EMBO/sd-character-level-ner huggingface dataset:
Detection of SCNAs in CSF is influenced by tumor grade, cfDNA concentration and contact between the tumor and CSF.Figure 2Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light grey Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL)
Figure 4The retinal vasculature of endothelial cell specific EVL knockout mice (EVLΔEC) and littermate controls (EVLfl/fl) on P5 was analyzed by Isolectin B4 staining. Radial outgrowth of the retinal vasculature; in global EVL-/- mice are shown for comparison; 4 - 5 independent experiments. (C) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front.(E) Filopodia numbers normalized to the length of the angiogenic front. (F) Endothelial cell proliferation analyzed by BrdU incorporation (red) of ERG positive (blue) endothelial nuclei.
Figure 1A) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells. B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon. C) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype. D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3 ) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.
Figure 5Control siRNA depletion (siCtrl) was conducted with siRNA oligoes specific to a firefly luciferase sequence; siCOPB = depletion of γ-COP; siRab18 = depletion of Rab18. C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted. N= 3, Error bars = S.D.
R2-P2 allows scalable and reproducible phosphoproteomics.Figure 2Different amounts (25 µg, 50 µg, 100 µg, 200 µg and 400 µg) of yeast protein extract were processed by R2-P2 or by in-solution digestion and reversed phase C18 SPE desalting, phospho-enriched by robotic Fe3+-IMAC followed by peptide analysis with DDA-MS. Sample preparation was performed in triplicate. Boxplot depicting MS1 signal intensity distributions for phosphopeptides detected in all samples. Line plot of log2 phosphopeptide intensity versus protein input amounts with each line representing an individual phosphopeptide. For every phosphopeptide a linear regression was performed and lines are colored according to the slope of the linear regression. Black line represents the linear regression of the median phosphopeptide intensities and the corresponding function is shown on top.   Boxplot depicting the distribution of slope values resulting from linear regressions of individual phosphopeptides shown in B.  Boxplot depicting the distribution of r squared values resulting from linear regressions of individual phosphopeptides shown in B.   Average number of unique peptides identified for R2-P1 performed on 5 different days (n=5).   Average number of unique phosphopeptides identified for R2-P2 performed on 5 different days (n=5).   Boxplot depicting CVs of peptide MS1 signal intensities for reproducibility analysis of R2-P1 within the same batch (n=5) and between different batches conducted on different days (n=5). For each peptide, one replicate per batch was randomly chosen for inter-batch comparisons.   Boxplot depicting CVs of phosphopeptide MS1 signal intensities for reproducibility analysis of R2-P2 within the same batch (n=5) and between different batches conducted on different days (n=5). For each peptide, one replicate per batch was randomly chosen for inter-batch comparisons.
– The CDKB1;1-CYCB1;1 complex phosphorylates RAD51.Figure 5A. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin, respectively, three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student's t-test).B. Image shows a wild-type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination.C. Image shows a wild-type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination. Images were taken 6 days after germination, i.e. 3 days after transfer to cisplatin. D. Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin. E. Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4 or >5 foci per cell. F. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ-32P]ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiograph (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK-cyclin complexes, protein blots indicate the relative amounts of the CDKs in the reaction (bottom panel).Abbreviation: p-RAD51 and p-Histone H1 for [32P]-phosphorylated MBP-RAD51-His6 and recombinant human histone H1, respectively, resulting from kinase assays with radio-labeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1-CYCA2;3, 3: CDKB1;1-CYCA2;3, 4: CDKB1;1-CYCD2;1, 5: CDKA;1-CYCB1;1, 6: CDKB1;1-CYCB1;1.
Figure 7(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer. (B-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.
ERK activity responses to multiple GF pulses.Figure 4A-CDouble‐pulse GF experiments. (A) 3′ GF/3′ pause/3′ GF. (B) 3′ GF/10′ pause/3′ GF. (C) 3′ GF/20′ pause/3′ GF. Cell‐averaged ERs, population average, and StDev range for n = 10 cells. D-GMulti‐pulse GF experiments. Population average ERs (n= at least 30 cells) for different EGF/NGF dosages. (D) 3′ GF/3′pause. (E) 3′ GF/10′ pause. (F) 3′ GF/20′ pause. (G) 3′ GF/60′ pause. HDecay kinetics of ERK activity maxima from (D-G). Boxplots with median, interquartile (box), and 1.5 IQR (whiskers) range are shown for at least n = 30 cells.
Myosin VI depletion affects the centriolar satellites via p53.Figure 5(A) A scheme of the IF analysis performed to calculate the total intensity of satellite staining that surrounds the centrioles. The centriole marker Cep135 or Cep164 are used to define the centre of a 3 μm circle, in which the intensity of the satellite marker PCM1 was calculated. (B) IF analysis of PCM1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1 intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=96 cell; MyoVI KD, n=98cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (C) IF analysis of PCM1 signal. hTERT-RPE1 cells were treated with Nutlin-3 for 24 hours and immunostained with anti-PCM1 and anti-Cep135 antibodies. Panels as in B. Mock, n=96 cells; Nutlin-3, n=100 cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (D) IF analysis of PCM1 signal. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep164 antibodies. Panels as in B. Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti-Cep164 staining. Mock, n=128 cells; MyoVI KD, n=114 cells from three independent experiments. ns, not significant by Mann-Whitney test.
Figure 3A. Top. Representative image of a radiograph of mature and immature bands of GluA2 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA2 band to the total GluA2 for each condition. For DHPG, n = 16; for DHPG + 2APB/dan, n = 11, p < 0.05, ANOVA. B. Top. Representative image of a radiograph of mature and immature bands of GluA1 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA1 band to the total GluA1 for each condition, For DHPG, n = 4; for DHPG + 2APB/dan, n = 4, ANOVA. C. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment. D. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.
Figure 5 A) Analysis of the autophagic flux (n=4/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rilmenidine, or rilmenidine plus colchicine. Colchicine alone increased LC3-II in WT but not Cox15sm/sm mice; rilmenidine had no effect on WT LC3-II levels, but reduced them in Cox15sm/sm mice. Rilmenidine plus colchicine increased LC3-II levels in both WT and Cox15sm/sm mice. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: **p=0.0093 (WT vs. WT+colchicine), ****p<0.0001 (WT+rilmenidine vs. WT+rilmenidine+colchicine), **p=0.0001 (WT vs. Cox15sm/sm), **p=0.0083 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine), *p=0.0112 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine+colchicine), ****p<0.000  B) Treadmill analysis (n=4/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM  C) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; GT: Gomori trichrome; PAS: periodic acid Schiff reaction  D) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ***p=0.0002, ****p<0.0001  E) Analysis of the number of centralized nuclei in muscle fibres (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0091
Figure 9Working model for the import route of Mcp3. The different steps (1-5) are described in the text.
PDI proteins are redistributed to the cytosol during ER stress.Figure 4Representative transmission electron microscopy images of the gold particles distribution (after immunogold labelling with PDIA3 antibodies) in A549 cells treated with DMSO or Tm. In the inserts, gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles. (Scale bar 500 nm). n represents the nucleus and m represents the mitochondria. (C) violin plots of the gold particles distribution from the electron microscopy experiment (immunogold labelling of PDIA3) as shown in Figure 4B and 4C. *P-value=0.0486. n=8 for DMSO and n=8 for Tm. Thick horizontal lines represent mean ± SD -lighter dashed lines. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad),
Figure 1A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed. B. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++). C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors. D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining). E. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy. G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.
Figure 3(A) NIH3T3 cells expressing nAC-mCherry were imaged for 120 min. Image frames represent maximum projections of 30 Z planes. Lower panel is a magnification of the indicated area (orange box); scale bars 10 µm. (B) NIH3T3 cells stably expressing H2B-mCherry were transfected with siCtrl or siACTN1 + siACTN4. After mitotic exit z-stacks (30 planes) were acquired every 5 min. Nuclei were 3D-reconstructed and volume was measured by Imaris. Data are mean ± SEM from 3 biological replicates with 22-27 (siCtrl) and 32-42 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by t-test at 90 min. (C) RPE-1 cells were analyzed as in B. Data are mean ± SEM from 5 biological replicates with 32-37 (siCtrl) and 31-39 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by Mann Whitney test at 90 min. (D) Cartoon illustrating experimental design of knockdown and reconstitution experiments in E, F and G to achieve cells lacking ACTN4 specifically in the nuclear compartment (left) versus cells expressing ACTN4-wt. (E) NIH3T3 cells stably expressing H2B-mCherry and siRNA-resistant ACTN4-NES were transfected with the indicated siRNAs and analyzed as described in B. Data are mean ± SEM from 5 biological replicates with 63-71 (siCtrl), 37-41 (siACTN1+4), 52-58 (siACTN1) and 52-55 (siACTN4) analyzed nuclei; ****p(siACTN4) < 0.0001; ****p(siACTN1+4) < 0.0001 by One Way ANOVA at 90 min. (F) NIH3T3 cells stably expressing H2B-mCherry and the indicated ACTN4 mutants were transfected with the indicated siRNAs and analyzed as in B. Data are mean ± SEM from 3 biological replicates with 63-69 (ACTN4 wt + siACTN4), 63-71 (ACTN4-NES + siCtrl) and 54-58 (ACTN4-NES + siACTN4) analyzed nuclei; ****p(ACTN4-NES + siACTN4) < 0.0001 by One Way ANOVA at 90 min. (G) NIH3T3 cells expressing siRNA-resistant ACTN4-NES were transfected with siCtrl or siACTN4; chromatin density was calculated from H2B-mCherry fluorescence intensities by total nuclear volumes (intensity/μm3) and normalized to t=0 (mitotic exit); Data are mean ± SEM from 5 biological replicates with 44-48 (siCtrl) and 44-47 (siACTN4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and *p < 0.05 by t-test at 90 min.
HSFA2 associates with HS memory-related genes transiently after an acclimatizing HS.Figure 5Figure 5: HSFA2 associates with HS memory-related genes transiently after an acclimatizing HS.Binding of HSFA2-YFP to the promoter regions of HSP18.2 (A), HSP22.0 (B), APX2 (C), HSP70 (D) and ACTIN7 (E) was determined by ChIP-qPCR. Quantifications represent the enrichment relative to input in %. Red-orange-yellow bars represent time points (0.5 h-4 h-28 h) after a ACC and grey bars represent the respective NHS control samples. Schematics show the promoter region of the respective gene with regions analyzed (arrow, TSS; grey bar, 5'UTR; black bar, exon; black boxes, heat shock elements (5'-nnGnAnnTnCtn-3'). Regions are drawn to scale. Intergenic control region 1 is 4610 bp (HSP18.2) and 4192 bp (HSP70) downstream of the 3'UTR and 2570 bp (HSP22.0) or 3123 bp (APX2) upstream of the TSS, respectively. One representative experiment out of at least four biological replicates is shown. Error bars are SD of three replicates.
Figure 6(A) The overall schematic diagram of the study design. The nasopharyngeal carcinoma xenograft model was established using HK1-LMP1 cells. (B) Representative images of xenograft tumors from different treatment groups. (C/D) The tumor volume (C) and tumor weight (D) of HK1-LMP1-derived xenograft tumors with various treatments. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01). (E) The mouse weight curve of the different treatment groups in a HK1-LMP1 cell xenograft model. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01). (F) Immunohistochemistry examination of the protein levels of Ki67 (scale bar, 50 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01). (G) Apoptosis was analyzed by TUNEL assay (scale bar, 50 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01). (H) Apoptosis was analyzed by Western blotting.
The hGID complex uses distinct substrate modules to target different substrates.Figure 1A. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3). Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3). Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3). D. Schematic representation visualizing the hGID E3 ligase complex using two distinct modules for substrate recruitment.
Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal.Figure 10 Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal. Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins. The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins (CtBP2, Calretinin and Homer1) fixed with glyoxal. None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation. Representative images show maximum intensity projections from z-stacks of inner hair cell ribbon synapses. N = 5 independent stainings from two animals per condition (PSD95, CtBP2, Otoferlin, Calretinin) and 10 - 15 images per condition (CaV1.3 and Homer1). Scale bar = 2 µm. ** p < 0.01, *** p < 0.001