EMBO/sd-character-level-ner huggingface dataset:
Figure 4Expression of YAP1 and downstream effectors in SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least three independent experiments with similar results is shown. Expression of YAP1 and downstream effectors in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least two independent experiments with similar results is shown. YAP1-responsive luciferase activity in SCP-1 cells transduced with FUS‑DDIT3. Relative luciferase activity is displayed as fold change relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition). Co‑IP of transiently expressed FUS‑DDIT3 and YAP1 from HEK293T cells. V5-tagged YAP1 was pulled down using an anti-V5 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown. Co-IP of endogenous FUS-DDIT3 and YAP1 from MLS 1765-92 cells. FUS-DDIT3 was pulled down using an anti-DDIT3 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.
Figure 1(A) Sequences of secreted Fc-tagged Nrxn1β variants analyzed. Mutated residues are shown in red, the HS modified serine in green, and cysteines forming the Cys-loop in blue. Right column ('HS?') summarizes the results from (B-C). Asterisks, colons, and periods indicate fully, strongly, or weakly conserved residues, respectively. (B) Representative immunoblot of Nrxn1β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against the Fc-tag and the 3G10 epitope (to detect the HS stub which reveal HS after heparinase digestion). (C) Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn1). Bar graph shows means ±SEM of 3 independent experiments.
Figure 5(A) Schematic illustration of barasertib preventive treatment protocol. Control and TGFα mice were treated with either vehicle or barasertib (40mg/kg; twice a day) for 4 wks while they were fed with Dox containing food. (B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm. (C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group). (D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group). (E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).
Strategy to perform ex vivo gene therapy from a single epidermal stem cell.Figure 1Schematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. (1) A biopsy is obtained from the patient to isolate epidermal stem cells that are then expanded under appropriate conditions (2). An aliquot of the culture is infected with the ad hoc recombinant shuttle virus (3). Single cells are then isolated (4) and clones expanded to built a frozen stem cell bank (5). In parallel, an aliquot of each clone is expanded to select for a clone fulfilling the criteria described in Table1. After validation (6), the approved clone is thawed and expanded to create master and working cell banks in a GMP facility (7). Genetically modified CEA are then produced (8) and grafts are transplanted onto the patient (9). MCB, master cell bank; WCB, working cell bank; CEA, cultured epidermal autografts; GLP, good laboratory practice; GMP, good manufacturing practice; GCP, good clinical practice.
Figure 5Difference in deuterium uptake by sE404r and sE419r trimers (D404-D419). Left panel, deuteration difference for each peptide reported against the 1-p value obtained via Student's t-test between the two different constructs. In the right panel, peptides are reported as short horizontal lines, with length and position corresponding to the peptide length and location along the protein sequence (x-axis) and with their deuteration delta on the y-axis. In both panels, red (positive) and blue (negative) indicate changes above the Student's t-test threshold, and in grey those not reaching this threshold. Four peptides, termed HDX-a, HDX-b, HDX-c and HDX-d, were identified as showing a strong increase in deuteration in sE404r compared to sE419r. The same pattern was observed in the comparison between sE404r and sE428r, whereas no difference deemed "significant" by the same test was seen when comparing sE419r with sE428r (see Fig EV3). Middle panel: ribbons representation of the sE trimer (PDB 1URZ) colored according to domains with high HDX regions a and c boxed and highlighted in light grey. The central box marks the domain I-II hinge. Residues 401 and 403, which occupy the
Validation of Chr 4 QTL on gene expression and chromatin accessibility implicate KRAB-ZFPs in trans regulation.Figure 6A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus. B. Gene expression (Riok3 and Rmc1) or chromatin accessibility (18:12.157 Mb) level measured in B6, D2, and (BxD) F1 hybrid mESCs. For this locus, both gene expression and chromatin accessibility are dominantly repressed in the F1 (mean ± SEM) C. Scatterplot of the additive versus dominant effect for each Chr 4 caQTL target locus. Points along the diagonal lines indicate dominant effects (red - caQTL targets found at the Riok3/Rmc1 locus. D. MA-plot of significant (FDR < 0.01) differential H3K9me3 level between wildtype (B6) mESCs and those with a 2.47 Mb deletion encompassed by the Chr 4 QTL (n = 2 replicates). Regions that overlap Chr 4 caQTL targets are indicated in magenta (Chr18:12.157 Mb locus from A in yellow). E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test). F. LOLA analysis of significant enrichment between sites that change between WT and Chr 4 KO mESCs and overlap QTL hotspot targets. For H3K4me3, H3K4me1, and H3K27ac there was only one ChIP experiment performed and therefore all sites with a log2 fold-change ≥ 1 were used for overlap enrichment. G. Box and whisker plot indicating direction of change for histone modifications between WT and Chr 4 KO cells. H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO. I. Model of trans-regulation of chromatin state for the Chr 4 QTL. Local variation at the Chr 4 QTL results in differential expression of KZFPs, leading to decreased expression of distal genes through recruitment of TRIM28 and formation of heterochromatin (H3K9me3).
Figure 6Browser representation and independent northern analysis and AGO1-loaded miR165/166. miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds. Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C). Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C). The principles of non-cell-autonomous miRNA-mediated gene regulation within a whole organ.
Elevated levels of Snai1 in in vitro organoids result in reduction of organoid growth and increase in cell deathVillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls.Figure 7VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002. Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.
Figure 3(A) Experimental plan. Mice were subcutaneously injected with LLC cells. When the tumor reached 80-100mm3 size, mice were treated by gavage with glufosinate (10, 20 and 40 mg/kg) for 14 days. Quantification of glufosinate (B) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS). Quantification of glutamine (GLN) (C) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS). Subcutaneous LLC tumor growth over the time (D) (pool of 2 independent experiments; 10 mice per condition in total). number of lung metastatic nodules in vehicle and glufosinate (10 and 20mg/kg) treated mice (E) (pool of 2 independent experiments; 10 mice per condition in total). Quantification and representative images of F4/80+ CD11c+ cells in the lung metastasis (F), F4/80+ CD206+ (G) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6). Quantification and representative images of F4/80 CD11c+ (H) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6). RT-PCR quantification of M2 (Ccl22, Arg1 and Ccl17) (I) markers in vehicle and glufosinate treated mice (n=4). RT-PCR quantification of M1 (Tnfa, Cxcl9, Nos2, Cd86 and Cd80) (J) markers in vehicle and glufosinate treated mice (n=4).
Figure 4A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2). B. Immunofluorescence of LC3 inVPS41WT/WT and VPS41S285P/R662* fibroblasts under steady state and starved conditions. At steady state conditions (0 hours), patient fibroblasts contain more LC3-positive compartments. Nutrient starvation (2 hours) increases the number of LC3-positive autophagosomes in control fibroblasts 13.4-fold, and in VPS41S285P/R662* fibroblasts only 3.8-fold, indicating a reduced responsiveness to starvation (quantified in B'). >54 Cells per cell line were quantified. Scale bars, 10µm. C. Western blot analysis of HeLaVPS41KO cells shows a 4-fold increase in LC3II protein levels in steady state conditions (0 hours) compared to HeLaWT cells. In contrast to control cells, nutrient starvation (2 hours) did not increase LC3II protein levels in HeLaVPS41KO cells, indicating irresponsiveness to nutrient availability (quantified in C') (n=3). D. Rescue experiments. HeLaVPS41KO cells transfected with EV-APEX2-V5, VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 and labeled for V5 and LC3 by immunofluorescence microscopy. Rescue with VPS41WT-APEX2-V5 decreases the number of LC3-positive compartments in steady state conditions (0 hours) and restores responsiveness to nutrient starvation (2 hours) and replenishment (2 hours starvation followed by 15 minutes restimulation). Neither of the mutant VPS41 variants rescues this autophagy phenotype (quantified in D'). >15 Cells per condition were quantified (n=3). Scale bars, 10µm. E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.
The Foxd1Cre::Pdgfrb+/J mice develop progressive renal anemia and are unable to regulate erythropoiesis under pathological stimuli Erythrocyte numbers (A), hematocrit (B) and hemoglobin content (C) decrease in Foxd1Cre::Pdgfrb+/J mice continuously.Figure 6Erythrocyte numbers in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point. hematocrit in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point. hemoglobin content (C) decrease in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point. EPO levels shown as delta from respective wt littermates. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point. RNA in-situ hybridization (RNAScope) of Epo (green) and Pdgfrb (purple) in renal kidney cortex of 35 week old mice shows that EPO-producing cells co-express Pdgfrb. Foxd1Cre::Pdgfrb+/J mice have less EPO-producing cells (arrows) than wt mice. After induction of anemia by blood taking, the erythrocyte numbers recovered after one week in wt mice, whereas Foxd1Cre::Pdgfrb+/J mice were not able to recover their erythrocytes numbers at all. Data are means ± SD of n = 5 per group.
Figure 1(A) Streptozotocin-induced diabetic C57BL/6 (H2b) mice were treated with mouse recombinant IL-33 daily for 5 consecutive days before islet transplantation. On day 0, mice were transplanted with BALB/c (H2d) islets. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected after two consecutive BGLs >16 mmol/liter (mM) after a period of normoglycemia. (B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle. (C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse. (D) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group). (E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001. (F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.
a–c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP–Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c).figf5(a-c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP-Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c). Single black and white inverted channels, merged channels and higher magnification insets are shown. (d) Immunoblots for indicated proteins in isolated fractions from starved mouse liver. Hom, homogenate; LE, late endosomes; Cyt, cytosol; APG, autophagosomes; APL, autophagolysosomes. (e) Immunoblots for different Cx in homogenates and late endosomes isolated from fed and 6 h starved mice. (f-h) Fluorescence images of NRK cells expressing GFP-Cx43 maintained in the presence of serum (f), absence of serum (g) or treated with lindane (h) and incubated with Alexa594-transferrin for 15 min before fixation. Top: single and merged channels and outlined area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the outlined areas. Nuclei are highlighted with DAPI. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Figure 3A volcano plot of proteins that showed significant up- or downregulation of pTyr levels (absolute fold change > 1.5, p < 0.05) in the whole brain of Ptprd-/- mice (P21). Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels. (n = 3 for WT and 3 for KO). See Dataset EV1, 'Phosphoscan Results' tab for a full list of significantly changed proteins. SynGO analysis (https://www.syngoportal.org/) of the 167 proteins that show significant (p < 0.05; fold changes were not considered for maximal inclusion of the proteins) up- or downregulation of pTyr levels in Ptprd-/- mice (P21) for their synaptic functions. See Dataset EV1, 'SynGO Ontologies' and 'SynGO Annotation' tabs for a full list of SynGO annotations and genes. SynGO analysis of the proteins with significant (p < 0.05) up- or downregulation of pTyr levels at their pre- and postsynaptic localizations. A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05). A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).
Figure 5A. Representative electron micrograph of a Wrb+/+:CreA IHC ribbon synapse. Scale bar: 120 nm. B-B'. In Wrbfl/fl:CreA (depicted are two consecutive ultrathin sections) IHCs, accumulations of large partially amorphous vesicles were observed close to ribbon synapses (red arrows), but also further away in the cytoplasm (cyan arrows). Moreover, cisternal structures resembling (r) ER are found unusually close to the ribbon (orange arrow in B'). Scale bar: 120 nm. C. 3D serial reconstruction of the ribbon synapse depicted in (B, B', using "Reconstruct" software). Scale bar: 100 nm. D. Schematic illustration of the quantitative analysis performed on random ultrathin sections, results shown in (E) and (F). E. Quantification in random ultrathin sections (Wrb+/+:CreA n = 28, from two animals; Wrbfl/fl:CreA n = 44, from three animals) of the numbers of membrane-proximal "MP" vesicles (in close proximity to membrane and ribbon); vesicles within a 100 nm range along the active zone membrane; vesicles at the lower "proximal" and the upper "distal" half of the ribbon as well as vesicle >70 nm ("large vesicles") in a radius of 800 nm around the ribbon. The vesicle number was significantly reduced in Wrbfl/fl:CreA IHCs at both ribbon parts but not at the presynaptic membrane.F. Average vesicle diameters of the membrane-proximal vesicles and the vesicles at both halves of the ribbon. The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl/fl:CreA IHCs. Data are represented as mean ± SEM. Student's two-sample t-test ***p<0.001
Quantification of total DCV pool in vitro and in vivo using super-resolution microscopy.Figure 2Representation of resolution limit in confocal imaging and dSTORM strategy to increase resolution. Electron micrograph of axon (green) and dendrite (red) containing DCVs, defined based on morphology. In conventional immunofluorescence confocal microscopy, individual DCVs cannot be separated (right zoom, green signal representing fluorescent signal using confocal). Super-resolution dSTORM imaging resolves the point-spread function (PSF) resulting in single DCV localizations. Scale bars: 100 nm. Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), axonal marker SMI312 (red) and dendritic marker MAP2 (blue). Areas of zoom (C) is indicated in (B). Scale bars: 20 µm (B), 5 µm (C). ChgB immunoreactivity of zoomed area indicated in (C) (top panel). Reconstructed ChgB localizations of dSTORM imaging (middle panel). Lower panel shows overlay of dSTORM ChgB localizations and ChgB immunoreactivity (confocal, 40x). Calibration bar represents no (0) to high (255) clustering of dSTORM localizations. Scale bar: 2 µm. Fluorescence intensity of ChgB immunoreactivity puncta (confocal) with corresponding number of dSTORM puncta for hippocampal (green) or striatal (red) cultured neurons. Squares show average ± SEM, dots represent individual observations. Hippocampus: N = 2 cells (89 puncta), striatum: N = 3 cells (37 puncta). Average histogram of fluorescence intensity of ChgB puncta (40x confocal) in neurons used for dSTORM imaging. Dotted lines indicate cut-off values of fluorescence intensity corresponding 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM, Hippocampus: N = 2 cells, striatum: N = 3 cells. Percentage of ChgB puncta per neuron with corresponding fluorescence value of 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM. Hippocampus: N = 2 cells, striatum: N = 3 cells. Analysis of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. (H) Representative confocal (100x magnification) image of mCherry labeled axon (blue) with ChgA confocal signal (red) and corresponding super-resolution STED ChgA signal (green). Zoomed areas are indicated by arrows. Scale bar merged image (left): 1 µm, scale bars zooms: 100 nm. (I) Percentage of single ChgA puncta within mCherry labeled axons with corresponding number of overlaying STED puncta. Data shows average ± SEM. N = 2 mice (7 STED images, 70 single observations).
β‐Catenin and TCF4 associate with the p62/SQSTM1 gene promoter.Figure 5(A) Chromatin immunoprecipitation (ChIP) demonstrates β‐catenin and TCF4 binding to the p62 promoter. HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies (IgG was used as a negative control). Binding of RNA Pol II, β‐catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin. During nutrient deprivation, binding of RNA Pol II to the p62 promoter increased; binding of β‐catenin to the p62 promoter decreased; TCF4 binding did not change (data are from one representative experiment of at least three independent experiments performed in triplicate). PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A. (B) Relative p62 mRNA expression by qRT-PCR increases ∼14‐fold after 24 h starvation in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). (C) Binding of acetyl‐Histone H3 to the p62 promoter increased under starvation conditions, suggesting p62 gene derepression (data are from one representative experiment of at least three independent experiments performed in triplicate). (D) Binding of β‐catenin to the p62 promoter (relative to β‐catenin binding to a non‐target gene promoter, GAPDH) significantly decreased under starvation conditions (mean±s.e.m., three independent experiments performed in triplicate, **P0.01).