA-Parkin cells treated with 1 µM of each of ABT-737 and S63485 for 3 h (BH3), antimycin A and oligomycin (AO) for 2 h or a combination of the two. Experiment was performed in the presence of 10 µM QVD.oph and immunoblots are representative of three independent experiments. (B) UBA enrichment of ubiquitinated proteins from HeLa+HA-Parkin cells, BAX-/- BAK-/- (DKO) HeLa cells or wild-type (WT) HeLa cells expressing HA-Parkin in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of QVD.oph (10 μM). Representative of three independent experiments. (C) HeLa cells expressing HA-Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non-targeting sgRNA control, treated with AO (2 h) to stabilise PINK1 expression and immunoblotted for PINK1. (D) Parkin activity induced by apoptotic mitochondrial damage is PINK1-dependent. UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of Q-VD.oph (10 μM). *, ubiquitinated protein. Representative of two independent experiments. (E) Schematic showing Parkin ubiquitination of BAK monomer in non-apoptotic cells, but not the BAK homo-dimer in cells undergoing apoptosis. In response to mitophagy stimuli, PINK1 phosphorylates ubiquitin to recruit Parkin, which in turn is phosphorylated by PINK1 to become activated to ubiquitinate monomeric BAK on K113. Mitochondrial outer membrane (MOM) permeabilisation driven by BAK oligomers during apoptosis provokes Parkin activity in a PINK1-dependent manner. Although Parkin can ubiquitinate mitochondrial outer membrane substrates (e.g. Mfn2) to promote mitochondrial clearance, it cannot ubiquitinate dimerised BAK on K113.
Figure 4A. Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes), Prospero (red nuclei of EEs) and Dronc-TurboID (green shows Streptavidin labelled proteins by Dronc-TurboID); note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells (white arrows indicate an intestinal progenitor cell upregulating Dronc). Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes) and Actin-Dronc-GFP-Myc (green, anti-GFP); note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells (arrow). High magnification of the picture shown in B. The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP+ EBs. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc. Transversal Z-section of the cells shown in C. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc. E. The enteroblast marker Su(H) (red, immunostaining against Beta-galactosidase) strongly co-localises with high levels of Dronc expression (green, immunostaining against GFP); white arrow indicates the enlarged area depicted in the insets. F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.
a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP–p27, YFP–p27T198A or YFP–p27T198D and selected with G418 for three weeks.figf2(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP-p27, YFP-p27T198A or YFP-p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3). (b) Flow cytometry analysis of MCF-7 cells transfected with YFP (C1) or YFP-p27, YFP-p27T198A and YFP-p27T198D for 48 h. Data are presented as mean ± s.d. (n = 3). (c) GFP-LC3 U2OS cells 48 h post-transfection with RFP, RFP-p27, RFP-p27T198A or RFP-p27T198D. Data represent results of three independent experiments, from which a total of ∼200 cells were counted (s.d., 10∼15%). The scale bars represent 50 μm in c.
Figure 1A. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates. B. Schematic summary of the assays used in this study. Preproteins (purified commonly in 6 M urea; except in Appendix Fig. S6B) were diluted in an aqueous buffer (final urea < 0.2 M) and their physicochemical properties and functional interactions with Sec system components analysed. Y: SecYEG; A: SecA. C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown. D. proOmpA translocation ATPase activity stimulation of SecA (as in C) in the absence or presence of TF2 or/and SecB4, added at the indicated molar excess over proOmpA1. The T/M ratios were calculated; the one in the absence of chaperones was considered as 100%, all other values were expressed as % of this. n= 2-4 biological replicates. Mean values (± SEM) shown. Unpaired parametric t-test, 95% confidence interval: ns: not significant (p = 0.1032); ****: p < 0.0001; **: p = 0.0037. E. Titrated TF-mediated inhibition (0-6 µM range) of proOmpA-stimulated translocation ATPase activity (as in C). Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 1-4 biological replicates; mean values ± SEM) versus the log10[TF2]. The Ki was determined using a variable slope fit (log[inhibitor] versus normalized response; GraphPad Prism). F. SecB-mediated relief of TF-mediated inhibition of proOmpA translocation ATPase activity(as in C). TF2 (1μM) and SecB4 (0-6 µM) were used. The T/M ratios were calculated, normalized (as in D) and plotted (n= 2-3 biological replicates; mean values ± SEM) versus the log10[SecB4]. An apparent relief constant (Krel) was determined (as in E).
Figure 5A HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were stained for 1 hour with fluorophore-labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated. B ACE2 competition experiments with selected VHHs from A. 50 nM RBD was mixed with indicated nanobodies (at 500 nM). Binding to ACE2 (immobilized on the sensors) was monitored by BLI.
Figure 6A. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). C C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm. F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm. G. The graph showing the ratio of intensities of JC-1 aggregates to those of monomers in (E). H, I. Box plot showing the ATP contents of mouse GV oocytes (H) or MII oocytes (I) from wild-type (WT) and Erk1/2oo-/- oocytes (N number was labeled in each graph).
Figure 3Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments. Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments. DC3.2-Rab39a cells were induced to express Rab39a overnight. Cells on coverslips were washed, fixed and stained according to the listed protocol and antibodies. Green = Rab39a (HA tag), Magenta = Lamp2. Arrows highlight punctate structures with colocalization. Scale bar = 10 µm. Representative image of two independent experiments. DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments. 5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments. 1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.
a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h.f6(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3) P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm. (b) The PF/EF ratio of the PtdIns(3) P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.
A model for division orientation in the follicular epithelium.Figure 8Orientation is achieved separately in the apical basal and planar axes. Pins/Mud in green, Apical actomyosin in red, microtubules in blue
cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.Figure 6cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.
Figure 4(A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis. (B) H&E and SOFG staining of spine in PGIS model.  (C) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 6 cages per group, Fisher's exact test. (D) Quantitative analysis of structural parameters of new bone by μCT analysis. n=5, one‐way ANOVA, Bonferroni post hoc.  (E) μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction). (F) Incidence of new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test. (G) Quantitative analysis of structural parameters of new bone by μCT analysis. n=9, one‐way ANOVA, Bonferroni post hoc. (H) H&E and SOFG staining of new bone in hind paws. Data information: CB: Cortical bone; NB: New bone (I) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection. (J) H&E and SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity. (K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc.