EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) UMAP plot of snRNA-seq data from 64,381 combined neural cells from GW7 to GW23. Cell cluster phenotypes are labelled and the red arrow indicates the EGFR-expressing glial cluster (left). Selected UMAP plots from different development stages (GW7, GW11, GW18, and GW23) represent the neural development trend (right). Gray represents non-specific cell types. (B) The stacked proportions of neural cell classes showing changes in stage-dependent differentiation trends with developmental stage. (C) Violin plot showing the expression of selected genes. EGFR+ Cluster 14 is circled with a red dotted line. (D) Visualization of selected top gene ontology (GO) enrichment terms of astrocyte clusters based on DEG analysis.
CIP2A and c-Myc are degraded in an autophagy-dependent manner.Figure 6.(A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0-24 h. (B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments. (C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control. (D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0-5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA. (E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm. (F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control. (G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (<100 kD), medium (>150 kD), and long (100-130 kD) exposures are shown. (H) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions (see G) and analyzed by immunoblotting for CIP2A and monoubiquitin. (I) Lysates from MCF7 cells transfected with the indicated ubiquitin-HA constructs were subjected to anti-CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA. (J) Lysates from MCF7 cells transfected with the CIP2A-FLAG construct (Junttila et al., 2007) and the indicated ubiquitin-HA constructs were subjected to anti-FLAG immunopurification in denaturing conditions with 0.1% SDS followed by immunoblotting with the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Ub, ubiquitin; WT, wild type.
a) HEK293 cells were transfected with either control shRNA or a PEX5 targeting shRNA, and lysates were blotted using pS6K (T389), S6K, pS6 (S235/236) and S6 antibodies.figf7(a) HEK293 cells were transfected with either control shRNA or a PEX5 targeting shRNA, and lysates were blotted using pS6K (T389), S6K, pS6 (S235/236) and S6 antibodies. (b) TSC2 functional assay was performed in HEK293 cells co-expressing myc-Rheb, Flag-TSC1 and Flag-TSC2 wild type (WT), or Flag-TSC2 mutants (RG, RQ and RW) and HA-S6K (left panel) or HA-4E-BP1 (right panel). Cells transfected with empty vector (EV) with or without myc-Rheb were used as controls. Lysates were further analysed for phospho-S6K (T389), S6K, phospho-4EB-P1 (S65), 4E-BP1 and 32P incorporation into S6. (c) Rheb GTPase activity assays were performed by co-immunoprecipitating TSC heterodimers from HEK293 cells expressing Flag-TSC1 and TSC2-TSC2 wild type, Flag-TSC2 mutants or the Flag-TSC2 GAP mutant (L1624P), for the indicated times. (d) TSC2 functional assay was performed using TSC2−/− MEFs co-transfected with Flag-TSC1, HA-S6K and Flag-TSC2 wild type or Flag-TSC2 mutants (RQ or RQ-9NT), with mock transfected cells as controls. Arrows denote the positions of Flag-TSC1 and Flag-TSC2. (e) Quantification of the ratio of phospho-S6K to total HA-S6K from Fig. 7d. (±s.e.m., n = 3 independent experiments). *P0.05;**P0.01. (f) In vivo guanine nucleotide loading of Rheb was measured using HEK293 cells overexpressing Flag-TSC1 and Flag-TSC2 wild type, or Flag-TSC2 mutants (RQ and RQ-9NT) with or without Rheb as indicated. (g) Graph shows quantification of the percentage of Rheb bound to GTP (indicative of Rheb GTPase activity, ±s.e.m., n = 4 independent experiments). *P0.05;***P0.001. (h) Quantification of axon number in hippocampal neurons co-transfected with GFP, Flag-TSC1 and Flag-TSC2 WT or Flag-TSC2 mutants. Quantification was performed on 150-250 neurons from each of the 3 independent experiment and the results are represented as polarity (%). All error bars represent s.e.m. *P0.05;**P0.01 compared with GFP-only transfected control. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.
Figure 6Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.
Systematic identification of secretion-related phosphorylation targets of PKCB.Figure 1A. Maximal IgE-induced beta-hexosaminidase release only releases part of the vesicle pool and is inhibited dose dependently by PKCB inhibitor, Ruboxistaurin (n = 9). Values are mean ± SEM. B. PKCB knockout in RBL-2H3 cells, achieved with genome editing using TALENs. Western blot validating complete loss of PKCB protein expression. C. IgE-induced beta-hexosaminidase release was inhibited in PKCB knockout cells and was rescued with stable overexpression of either PKCB1 or PKCB2 (n = 24). Wild type RBL cells in pink and PKCB knockout cells in blue. Values are mean ± SEM; ***P < 0.001. D. Phosphoproteomics analysis of PKCB targets during mast cell secretion. Intensity for each phosphopeptide compared between biological replicas of wild type cells challenged with antigen. E. Schematic of how candidate phosphorylation sites were narrowed down to 179 secretion connected phosphopeptides. F. Mean intensity for each phosphopeptide with WT compared to PKCB inhibitor versus WT compared to PKCB knockout cells. Dotted line represents threshold for 2-fold change of intensity. The hits (179 phosphopeptides) are highlighted in blue and the 4 phosphopeptides from VAMP8 are highlighted in red: T53 (above both thresholds), T47 (above one threshold), S54 (below both thresholds) and S17 (below both thresholds and site not perfectly conserved in mammals). G. VAMP8 immunocytochemistry imaged with a spinning disk confocal microscope. VAMP8 localized to vesicles in resting cells and expression was reduced after VAMP8 siRNA knockdown. VAMP8 translocated to the plasma membrane following antigen stimulation (4 minutes) (n = 3, >50 cells per condition). Scale bar 10 µm.
Figure 9In the absence of injury, PGE2-EP4 signaling does not reach the signal threshold required for WAE cell formation, and enterocytes differentiate from intestinal epithelial stem cells in accordance to their normal program of differentiation. Upon mucosal injury, Ptgs2-expressing mesenchymal cells become localized to the wound bed in close proximity with the migrating WAE cells. High local production of PGE2 signals to the EP4 receptor on differentiating intestinal epithelial cells to induce formation of WAE cells, which migrate to cover and seal the wound bed, thereby re-establishing the epithelial barrier.
Figure 1A Domain architecture of CALCOCO paralogs showing the SKICH domain, a conserved LIR motif (LVV), coiled-coli regions (CC) and zinc finger domains (ZF). Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In C endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant. Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant. F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 μm and 2 μm (zoomed inset). G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans. H CALCOCO1, p62 and LC3B puncta in in the indicated conditions, counted using an automated system. The error bars represent mean±SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001. I Percentage of co-localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean±SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001. J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.
HGSOC organoids match tumor tissue in mutational profile and gene expression biomarkers.Figure 2Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected. Representative western blot showing loss or overexpression of p53 in individual organoid lines. Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid). Multi-Dimensional-Scaling (MDS) plot based on the gene expression profiles (microarrays) of 3 healthy FT and 8 tumor tissue samples and their respective organoid cultures shows 4 clusters: Normal FT tissue, normal FT organoids, cancer organoids and cancer tissue. Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors. Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.
Figure 5In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM10, TMPRSS2 (A) for 24 hours at a 1:1 ratio and analyzed by Western Blot. Camostat mesylate or BB94 (10 µM) were pre-incubated with TMPRSS2 or ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag. B. In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM17 (B) for 24 hours at a 1:1 ratio and analyzed by Western Blot. BB94 (10 µM) were pre-incubated with ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag. C. Representative images and quantification of a proximity ligation assay (PLA) between the SARS-CoV-2 Spike and ADAM17 in A549-ACE2 cells. Cells were infected with SARS-CoV-2 and placed on ice (4°C). After 2 h, cells were shifted to 37°C for 15 min or left on ice. Images are presented as overlay between the Nuclei (Hoechst, blue) and the PLA (pseudo-color: red) channel. Scale bar, 20 µm. The number of PLA foci per cell was determined for the acquired images (N = 28-30). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. D. A549-ACE2 cells were treated for 48 h with BB94 (10 µM), DPC-333 (5 µM) or DMSO. PMA (25 ng/ml) was added for 3 h before harvest to stimulate ACE2 shedding. Concentration of the soluble ACE2 fragment was determined in the conditioned medium by ELISA and is shown relative to the concentration in the DMSO control (N = 6). One-way ANOVA with Tukey's post-hoc multiple comparison test. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. E. GFP-encoding spike pseudoparticles (VSV pseudotyped with SARS-CoV-2 spike protein (VSV-S) were incubated with indicated amounts of recombinant, soluble ACE2 (sACE2) or bovine serum albumin (BSA) as a control prior to infection of A549-ACE2 cells. At 16 hpi, infection was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without sACE2 or BSA (N = 3). Two-sided independent Student's t test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
TrkB trafficking is impaired in CDKL5 KO dendrites.Figure 5 A, B Representative stills (A) and kymographs (B) of TrkB-RFP overexpressed in dendrites of WT and CDKL5 KO DIV14 cortical neurons. Scale bar is 5 μm. C, D No significant change is observed in the ratio of dynamic vs stationary vesicles (C) or anterograde vs retrograde runs (D). S = stationary, D = dynamic, A = anterograde, R = retrograde. Vesicles with a net distance > 5 μm were considered dynamic. E, F Both anterograde and retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons (E). Anterograde run lengths are significantly reduced in KO compared to WT neurons (F)
Figure 2(A) Subjective assessment of hair depigmentation (score 0-5, with 0 = completely black and 5 = completely white) at 2 doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (mean ± SEM, n=8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal-Wallis with Dunn's post-hoc test. (B) Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro.
Performance evaluation of JSTA using simulated data.Figure 2a. Representative synthetic dataset of nuclei (black) and mRNAs, where each color represents a different gene. b. Ground truth segmentation map of the cells in the representative synthetic dataset. Each color represents a different cell. c. Average Accuracy of calling mRNA spots to cells at different cell type resolutions using 83 genes across 10 replicates. Accuracy was determined by the assignment of each mRNA molecule to the correct cell type. JSTA (solid line) is more accurate than watershed (dashed line) at assigning mRNA molecules to the correct cells (FDR < 0.05). Statistical significance was determined with a Mann-Whitney test and false discovery rate correction. d. Accuracy (as described in c) of calling mRNA spots to cells when using JSTA to segment cells with a lower selection of cell type marker genes (8-44 genes tested). The color of the line gets progressively darker as the number of genes used increases.
Figure 6A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected. B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo. D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9). E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8).
a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time.figf4a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time. Homogenates of P9, P18 and P56 brains from Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice were immunoblotted with an anti-ubiquitin antibody. An anti-actin antibody was used as a loading control. Data shown are representative of three separate experiments. We observed no sex difference in the accumulation of ubiquitin in Atg7flox/flox; nestin-Cre mice. b, Peptide hydrolysis activity of 20S and 26Sproteasomes. Homogenates from P28Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glyceroldensity gradient centrifugation (10-40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads. Note that whereas 26Sproteasomes exist in active forms in tissues, 20Sproteasomes are latent and are activated artificially by a low concentration of SDS. c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. d, Immunoblot analysis of 26S proteasome components. Homogenates from P28 Atg7flox/flox, Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains were immunoblotted with antibodies against the indicated proteins. Data shown are representative of three separate experiments. There was no change in proteasome status for the different genotypes.
PGAM5 is an IMM protein that protects PINK1 from degradation by mitochondrial proteases including PARL.f3PGAM5 is an IMM protein that protects PINK1 from degradation by mitochondrial proteases including PARL. After CCCP treatment, full-length PINK1 protected by PGAM5 will move to the OMM, while PGAM5 remains on the IMM. In the absence of PGAM5, PINK1 loses the protection from PGAM5, and will be cleaved and degraded. CCCP can trigger PGAM5 stabilized PINK1 translocation from IMM to OMM where it associates with parkin leading to ubiquitination events associated with p62 recruitment and culminating in LC-3-mediated mitophagy.
Figure 10(A) Representative immunofluorescence images of ZW10 streaming from KTs in Drosophila S2 cells in metaphase expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. ZW10 streaming is also shown in S2 cells expressing SpindlyWT- or SpindlyS499A-EGFP in a dynein-depleted background. Insets display magnifications of the outlined regions, which highlight streaming robustness. (B) Graph represents the percentage of cells in metaphase showing different levels of ZW10 streaming (n≥35 cells for each condition, n=2 independent experiments). (C) Graph represents the percentage of ZW10 levels at aligned KTs normalized to ZW10 levels at the spindle region. All values were normalized to the mean fluorescence intensity quantified in SpindlyWT-EGFP expressing cells, which was set to 100% (n≥470 KTs from at least 35 cells for each condition, n=2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (D) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing SpindlyWT-, SpindlyS499A-, SpindlyS499D- or SpindlyS499D-EGFP in a ZW10-depleted background. Insets display magnifications of the outlined regions which highlight different attachment configurations (E - end on; L - lateral; M - merotelic). Cartoon depicts the attachment configuration of the respective KT pair. Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber. Plotted profiles show the overlap between CENP-C and tubulin signals for the highlighted KT. CENP-C was used as a KT reference. (E) Graph represents the percentage of metaphase cells showing only end-on attachments or at least 1 KT with lateral or merotelic attachment, as shown in D (n≥44 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (F) Proposed model for Polo-mediated regulation of RZZ-Spindly and its impact on KT-MT attachment. High levels of active Polo during early mitosis render Spindly phosphorylated on Ser499, thus weakening its interaction with the RZZ complex at KTs. On laterally attached KTs, the presence of minus end-directed motor dynein allows the removal of phosphorylated Spindly while the RZZ complex is retained at high levels to inhibit premature formation of stable end-on attachments and therefore avoid merotely. As protein phosphatases are recruited to KTs, Polo activation status declines and Spindly is eventually dephosphorylated. Spindly is now able to bind Zwilch and the Spindly-RZZ complex is stripped from KTs through dynein, which enables the stable conversion from lateral to end-on attachments and SAC silencing.