EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 3(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm. (B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm. (C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates. RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (E) Rab8A pT72 phosphorylation was analysed by Western blot. (F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (H) Quantification of E and F.
Figure 7A, Diagram showing the Wbp2 molecular pathway, including its downstream targets and their functional relationship. The blue arrows, light blue lines and green lines link data from the literature (in vivo and in vitro); the orange squares and red arrows indicating up- or downregulation show our experimental observations, as reported in this study. B, Quantitative real-time PCR showing reduced mRNA levels for Esr1, Esr2 and Pgr and upregulation of Shank3 and Psd-95 in cochleae of 4 week old Wbp2-deficient mice compared to littermate controls (n=3 for each genotype). Hprt is used as relative control. Error bars: ±SD. Two-tailed student t test: p=0.03 for Psd-95; p=0.007 for Shank3; p=0.03 for Esr2; p=0.0016 for Esr1; p=0.037 for Pgr. C, Synapses from one mutant and one control IHC at 4 weeks of age after Psd-95 and CtBP2 labelling, showing stronger Psd-95 expression in the mutants compared to controls, representing double labelling experiments performed on 3 mutants and 3 controls. P indicates direction of pillar cells. Scale bar: 10μm.D, Quantification of Psd95 fluorescence in IHC synapses, representing expression in the apical (9kHz best frequency region of the cochlea) and basal (24kHz best frequency region of the cochlea) regions at 4 weeks old . Data from 2 wt and 2 homs were analysed (16 synapses per cochlear region per mouse). AU, Arbitrary Units.Wt: 24kHz 22.41±10.70, 9kHz 11.045±2.128; Mutants: 24kHz 66.73±13.70, p=0.069; 9kHz: 26.02±5.79 p=0.075.
Figure 8FRAP of Tau accumulations show no recovery of NUC Tau (NUC: <10% recovery; t1/2 ≈11 s), ~20% recovery of CYT and NE Tau with a faster recovery rate for NE Tau (NE: t1/2 ≈2.5 s; CYT: t1/2 ≈7 s). N=15-20 Tau accumulations per type. Data shown as mean±SD. Example FRAP image series for cytosol and the different Tau accumulation types in HEK sensor cells treated with AD brain lysate (pre-bleach=-2s, immediately after bleaching=0s, after recovery=40s). Arrows indicate position of bleached ROIs. Scale bars=5 or 10 μm as indicated. High-resolution CFP FLIM of HEK sensor cells expressing CFP/YFP-TauRDP301L and treated with Tau condensates. FRET between CFP and YFP induces CFP-lifetime reduction in densely packed CFP/YFP-TauRDP301L accumulations. Fit-free analysis of the lifetime distribution in phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, LT-4=fully aggregated CYT Tau. LT-1 corresponds to soluble CFP-TauRD in the cytosol of cells without accumulations. Asterisks indicate position of individual cell nuclei. Scale bars=10 μm. CFP-FLIM of seeded HEK sensor cells expressing only CFP-TauRDP301L. The dense packing of CFP-TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP-lifetime. Fit-free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, and LT-1=soluble CFP-TauRD in the cytosol of cells without accumulations. Graphs show lifetimes of CFP-lifetime components assigned to soluble, NE, and CYT+NUC Tau in 6 different images (left graph, RM one-way ANOVA with Tukey post-test) and lifetime reduction relative to LT-1 in each respective image (right graph, one-way ANOVA with Tukey post-test.). N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. Scale bars=10 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. FLIM of 1h- and 24h-old CFP-Tau (full-length) condensates and heparin induced CFP-Tau aggregates. Lifetime components are indicated in phasor plots. Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP-Tau. N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. One-way ANOVA with Tukey post-test. Scale bar=5 μm.­ *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Bottom right: Schematic of lifetime quenching in condensates and aggregates.
Figure 6(A) Crystal structure of the catalytic subunit of NatA from yeast complexed with a bisubstrate inhibitor (PDB code 4KVM). The interaction of the α1α2 and β6β7 loops restricts access to the binding site of the peptide promoting N-α acetylation of substrate peptide. β6β7 loop in are coloured in cyan. Peptides and the CoA moiety are displayed as green and orange sticks, respectively. (B) Crystal structure of the HAT domain of Tetrahymena GCN5 bound with both peptide ligand and CoA (PDB code 1QSN). The absence of a β7 strand and the different C-terminus creates an accessible groove at the surface of the HAT protein promoting internal N-ε lysine acetylation of the substrate peptide. The main chain (top) and solvent accessibility (bottom) of both proteins are displayed as grey ribbon or grey surface, respectively). α1α2 loop, β4 strand, β5 strand are colored in red, yellow and pink. β6α4 loop in B are coloured in cyan. Peptides and the CoA moiety are displayed as green and orange sticks, respectively. (C) Models of GNAT4 obtained from structure homology-modelling server SWISS-model using the pdb codes of two GCN5-related N-Acetyltransferases (2x7b, left/model 1; 4H89, right/model 2) as template. The GNAT4 models show different α1α2 and β6β7 loops conformation, suggesting a mobile loop allowing GNAT4 to ensure a KA/NTA dual activity.
Effect of Gata6 loss in K14ΔNLef1 sebaceous tumors.Figure 4(A) Sections of mouse skin tumors were stained with antibodies to Gata6 and Krt14. A papilloma tumor found in WT mice treated with DMBA and TPA (left panel), is compared with sebaceous tumors found in K14ΔNLef1 mice (right panels). Data information: Scale bars: 100 μm. (B) Tumor burden and tumor incidence in DMBA-treated K14ΔNLef1 (n=11) and K14ΔNLef1:cKO (n=8) mice. (***) p-value < 0.001; Wilcoxon matched-pairs signed rank test. Data information: Data are means ± SEM. (C) Representative H&E-stained tumors of K14ΔNLef1 and K14ΔNLef1:cKO mice with quantification of each tumor type relative to the total number of tumors in each group (middle panel) and as the average absolute number of tumors per mice (right panel). A total of 268 tumors were analyzed. 143 tumors were found in 7 K14ΔNLef1 mice (103 sebaceous tumors and 40 papillomas), while 125 were found in 5 K14ΔNLef1:cKO mice (55 sebaceous tumors and 70 papillomas). (**) p-value < 0.005, Student's t-test. Data information: (C) Scale bar: 1 mm. (D) Representative images of mouse skin tumor sections labelled with antibodies to Plet1 and Atp6v1c2. Data information: D) Scale bars: 100 μm.
Establishment of 4D quantitative analysis for MTOC dynamics(A) Live imaging of MTOC dynamics during meiosis I.Figure 1(A) Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points. (B) Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume). (C) Average density maps of 21 oocytes mapped as in (B). (D) Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time. (E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments). (F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.
a) Schematic diagram of human NDP52, the domain-deleted constructs and mouse.f9(a) Schematic diagram of humanNDP52, the domain-deleted constructs and mouseNDP52. (b) Naive cortical cells were co-transfected with the plasmids expressing HA-hNDP52 or HA-tagged domain-deleted mutants of human NDP52 together with GFP-tau. Twenty-four hours later, cell lysates were prepared and transfected tau was immunoprecipitated and blots probed for both the HA-hNDP52 constructs and GFP-tau. Homogenates (input) used for co-immunoprecipitation were analysed by immunoblotting using anti-HA or a polyclonal tau-specific antibody (Tau). (c) Stably transfected CN1.4 cortical cells were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h. Homogenates from CN1.4 cortical cells were used for the co-immunoprecipitation of mouse NDP52 (mNDP52) using a Tau5 antibody. Co-precipitation of mNDP52 was examined by immunoblotting using anti-NDP52 antibody. Input of the homogenates used for co-immunoprecipitation was analysed by immunoblotting using anti-NDP52 or Tau antibody. (d,e) CN1.4 cortical cells were transfected with the mock or HA-mNDP52 plasmid (d) or with mNDP52 siRNA or scramble RNA as a control (e). The cells transfected with siRNA or scramble RNA were treated with SFN (10 μM) for 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot.
Figure 2DNA and crRNA sequences used for multiple PAM experiments with indicated labeling positions. Di-nucleotide GG PAMs are shown in boxes. Example time traces when DNA containing 3 PAM sequences is added (top) and when DNA containing a single PAM is added showing both short and long binding events (bottom). FRET histograms of all binding events for each construct. Y axis represents total number of counts, divided by the number of immobilized molecules in the field of view. Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences. The times shown are mean values of dwelltimes obtained during four different experiments on four different days. Error bars represent standard error of the mean. Example dwelltime histograms from negative control and a construct containing a single PAM. Negative control dwelltime distribution is characterized by a single exponential decay (top) and dwelltime distribution for PAM-containing DNA is fitted by a double-exponential decay (blue line) (bottom). Equation for the double exponential decay fit: y=A1e-t/τ1 + A2e-t/τ2; ΔA1= 796.4 ΔA2= 28.3. Red line shows what a single-exponential decay fit for such distribution would be. Errors represent standard error of the mean. Scatter plot showing the average dwelltime (Δτav) values for the cases of 2 (d2) and 4 (d4) nucleotide separation between the PAM sites. The values are averages of four measurements over four different days for each case. Error bars represent the standard error of the mean. The average dwelltime was obtained using the equation τav=(A1Δτ12+A2τ22)/(A1Δτ1+A2Δτ2) Example trace showing direct FRET transitions for a 5-PAM containing DNA construct with separation between PAM sites being 4 nucleotides. Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides. The values are averages of four measurements over four different days. The error bars represent standard error of the mean.
Figure 2A. IHF of GASZ and Cytochrome C (Cyt C) of testical sections from mice at day 7 postnatal, or MILI and MVH of gonad sections from wild-type embryos or embryos with MLS deletion at 16.5 dpc. Scale bars: 5 m. B. Western blots of GASZ expression of testes at day 5 postnatal. C. Gross morphology of testes from adult mice at 3 months postnatal with mutated MLS compared to their wild-type littermates. D. Histological studies of testis sections from wild-type mice or mice with MLS deletion at different time points after birth. Scale bars: 50 m. E. EM of testis sections from wild-type neonatal mice or mice with MLS mutation. Red arrows point to IMC among mitochondria. F. Expression of transposable elements analyzed by real-time RT-PCR on testes from wild-type mice or mice with mutated MLS at various time points after birth. Data were shown as mean (of expression from three biological replicates) ± one s.e.m. n=3 for each genotype. **: p<0.01. *: p<0.05.
Fig.Figure 2(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm. (C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm. (D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies. (E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.
Increased accumulation of autophagosomes and lysosomes in BI‐1‐deficient cells.Figure 1(A) BI‐1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5-GFP to visualize the ER (green). Then cells were stained with lysotracker (red) and observed with a confocal microscope. Nucleus was stained with Hoechst (blue). Scale bar: 30 μm. (B) BI‐1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy. Acidic vesicles were visualized with a confocal microscope after lysotracker staining. Data represent the results of three independent experiments. Scale bar: 50 μm. (C) (a) Epifluorescence imaging of BI‐1 KO cells stained with lysotracker; (b) electron micrograph of the same field of cells shown in (a); (c-e), magnification of lysotracker‐positive vesicles in BI‐1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope (c) and the same field subsequently imaged by EM (d). Overlapping images are presented (e). Left panel: analysis of vesicular structures by EM with morphologies resembling early (f), intermediate (g) and late (h) autophagy vesicles. (D) Left panel: the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI‐1 WT and KO MEFs cells at basal conditions (NT) or after exposure to EBSS for 3 h. Scale bar: left 15 μm and right 10 μm. Right panel: quantification of the number cells containing three or more LC3‐positive vesicles (N=160 cells). Mean and standard deviation are presented (N=4). Student's t‐test was used to analyse statistical significance, **P0.001 and *P0.01. (E) BI‐1 WT and KO cells were transiently transfected with an expression vector for a monomeric‐tandem LC3-RFP-GFP construct. After 24 h, cells were exposed to EBSS for 3 h. Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3‐positive dots in the red and green channels using a confocal microscope. Scale bar: 10 μm. Right panel: quantification of the ratio between red and yellow dots per cell is presented. Mean and standard error of the analysis of 15 cells are shown.
Figure 4GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test. GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test. TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA. TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA. GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs. GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.
Figure 2A Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 d or SC/−Leu/−Trp/−His/−Ade medium for 6 d. OST1-BD/AD and BD/EGR2-AD were used as negative controls. B OST1 interacts with EGR2 in vitro. Purified recombinant MBP-His-EGR2 or MBP-His proteins from E. coli were immunoprecipitated with MBP beads, and then incubated with purified recombinant GST-OST1. Precipitated proteins were detected with anti-His and anti-GST antibodies. C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively. D Bimolecular fluorescence complementation (BiFC) assay. Wild-type Arabidopsis protoplasts transformed with OST1-YFPC and EGR2-YFPN proteins were incubated for 18 h. The interaction signal was detected by confocal microcopy. The combinations of OST1-YFPC/GUS-YFPN and GUS-YFPC/EGR2-YFPN were used as negative controls. Scale bars: 100 μm. E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining. F, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress. Data information: the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test). H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).
BRAVO and WOX5 have a joint role in repressing QC divisions.Figure 5Computational estimation of the contributions of BRAVO-mediated (TBWT), WOX5-mediated (TWWT) and BRAVO-WOX5 joint (TBWWT) regulations of QC divisions in the WT, as a function of the attenuating factor of BRAVO contribution in the wox5 mutant, qBwox5. Continuous lines represent the best estimated values, while dashed lines are the enveloping confidence intervals (e.g. TBWT ± δTBWT). The horizontal grey dashed lines mark the zero lines. For a wide range of qBwox5 values, the joint contribution of BRAVO and WOX5 is the only one relevant. The individual contribution of BRAVO becomes significant only for small values of qBwox5. In all three panels, we set qWbravo=0.8. Positive contributions correspond to repression of QC divisions, while negative contributions correspond to activation of QC divisions. Sketch representing the spatial distribution of BRAVO, WOX5 and their product BRAVO x WOX5, which can be interpreted as the protein complex. Their joint interaction peaks at the QC, where repression of cell division occurs.
PSAT1 expression in breast cancer is associated with IKKε expression and immune infiltration.Figure 5PSAT1 expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the PSAT1 staining grade are shown. Scale bar: 50 ­μm. Distribution table of PSAT1 expression in the cohort of 41 human breast carcinomas according to the staining grade. Correlation between PSAT1 and IKKε expression ("0": no staining; "1": weak; "2": moderate; "3": strong). Bubble plot of PSAT1 and IKKε staining grades, size shows the number of tumours falling in the category. Correlation between PSAT1 and and immune cell infiltration ("0": no inflammatory cells; "1": weak; "2": moderate; "3": strong; "4": very strong). Bubble plot of PSAT1 and immune infiltration grades, size shows the number of tumours falling in the category. Correlation of mRNA levels of the SBP enzyme genes PSAT1 (E), PHGDH (F), PSPH (G) with the immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patient (Curtis et al, 2012).