EMBO/sd-character-level-ner huggingface dataset:
Fibroblast proliferation and ECM changes during wound healing.Figure 5Experimental design of wound healing time course analysis. (A) Wound healing time course. Skin was collected at the indicated time points. (B) Quantification strategy is schematically illustrated. Red boxes upper dermis; black boxes lower dermis; solid lines inside and dashed lines outside the wound site. Fibroblast activation during wound repair. Immunofluorescence staining for α−sma (red) of PDGFRαH2BEGFP (green) wound sites at indicated post wounding (PW) times Nuclei were labelled with DAPI (blue) Fibroblast activation during wound repair. quantification of α−sma fluorescence intensity in pixel per area inside the upper and lower wound bed (n= 4 PW0, PW2, PW7, PW21; n= 3 PW10, PW14; n= 2 PW4 biological replicates) Changes in fibroblast density, proliferation and collagen deposition during wound repair. Immunostaining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin sections at indicated wound healing time points. Nuclei were labelled with DAPI Changes in fibroblast density, proliferation and collagen deposition during wound repair. Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates). Quantification of fibroblast density during wound repair inside and outside the wound bed (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates). Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding. Quantification of proliferating (Ki67 positive) keratinocytes and fibroblasts during wound repair over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).
Figure 4A The genomic structure of Gr28b. It is composed of five different forms by alternative splicing. The arrow head indicates the P-element insertion. White boxes indicate predicted untranslated regions in flybase. Arrows indicate primers for RT-PCR. B RT-PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control. From left to right, the band of tubulin of wild-type, tubulin of Gr28bMi, DNA ladder, Gr28b of wild-type, and Gr28b of Gr28bMi. Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4/UAS systems. (n ≥ 4). D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).
a, Western blot analysis of flag-tagged beclin-1-transfected.figf3a, Western blot analysis of flag-tagged beclin-1-transfected MCF7 clones. C, MCF7.control cells (clone 38); St, MCF7.beclin1stop cells (clone 70). Mr, relative molecular mass. b, Western blot analysis of flag-tagged beclin1stop-transfected MCF7 clones. C, MCF.control cells (clone 38); B, MCF7.beclin1 cells (clone 1). c, Photomicrographs of MCF7.control cells (left panel, clone 38), MCF7. beclin1 cells (middle panel, clone 17) and MCF7.beclin1stop cells (right panel, clone 70) 48 h after seeding at similar densities. d, Proliferation of MCF7.control cells (clones 37 and 38; closed squares and circles, respectively, solid lines), MCF7.beclin1 cells (clones 1, 6, 17; open squares, circles, triangles, respectively, solid lines) and MCF7.beclin1stop cells (clone 70, closed triangles, dotted line). Results represent mean Absorbance (A) (±s.e.m.) for six wells; similar results were obtained in three independent experiments. e, Cell viability of MCF7 clones determined by trypan blue staining. Symbols for each clone are as in d f, Clonigenicity in semisolid medium (soft agar) of MCF7 clones. Results are mean (±s.e.m.) for pooled triplicate wells from 3-4 independent experiments. g, Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones. Numbers on top of bar indicate number of autopsy-confirmed tumours at 8 weeks per number of mice injected with each clone.
Figure 8CD8+ LY CAR-T cells generated from human PBMCs were treated with PTPN2-inhibitor (+) or vehicle (-) followed by incubation with plate-bound α-LY, LY-negative MDA-MB-435 cells and LY-expressing OVCAR-3 cells. The proportion of IFNγ+ versus IFNγ+TNF+ CD8+CAR-T cells was determined by flow cytometry. HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs and intracellular PTPN2 levels were determined by flow cytometry. c-d) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored. HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored. CD45+CD3+CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 positive tumours and spleens by flow cytometry 21 days post adoptive transfer. HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with Cas9 and control or Ptpn2 sgRNAs using the Lonza 4D-Nucleofector and after two days intracellular PTPN2 levels determined by flow cytometry. HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 control HER-2 CAR-T cells or those in which PTPN2 had been deleted by CRISPR RNP. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and tumour growth monitored. HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 control HER-2 CAR-T cells or those in which PTPN2 had been deleted by CRISPR RNP. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and tumour growth monitored. g) CD45+ CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 tumours by flow cytometry 19 days post adoptive transfer.
a) Outline of freeze-fracture EM.f2(b) QF-FRL in wild-type yeast in log phase. PtdIns(3) P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm. c) Quantification of the labelling intensity of PtdIns(3) P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images. (d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.
Figure 5Bacterial inoculation promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B. subtilis. (A), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results. Bacterial inoculation promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B. subtilis. (B), The seedlings were harvested, and the shoot fresh weight was measured 4 weeks after inoculation. The data shown indicate the means ± SDs (n = 30, n refers to the number of seedlings per group), Central bands: median, boxes: interquartile range, whiskers: max./min; n.s., not significant; *, p < 0.05, **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. (C) Pto DC3000 and B. subtilis upregulate phosphate absorption gene expression. Col-0, phr1 and fer-4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B. subtilis in Pi-sufficient or Pi-deficient condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.
Loss of Grk2 impairs Hh signal transduction in zebrafish.Figure 2A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed. B Phenotype of wild type, MZgrk2, and grk2-GFP mRNA injected MZgrk2 embryos at 24hpf (n=20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200μm C In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild type, MZgrk2 and grk2-GFP injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right. (n=30 for each sample). Scale bars, 200μm (whole mounts), 50μm (sections). D Expression of Prox1a and Engrailed proteins (Eng) in somites of wild-type, MZgrk2 and grk2-GFP mRNA injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merge images with DAPI staining in blue. (n=10 for each sample). Scale bar, 50μm.
Quantitative assessment and xenograft tumor formation potential of progenitor-derived carcinoma cells.Figure 7A P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice. B Cell duplication rate of primary hepatocytes from P240 PR-SET7ΔHepA livers in culture. C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers or Hepa 1-6 cells. Right panel shows average tumor volumes (n = 3) dissected at the indicated time points following injection. D, E Hematoxylin and eosin and immunohistological staining with A6, HNF4, Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers.
Figure 3qRT-PCR measurement of of Ifnb1, Il6 and Il1b mRNA transcripts in mouse peritoneal macrophages left unstimulated or stimulated with LPS (0.1 μg/mL) (A-C) or transfected with Poly(I:C) (1μg/mL) The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. ns, not significant. qRT-PCR measurement of of Ifnb1 mRNA transcripts in mouse peritoneal macrophages left unstimulated or stimulated with LPS (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. ns, not significant. qRT-PCR measurement of Il6 and Il1b mRNA transcripts in mouse peritoneal macrophages left unstimulated or stimulated with LPS (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. ns, not significant. (G) qRT-PCR measurement of IFNB1 transcripts in HEK293 cells stably expressing Flag-STING that were left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) for 4 h in the presence of DMSO or Palbociclib at indicated concentrations. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. ****, P<0.0001; ns, not significant. (H-J) Fitted dose-response curves illustrating the relative level of the transcripts of Ifnb1 (H), Cxcl10 (I) and Il6 (J) in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1 μg/mL) in the absence or presence of indicated concentrations of Palbociclib and H151. The data shown are mean ± SEM of n=3 biological replicates. The IC50 was calculated and shown.
Working model.Figure 4Working model depicting the function of CaMKII and CaN in APC/C activation at meiotic exit in Xenopus laevis oocytes. Green and red connections indicate activating and inhibitory, respectively, relationships.
Figure 7 Figure 7. Model illustrating the possible mechanism induced in the prefrontal cortex by chronic consumption of THC during adolescence that leads to long-lasting cognitive impairment. Left panel: physiological condition. Right panel: chronic consumption of THC during adolescence induces an overactivation of prefrontal CB1 receptors that are mainly located on GABAergic terminals. As previously established in adult mice that received an acute THC treatment (Puighermanal et al., 2009), this might lead to a decrease in GABA release and, subsequently, to an increase in the glutamatergic tone. Likewise, adolescent THC consumption might also increase serotoninergic tone leading to a sustained activation of 5-HT6 receptor located on pyramidal neurons which is prerequisite for the long-lasting stimulation mTOR signaling pathway. This in turn leads to an alteration in intrinsic neuronal properties and cognitive deficits in adulthood. The non-physiological activation of mTOR and resulting imbalance in the excitatory/inhibitory equilibrium during adolescence might also interfere with maturational events occurring in the adolescent prefrontal cortex leading to a persistent rearrangement of cortical networks affecting the serotonergic system itself and, ultimately, to a persistent mTOR activation. For the clarity of the figure, CB1 receptors were only represented on GABAergic terminals. The presence of GABA-A receptors onto glutamatergic and serotoninergic terminals were previously suggested in (Alle and Geiger, 2007; Ruiz et al., 2010; Yamamoto et al., 2011) and (Cerrito et al., 1998), respectively. Expanded View Figure Legends
Figure 3Gene expression profile of MDA231-LM2 cells grown as oncospheres or as a monolayer. Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red. GSEA graph showing enrichment of JNK signature in MDA231-LM2 cancer cells grown as oncospheres. NES, normalized enrichment score. Immunoblot of phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in oncospheres from SUM159-LM1 cells. Tubulin was included as a loading control. GSEA of a basal-like breast cancer signature induced in MDA231-LM2 oncospheres. Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells. Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD. Oncosphere formation in SUM159-LM1 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD. Representative images of spheres Oncosphere formation in JNKi-treated cancer cells from breast cancer patients (pleural effusions or ascites). (I) Quantification of oncospheres per well in 4 different patients. Values are mean from 10 wells ± SD. (J) Representative oncospheres from patient samples. Scale bar, 50 μm. Subcutaneous injection of limiting numbers of JNKi-treated MDA231-LM2 cells in NSG mice. Analysis of tumor initiation ability of vehicle- and JNKi-treated cells from experiment described in panel (K); n = 3 mice per group, two implantations per mouse. Matrigel invasion assay of MDA231-LM2 cells treated with JNKi. Left, examples of invading cancer cells stained with crystal violet (arrows). Scale bar, 50 μm. Right, quantification of invading cells per field of view (FOV) showing mean from 8 fields ± SEM.
Inhibition of fission or autophagy results in increased protein oxidative damage.Figure 7(A) Level of oxidized mitochondrial protein in mitochondria purified from FIS1 RNAi (n=3), DRP1‐DN (n=4) and their respective control cells. The blot detects carbonyl groups on amino‐acid side chains (Oxiblot analysis, see Materials and methods). Loading control CIII, mitochondrial ComplexIII Core I (*P0.03, **P0.04). (B) COS7 cells coexpressing DRP1‐DN and mtDsRed (red) were fixed, permeabilized and stained with a monoclonal antibody against nitrotyrosine (green). Control cells were infected with mtPA‐GFP lentivirus. DRP1‐DN cells show increased nitrotyrosine immunoreactivity in mitochondria, particularly in the distal and enlarged ends. Scale bar, 15 μm (see also Supplementary Figure S9). (C) Knockdown of FIS1 does not increase ROS production. ROS production in FIS1 RNAi and control cells as measured by DHE FI under different glucose levels. Cells were loaded with dye and FI was measured before and 10 min after changing of glucose concentration (NS, non‐significant; P=0.5; *P0.001). (D) Inhibition of autophagy results in increased protein oxidative damage. Treatment of INS1 cells with bafilomycin (blocking autophagy by preventing fusion with lysosomes, 0.1 μM for 3 h) results in the accumulation of oxidized proteins in mitochondria, which is shown by increased level of carbonylated amino acids in isolated mitochondria. (E) Inhibition of autophagy generates Δψm heterogeneity. INS1 cells were treated with 3‐MA (2 mM for 5 days) and then stained with TMRE and mitotracker green. Ratio image of red/green represents Δψm and is shown in pseudocolor where depolarized mitochondria appear blue. Note the increase in subcellular Δψm heterogeneity and the appearance of depolarized units with low TMRE (see also Supplementary Figure S4A-C). Scale bar, 5 μm.
Effect of antagomirs on MIR376A target expression.Figure 5(A and B) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF-7 cells transfected with control antagomirs (CNT-Ant) or antagomir-376a (Ant-376a) (mean ± S.D. of independent experiments, n = 3, **p<0,03 for ATG4C, and n = 3, **p<0,03 for BECN1). Results were expressed as fold changes against GAPDH mRNA levels. (C and D) ATG4C and BECN1 protein levels were increased following antagomir-376a (Ant-376a) transfection. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.
Figure 2Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 and GPN. Arrows serve as event markers that denote the points of time at which the drugs were added. Grey lines on top represent the time at which the bath Ca2+ was removed. All values represent mean±SEM from 3 biological replicates. Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release in response to the application of ML-SA1. All values represent mean±SEM from 3 biological replicates. Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to GPN. the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release Ca2+ release was evoked using GPN. Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 (E) All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to GPN (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Ratio of the amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Total Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Schematic representation of the notion that an increase in the expression of MCOLN1 results in a proportional increase in ML-SA1-induced Ca2+ release in HN31 cells.
Figure 4(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (B) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65, p65 and zonulin] in vitro (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.