EMBO/sd-character-level-ner huggingface dataset:
Figure 2A) ATP content in ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 4 independent biological replicates. B) Extracellular acidification rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser. C) Glucose uptake rates in Zscan4+ (red) and 2-cell-like cells (green) were measured using a luciferase-based assay across 4 independent biological replicates and are represented relative to those of control ES cells (blue). D) Schematic representation of measured fluxes (left). In order to ascertain whether the increased glucose uptake observed leads to higher flux into the hexosamine biosynthesis pathway (HBP) or the pentose phosphate pathway (PPP), one enzyme of each pathway was disrupted through siRNA-mediated knockdown (right). E) Experimental design. ESC cultures were transfected with siRNAs targeting Gnpnat1 (HBP), G6pdx (PPP) or a negative control siRNA (Neg). After 48 hours of culture cells were FACS-sorted into a 96-well plate based on their fluorescent reporters and glucose uptake rates were measured using a luciferase-based assay. F) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4+ and 2-cell-like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each. G) Glucose-6-phosphate dehydrogenase activity was measured in ES (blue), Zscan4+ (red) and 2-cell-like cell (green) lysates using a fluorometric assay. Measurements were obtained from 3 independent biological replicates, performed in 3 technical replicates each. n.s. - not significant; one-way ANOVA. Data information: In panels A-C and G, boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate.
Figure 5(A, B) Retinal sections from 5-month-old mice were stained with indicated antibodies. Thickness of each layer, number of Brn3a positive cells (retinal ganglion cells) per section and length of outer segment as indicated by opsin staining are quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. (C Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. D) Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. (E Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. F) Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control. (G, H) Increase in microglia activation in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by IBA1 staining. The number of IBA1 positive cells per section was quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.
Figure 1A. Schema of the experimental paradigm used for drug administration. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC- or vehicle. Biochemical and behavioral experiments were performed from PND 60. B. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected WT mice. They are the means ± S.E.M. of results obtained in five mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test. C. Top: schemas illustrating the behavioral tasks examined in WT mice. Bottom: the plots represent the discrimination index for the novel object recognition task (Vehicle N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12), the sociability index (Vehicle: N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12) and the discrimination index for the social discrimination task (N=8 for each group), measured in each condition. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line to a discrimination index (exploration of novel object - exploration of familiar object/total object exploration) equal to zero. D. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5-HT6-/- mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected 5-HT6-/- mice. They are the means ± S.E.M. of results obtained in four mice per group. n.s. p > 0.05, one-way ANOVA followed by Newman-Keuls test. E. Top: schemas illustrating the behavioral tasks in 5-HT6-/- mice. Bottom: the plots represent the discrimination index for the novel object recognition test (discrimination index: 0.30 ± 0.05, N=10 and 0.31 ± 0.03, N=11, for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), the 3-chamber social preference test (sociability index: 0.49 ± 0.04, N=10 and 0.48 ± 0.07, N=11 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05) and the social discrimination test (discrimination index: 0.15 ± 0.06, N=8 and 0.21 ± 0.06, N=8 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), measured in each condition. One-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line corresponds to a discrimination index equal to zero).
Figure 1(A) General sequence alignment of SOX9 high affinity CDC4 phosphodegron (CPD) motif against other affinity motifs present in known FBW7α substrates including Cyclin E, cMYC, and c-Jun. (B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments. (C) FBW7α immunoblot eluted from the agarose bead-bound peptide, which had been incubated with the recombinant SCF-FBW7α for 1 hour at 37◦C. The agarose bead-bound peptide contains either the non-phosphorylated SOX9 amino acid sequence spanning from residue number 231 to 245 (SOX9 peptide), or those with the threonine corresponding to residue number 236 and 240 being phosphorylated (pSOX9 peptide). The input (10%) showed the level of the supplemented recombinant SCF-FBW7α in the in vitro binding reaction. Shown blot is representative from 2 independent experiments. (D) Representative Western blotting set from 3 independent repeats of pT236-SOX9 from lysates of HEK293 transfected with either HA-EV or HA-SOX9. Each lysate were immediately divided and left untreated or subjected to lambda phosphatase (λ-PPase) treatment for 1 hour at 37◦C prior to gel electrophoresis. SOX9 blot showed the presence of SOX9 protein in both the untreated and the phosphatase-treated SOX9-transfected cell lysates. (E) Bead-immobilized in vitro translated (IVT) HA-SOX9 wild type (WT) or T236/240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37◦C prior to elution and gel electrophoresis. The SOX9 blot showed total SOX9 protein eluted from the beads from each reaction. Shown immunoblots are single representative experiment from 3 independent repeats. (F) GSK3-mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF-FBW7α in vitro. Bead-immobilized IVT HA-SOX9 were either untreated or phosphorylated with GSK3 as described in (E) prior to further incubation with recombinant SCF-FBW7α for in vitro binding assay. Immunoblot showed FBW7α protein in the in vitro binding reaction (input) as well as those which had been eluted from the untreated and GSK3-treated immobilized SOX9. The set of blots shown is representative from 3 independent experiments.
Figure 5(A) Percentage of single LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice that completed first and second division up to 48 hours post initiation of culture. (192 sorted HSCs cells were analysed, error bars indicate mean±SD). (B) Percentage of single LT-HSCs septin 7fl/fl of septin 7∆/∆ dying up to 48 hours (192 sorted HSCs were analysed, error bars indicate mean±SD). (C). Frequency of colonies with distinct cell size 5 days after initiation of culture of individual LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice (192 sorted HSCs were analysed, error bars indicate mean±SD).
Figure 2Left image: crystal structure of the complex between Mac2 (orange) and Paip1M (light blue). Mac2 possesses a three-layer α/β/α fold. α1, 2, and 3, as well as η1 and α4, 5 form the two outside layers, whereas the β sheet (β3−β4-β2−β5−β6) forms the central layer. The first 16 residues of Mac2 are shown in green. Paip1M comprises ten helices within two layers. α1, 3, 5, 7, and 9 form one layer while α2, 4, 6, 8, and 10 form the other. The long loop region (Ile204−Thr212) is displayed in purple. The N and C termini of Mac2 and Paip1M (in italics) are marked. Right image: space-filling representation showing the tight interaction between Mac2 and Paip1M. Left image: Fo-Fc difference density omit map (2.5σ; blue) for the region Lys389−Thr408 of Mac2 (orange) in this complex. Right image: Conformational change of this region between free Mac2 (blue; PDB entry: 2W2G, Tan et al, 2009) and Mac2 in this complex. The crystal structures of Mac2 (C; blue), Paip1M (D; red; PDB entry: 3RK6, Lei et al, 2011), and Mac2:Paip1M (E) fit the shape of envelopes derived from the small-angle X-ray scattering (SAXS) data. The blue and red dashed lines indicate the four (GSHM) and seventeen (GSHMASMTGGQQMGRGS) artificial residues remaining from the expression vectors in Mac2 and Paip1M, resp.
Figure 4A. Protein sequence alignment of the HEAT8 hairpin and TAF1-TAND1, and the HEAT19 loop and TAF1-TAND2 (numbers represent amino acid positions). α-helices were assigned based on crystal structures and are depicted as blue cylinders. The Phe57 anchor residue of TAND2 is indicated 24[]. Black color highlights amino acid identity and gray shading indicates amino acids with similar properties. B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.
a, Western blot analysis of yeast cell lysates with polyclonal anti-flag.figf1a, Western blot analysis of yeast cell lysates with polyclonal anti-flag antibody to detect flag-tagged human Beclin 1 expression. Two-hundred micrograms of protein per lane was loaded. SEY6210, wild-type yeast; JCY3000, SEY6210 disrupted in apg6/vps30; (+) control, lysates of mammalian cells transfected with flag-beclin 1. b, Autophagicbody formation in yeast deprived of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows denote cells that would be scored as positive in the experiment shown in c. c, Quantitative effects of apg6/vps30 and beclin 1 transformation on autophagicbody formation in apg6/vps30-disrupted yeast in the presence of PMSF. Cells with one or more autophagic bodies within the vacuole were scored as positive (see arrows in b). A minimum of 100 cells was counted for each sample. Results represent the mean (±s.e.m.) percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments. d, Sorting of vacuolar protein carboxypeptidase Y, (CPY). p2CPY, precursor form; mCPY, sorted mature form. Mr, relative molecular mass.
Fig.Figure 1(A) Oxygen consumption ratio, , in the indicated VSMC types. (B) mitochondrial ATP synthesis in the indicated VSMC types. (C) mitochondrial membrane potential (MP), in the indicated VSMC types.
Figure 6Computer simulations indicate that changes to homogeneous resting areas are able to alter cell topology. A) Diagram resulting from a vertex model simulation with homogeneous parameters for contractility, line tension and ideal area. B) Diagram resulting from a vertex model simulation where in ten percent of the cells (grey) the line tension and ideal area parameters were reduced C) Control diagram where in the ten percent of the cells (blue) only line tension parameter was reduced D) Comparison of polygon distribution for the 10% smallest cells from BCA (green bar) and Control Simulation (grey bar) with the sick cells from BNA (light blue bar) and Atrophy Simulation (grey cells from (B), purple bar).
Representation of active human kinases in the yeast array.Figure 3A. Kinase activity in yeast. Kinase activity is played on the human kinome tree showing that all kinase families contain highly active kinases members. B. Cancer kinases. The color-coded annotated cancer kinases overlap with of highly active kinases (larger node size) in the array. 168 kinases in the array are linked to at least one cancer type (Piñero et al, 2021). C. Dark kinases. Dark kinases (Berginski et al, 2021) in the kinome are color coded in purple, the tree shows the overlap of 35 highly active kinases (larger node size) and the total of 72 dark kinases represented in the array. D. Tissue specific kinases. Normalized proteomics data (Wang et al, 2015) from 31 tissues show that some kinases are only expressed in a few tissues. Many tissue specific kinases (darker color) belong to the highly active kinases (larger node size) in the yeast array. Low abundance kinases. Average relative expression levels over 31 tissues from PaxDB are color coded. Many kinases with low expression in mammalian cells (darker color) belong to the highly active kinases (larger node size) in the yeast array.
Figure 7(a) Transverse cryosection of E10.5 Venus::HES5 spinal cord. Venus::HES5 endogenous signal, OLIG2 - motorneuron progenitor marker, NGN2 - early marker of neuronal commitment; scale bar 20 μm. (b) Relative nuclear intensities of Venus::HES5 and NGN2 in motorneuron and interneuron progenitors; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments; Kruskal-Wallis with Dunn's multiple comparison test adjusted p-values **p=0.0032, ***p<0.001. (c) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity within motorneuron (MN) and interneuron (IN) domains, corresponding to segmented image in (a). (d) Dimension of microclusters in DV axis for MN and IN domains; microclusters counted contained cells with high and similar levels of HES5 (Materials and Methods); bars show mean ±SD; data consists of 34 microclusters measured from 5 sections and 3 independent experiments; 2-tailed Mann-Whitney test. **** p<0.0001. (e) Peak to peak distance in auto-correlation plots of de-trended Venus::HES5 spatial profile in MN and IN domains; this is a measure of spatial period in Venus::HES5 profile along dorsal-ventral axis of spinal cord; grey data points represent mean peak to peak distance of at least 3 slices with left and right ventricle analysed separately in 6 experiments; black dots show median per experiment and line shows overall median; 2-tailed Mann-Whitney test p-values ****p<0.00001. (f) Cell-cell concentration differences in HES5 between neighbours, normalised to mean concentration of HES5 in that domain; grey data points represent normalised concentration difference between a pair of neighbours, bars shows mean and SD; 2 independent experiments; 2-tailed Mann-Whitney test with p-values ***p = 0.003, ****p<0.00001.
Figure 8A-C We overexpressed GFP-tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts. (B) We analyzed the proliferation at 1, 3 and 5 days, and proliferation rate in patient-LV-NICD-GFP reached the level observed in control-LV-GFP, which were higher than in patient-LV-GFP. (C) However, the percentage of PAX7+cells in patient-LV-NICD-GFP showed the same reduced value as in patient-LV-GFP, which were significantly lower than in control-LV-GFP. The majority of control myoblasts were proliferating cells (PAX7+ MyoD+, arrows), with no quiescent cells and few differentiating cells detected. Data were collected from n = 2 independent assays (5-8 images per condition). Mean ± SEM is shown; One-way Anova with Tukey multiple comparisions test (B) and Kruskal-Wallis with Dunn´s multiple comparisons test (C); Scale bar, 50 µm). D-F As previously described in patient's primary myoblasts, patient-LV-GFPmyoblasts showed a striking facilitated differentiation compared to control-LV-GFP-myoblasts. This phenotype fully disappeared in patient-LV-GFP-NICD myoblasts, as quantification of the fusion index (E), and myogenin expression (F) demonstrated. The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients. Data were collected from n = 3 independent assays (13-17 images per condition). Mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).
Figure 3De novo identification of DNA consensus motifs enriched in the 1000 IL-2 pDHSs determined by HOMER. Jund mRNA levels in TB IL-2 and TB IL-2nil. Standard deviation is shown from the mean of 3 samples. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing. Western blot analyses showing JUND and pSTAT5 protein levels in TB IL-2 and TB IL-2nil (left panel) with the signal calculated from 2 replicates relative to TB IL-2 in the right panel. DNA motif density plots (right) showing the location of binding motifs for RUNX, ETS, STAT and AP-1 transcription factors at the 32694 DHSs detected in TB IL-2 and TB IL-2nil, as depicted in Figure 2A, ordered according to fold enrichment of DNase-Seq tag counts in TB IL-2 compared to TB IL-2nil (left). DNA sequence tag density plots from ChIP-Seq assays of RUNX1, ETS1, STAT5 and JUND in TB IL-2, and STAT5 and JUND in TB IL-2nil ordered as in (D). UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.
Figure 2A) Representative histology images of cerebella from 3-months-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1-/-, Ttll7-/- and Ccp1-/-Ttll1-/- mice, in contrast to the complete absence of these cells in Ccp1-/- and Ccp1-/-Ttll7-/- mice. B) Histology of 25-day old mice with partial degeneration of Purkinje Cells. Note that both, Ccp1-/- and Ccp1-/-Ttll7-/- brains, show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration). C) Representative immunoblots of cerebellum extracts of wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 lead to a loss of glutamylation from β-tubulin (β‑monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both, α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE).
Figure 3a.Vero E6 cells were infected with an moi of 0.05 with the fluorescent mCherry D614 strains, wild-type ISA D614 or mock infected. Pictures were taken at 48h pi (20x). Scale bar, 100 µM. b. After 5 passages (p2, p3, p4 and p5) on VeroE6 cells, the mCherry reporter gene in ISA mCherry D614 supernatant medium was RT-PCR amplified and analysed using gel electrophoresis. (c) Twenty-four human sera were then two-fold diluted and incubated with the ISA D614 and mCherry D614 strains and nAb titers were recorded at 5 days post-infection. nAb titers were defined as the highest dilution that inhibited the production of distinct cpe with the ISA D614 SARS-CoV-2 or fluorescence with the fluorescent mCherry D614 SARS-CoV-2. Each black dot represents results from a given number of sera. Statistical analyses were performed using univariate linear regression. The error band (in grey) represents the 95% confidence interval of the regression line. The Pearson correlation coefficient (R2) and P value analyses are shown. (d) Representative neutralizing curves of the nAb fluorescence-based assay. The four-parameter dose-response curve was fitted using the nonlinear regression method and nAbs were calculated in the software Prism 7.0. For negative serum samples, an arbitrary value of 10 was assigned (detection threshold for both methods).