EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points. (C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (E) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM) and AT-406 (1 μM) for the indicated timeframes. The levels of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates were analysed by immunoblots. Results are representative of three biological replicates. (F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test. (G, H) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice (n = 5) were injected i.v. with TNF (1.5 mg/kg), before recording body temperature for the next 6 h (G), and collecting serum samples at 3 h to quantitate LDH release (H). *P < 0.05 as determined by two-way ANOVA (G), or P values calculated by unpaired t-test with Welch's correction (H).
Figure 2A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm. Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm. Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm. G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test. H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test. I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm. Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm. Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm. O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test. P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.
Figure 2A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. A. Representative carmine-alum stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4 - 15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7 - 9 glands, 7 weeks old mice). A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. D. Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μm A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. E. Representative carmine-alum stained images highlighting terminal end buds (TEBs) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F. quantification of the number of TEBs per gland (n = 9 - 10 glands). Scale bar represents 500 μm. A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 µm. Mean ± SEM.
Models of hormonal stem cell control and Wnt4 action during mammary gland developmentControl of mammary stem cell activity by Wnt4 during development.Figure 6A Control of mammary stem cell activity by Wnt4 during development. Schematic representation of mammary gland development (bottom) and control of mammary stem/progenitor cells by hormones (top). Model showing Wnt4 expression during mammary gland development. Wnt4 is important for stem cell activation throughout postnatal mammary gland development. Perinatal Wnt4 expression is independent of ER and PR, yet unknown factors control it. PR signaling is required for Wnt4 expression in puberty and adulthood. PR expression is induced by ER signaling. B Model of Wnt4 action in the mammary epithelium. Progesterone stimulation results in Wnt4 induction in the PR+ luminal cells (LC), the 'sensor cells.' The secreted Wnt4 acts on adjacent basal/myoepithelial cells (MC). In the myoepithelial cells, Wnt4 activates canonical Wnt signaling which induces changes in gene expression. This results in the secretion of factors and changes in the ECM (light blue arrows) that in turn impinge on stem cells (SC), luminal‐restricted stem cells (L‐RSC), and basal‐restricted stem cells (B‐RSC). Wnt4 may also act directly on stem cells that are found within the basal layer.C Model for the tumorigenic effects of progesterone and Wnt4 in the mammary epithelium. Repeated activation of this intercellular signaling cascade downstream of PR signaling may promote tumorigenesis by expanding luminal progenitor cells with oncogenic mutations and by expanding the stem/progenitor cell compartment.
Figure 8A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm. C-D. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test). E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm. F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test). H. Summary diagramme: following central nervous system (CNS) spinal injury, protein phosphatase 4/2 activity is not induced since calcium levels remain unchanged compared to uninjured conditions. HDAC3 remains phosphorylated and occupies deacetylated chromatin contributing to its compaction inhibiting gene expression. Following peripheral nervous system (PNS) sciatic injury, protein phosphatase 4/2 activity is induced by calcium. HDAC3 is dephosphorylated leading to its inhibition and release from chromatin sites contributing to increase in histone acetylation and in the expression of regeneration associated genes (RAGs).
Model of mechano-sensing of metabolic status during liver regeneration.Figure 7Model of YAP regulation by mechanical stimulation through expansion of the BC. Arrows denote reactions or transport steps. Blue numbers label individual processes and associated parameters as listed in Appendix Table S4. The pathway combines a signaling cascade with regulated cytoplasmic retention and is here illustrated based on inactivaton by a mechanosensory, SENSOR; inactSENSOR, inactivated mechanosensor; inactYAP, inactivated YAP; SF, sequestration factor; but alternative molecular states are possible. See Experimental Procedures for model equations and analysis. Model prediction (solid black line) of cortical tension and nuclear YAP levels reveals a sigmoidal stimulus-response curve. Experimental data (symbols) of nuclear YAP is reproduced from Fig.3D and mapped to cortical tension levels using experimental data from Fig.1B and Fig.2D. Schematic drawing of YAP regulation by BA through canalicular cortical tension. Tissue resection by partial hepatectomy induces a BA overload which elevates osmotic pressure and cortical tension of bile canaliculi. Concomitant changes of apical acto-myosin properties recruit YAP to the apical cortex where it is activated for nuclear translocation.
Figure 4A) High throughput screening of SARS2 PLpro was performed against 5576 approved drugs and late-stage clinical compounds, in 1536-well format using Ub-Rhodamine (see Methods). Two replicates out of three are shown; hit compounds were those that inhibited PLpro activity by more than 40% in all three replicates. Correlation (R2) between all screens exceeded 0.8­9. See Fig EV4 for assay design and quality control, and Methods. B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.
interacts with MAVS (A) Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING or Flag-TBK1.Figure 2(A) Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING or Flag-TBK1. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (B) Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53+pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34+pACT-2 was used to exclude the self-activation of RNF34. (C) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. (D) GST-tagged RNF34 was subjected to a pull-down assay with HEK293T cell lysates. Immunoblot with an anti-MAVS antibody are shown in the top panel. Loading of the GST proteins assessed using Coomassie blue staining is shown in the bottom panel. GST was used as a negative control. (E) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-MAVS or Flag-vector-expressing plasmids were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-RNF34 (upper panel) or GST (middle panel) for 2 hrs and then analyzed with anti-Flag antibody. (F) Representative confocal images of immunofluorescence staining for Flag-RNF34 colocalization with endogenous MAVS in THP-1 cells infected with VSV for 12 hrs. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results. (G) Immunoblot showing the levels of the RNF34 protein in THP-1 cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control. (H) In situ PLA assay of the RNF34-MAVS complex in HEK293T cells infected with VSV (MOI =1.0) for the indicated times using an anti-RNF34 or anti-MAVS antibody. RNF34-MAVS complex, red; nuclei, blue. Scale bar, 5 μm. Cell-based studies were performed independently at least three times with comparable results. (I) One hundred cells were counted, and the quantification of PLA signals per cell is shown. Cell-based studies were performed independently at least three times with comparable results. (J) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-mMAVS. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibody.
Tumor metastasis is abrogated in Asm-deficient mice and restored by transplantation of wild-type platelets.Figure 1A, B 1 × 105 B16F10 melanoma cells were intravenously injected into C57BL/6 wild-type (WT) or Asm-deficient (Asm−/−) mice, and the number of lung metastases was determined after 14 days. The photographs (A) show two representative results. The graph (B) shows the mean ± SD number of pulmonary metastases in 15 WT and 14 Asm-deficient animals. C, D B16F10 melanoma grow as fast or slightly faster in Asm-deficient mice than in wild-type mice after subcutaneous injection at the flank (C) or transcutaneous direct intrapulmonary injection (D), excluding a general growth defect of the tumor in Asm-deficient mice. The size of tumors was determined 14 days after local injection at the flank or 10 days after injection into the lung. E The number of lung metastases 25 days after intravenous injection does not differ from the number observed after 14 days in (B). F 5 × 108platelets isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice were injected intravenously into Asm-deficient or wild-type mice, respectively. After 120 min 1 × 105, B16F10tumor cells were intravenously injected. The number of lungmetastases was determined after 14 days. Displayed are the mean ± SD of the number of pulmonary metastases, n = 3 in WT platelets in WT mice, n = 5 in WT platelets in Asm−/−mice, and n = 4 in Asm−/−platelets in Asm−/−mice.
Figure 5(A) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2 (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay. (B) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.
High-affinity cooperative binding of activated full-length UVR8 to the COP1 WD40 domain is mediated by its UV-B-activated β-propeller core and its C-terminal VP-peptide motif.Figure 2A. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation). B. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant. C. The domain organization of Arabidopsis UVR8. It consists of a UV-B-sensing β-propeller core (residues 12-381) and a long C-terminus containing the VP motif. Constructs and peptides used and their residue endings are indicated. D. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant. E. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant. F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.
Directionality, processivity and initiation of DNA processing events for the different protein combinations A Mean duration of bursts (violet bars) (N > 40) and pauses (green bars) (N > 40) for the different enzyme combinations.Figure 5A Mean duration of bursts (violet bars) (N > 40) and pauses (green bars) (N > 40) for the different enzyme combinations. Error bars represent standard error of the mean. B Mean duration of a unidirectional unwinding event (N > 350). Error bars represent standard error of the mean. C Mean processivity of unidirectional DNA unwinding (N > 350). Error bars represent standard error of the mean.
Figure 4(A) Quantitative RT-PCR of Chrne, Musk, Lrp4, and Ctgf in C2C12 myotubes treated with 10 ng/ml BSA or recombinant human CTGF. (B) Quantitative RT-PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng/ml agrin and/or 10 ng/ml neuregulin-1. (C) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18.5. Co-localization of AChR (red) and CTGF (green) is indicated in the merged images. Red arrow points to an AChR cluster. Scale bar = 20 μm. (D) Quantitative RT-PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse. Mean and SD (n = 3 mice) are indicated. p-value < 0.05 by two-way repeated measures ANOVA. p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.
Figure 2(A) H&E of hearts from WT and miR-574-/- mice with or without isoproterenol (ISO) treatment for 4 weeks. Mice were between 8-10 weeks old females. (B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO). (C) WGA (wheat germ agglutinin) staining in WT and miR-574-/- mice. Cross-sectional area (CSA) of CMs was measured and quantified (n≥500 cells). Scale bar: 50 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group. (D) Picrosirius red staining in WT and miR-574-/- mice (n=6 per group). Scale bar: 1 mm. (E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with ISO treatment (n=3 per group). ANF, atrial natriuretic factor; BNP, B-type natriuretic peptide; Myh6/Myh7, myosin heavy polypeptide 6/7; Col1a2/Col3a1, procollagen, type I, α2; type III, α1. (F) TUNEL assay for heart tissue sections from WT and miR-574-/- mice under ISO versus vehicle treatment (n=6 per group). Scale bar: 10 μm.
Figure 7A. Schematic representation of generation of expanded CD4+ T cells and reactivation. CD4+ T cells were sorted from PBMCs of ARVs suppressed HIV-infected individuals and expanded in presence of PHA, IL-2, and autologous feeder PBMCs from healthy donor. B. Expanded CD4+ T cells from 3 patients were cultured in presence of IL-2 and ARVs, with and without 25 ng/µL Vs for 21 days. Vs treatment was given for 15 min every 3rd day. HIV transcripts were quantified by RT-qPCR at day 14, day 21, and at 24 h post-stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT-qPCR was 3 viral transcripts per million cells. C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C). E. Total HIV-1 DNA was determined up to 21 days in cells treated with ARVs or Vs + ARVs.