EMBO/sd-character-level-ner huggingface dataset:
Figure 4T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.m Significant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group).
Figure 5A Cell morphology and the mRNA levels of inflammatory cytokines, including and TNF-α, IL-6, and IL-1β in mouse primary macrophages treated with LPS and ANGPTL4 (100 ng/ml) using qPCR and RT-PCR, and levels of TGF-β and NO in the LPS- and ANGPTL4-treated primary macrophages (n = 5). Scale bar represents 100 µm B Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2-well silicone culture inserts that were placed in a cell culture μ-Dish and transwell at 24 h (n = 5). Scale bar represents 50 µm C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes (macrophages, red and acinar cells, green). Co-cultured cells treated with 100 ng/ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h. Scale bars represent 80 µm (red) and 40 µm (white).
Fig.Figure 7A The nuclear lamina is visualized using a GFP-tag inserted into the endogenous locus of the Kugelkern (Kuk) gene in animals expressing the indicated overexpression or RNAi constructs in the epidermis. B A GFP-tagged transgenic construct of Kugelkern is co-expressed with the indicated overexpression or RNAi constructs. High levels of Kuk induce lobulation and other nuclear defects, which are ameliorated if TOR is downregulated or autophagy upregulated, but not if other branches of the TOR signalling pathways (S6K or rictor) are modified.
Figure 3Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2. Two core components of the INO80 complex were confirmed by immunoblotting. Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates. HAND2-AS1 was visualized by RNA FISH, followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells. Scale bar, 100 μm. Mapping analysis of INO80-binding domains of HAND2-AS1. Schematic diagram of HAND2-AS1 full-length and truncated fragments (top panel); Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2-AS1 fragments (middle panel); Different HAND2-AS1 fragments (bottom panel). EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein. Binding affinity of HAND2-AS1 with INO80 was determined by five independent EMSA assays. Non-linear regression curves were generated by GraphPad Prism. Results are shown as means ± SD. Interaction regions of INO80 with HAND2-AS1 mutations at nt 83-242 of exon 2 were detected using RNA pulldown assays. LR, loop region. Representative whole-body imaging of Huh7-Luc cells transduced with control or INO80 shRNA #1 or shRNA #2 vectors. Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD (n = 5 mice per group). One-way ANOVA with Dunnett's correction for multiple comparisons. Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).
ADAP-deficient mice enhance DC-based vaccine for prevention of tumors in vivo.Figure 5A The same procedures as in Fig4A were performed with WT and ADAP−/− C57BL/6 mice. HE staining of lung tissue was examined on day 26 (representative graph of 5 mice). B, C PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells (mean ± SD, n ≥ 5 mice per group) or granzyme B expression in CD44hiCD8+ T cells were examined from the lungs at day 26 (representative data of 3 mice per group). D, E CTLA-4 levels in lung infiltrating CD44hiCD8+ T cells and the percentages of CD4+Foxp3+ T cells in the lungs or MLN were measured at day 26 (mean ± SD, n ≥ 5 mice per group).
Intestinal barrier dysfunction in Vil-Ogt KO mice.Figure 4 A, B Intestinal barrier functional assays measuring serum FITC-dextran (A) and albumin from fecal sample (B) of male wildtype and Vil-Ogt KO mice. (A: WT n = 5, KO n = 6, P = 0.0015; B: WT n = 9, KO n = 6, P = 0.0053  C Representative EM pictures of ileum epithelial cells. Arrows, arrowheads, and stars indicate tight junctions, adherens junctions, and desmosomes, respectively. Scale bars = 1 μm  D Immunoblotting of protein markers of tight junction (ZO-1), adherens junction (β-CATENIN, α-E-CATENIN and PLAKOGLOBIN), and desmosome (PLAKOGLOBIN and DESMOPLAKIN) in colon E Densitometric analysis of Plakoglobin protein levels in (D) (WT n = 6, KO n = 5, P = 0.021)  F Representative images of ZO-1, ZO-2, PLAKOGLOBIN and β-CATENIN immunofluorescent staining in the ileum tissue of male wildtype and Vil-Ogt KO mice. Arrow and arrowhead indicate ZO-1 and PLAKOGLOBIN, respectively. Scale bars = 10 μm
Figure 2 A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test  C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated  E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;
by amino acids stimulation A.Figure 5A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B) C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of Exo-2 (10 μM) (C). D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation. E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm. F. Statistical analysis of the Pearson's correlation coefficient of COPA and lysosomes in Figure 5E (n=10-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.
Alterations of SMN, HDAC1 and eIF2α in FusΔNLS/ΔNLS mice.Figure 8Representative images of SMN (green) immunofluorescence in spinal cord. Nuclear gems, corresponding to SMN-immunoreactive foci in nuclei are marked by arrows. HDAC1 immunoreactivity in spinal cord sections of Fus+/+ and FusΔNLS/ΔNLS mice. Representative images of immunofluorescent staining of motor neurons, labelled with ChAT (red) and HDAC1 (green). Examples of HDAC1 immunoreactive foci in motor neurons are indicated by arrows. Representative images of immunofluorescent staining of motor neurons labelled with ChAT (red) and phosphorylated eIF2α (green), a general translational stress response marker. DRAQ5 (cyan) was used to label nuclei.
Figure 2(A, B) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot. (C) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β-catenin phosphorylation by western blot. (D) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers. (E) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay. (F) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors. In vitro formation of MDMX-CK1α complex was detected by MDMX IP followed by CK1α western blot.
suppresses mucinous tumor emergence and overall tumor progression.Figure 6(A) H&E, PAS and AB staining of lung sections in KLN mice with vehicle or a combinational therapy of phenformin (Phen) and LY-411575. Representative images were shown. Scale bar, 150 μm (left panel), 50 μm (right panel). Statistical analysis of mucinous tumor incidence (B) in KLN mice with vehicle or combined treatment (n = 7 per group). Results were shown as mean (B). Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. **p=0.0078 (B) Statistical analysis of total tumor number (C) in KLN mice with vehicle or combined treatment (n = 7 per group). Results were shown as mean ± SD Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. , *p=0.0490 (C) Statistical analysis of tumor size (D) in KLN mice with vehicle or combined treatment (n = 7 per group). Results were shown as mean ± SD Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. , *p=0.0134 (D). (E, F) Statistical analysis of Ki67 (E) and CC3 (F) reactivity in lung sections of KLN mice following vehicle, LY-411575 treatment or a combinational therapy of phenformin and LY-411575. The numbers of tumors analyzed over vehicle, phenformin and combinational therapy groups were 20, 30, 13 for Ki67, 14, 11, 9 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0014 (E), ***p=0.0001 (F). (G) IHC staining for Ki67 and CC3 in lung tumor tissues of KLN mice in vehicle, LY-411575 treatment group and combinational group. Representative images were shown. Scale bar, 50 μm. (H) Schematic illustration shows that Nanog is a barrier for KL ADC to IMA transition. Notch signaling is up-regulated in IMA and promotes mucinous differentiation which could be reversed by γ-secretase inhibitor LY-411575. LY-411575 or Phenformin treatment has little effect on mucinous tumor progression. Combination therapy overcomes the treatment resistance imposed by mucinous lineage transition through induction of cell apoptosis.
Figure 1Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM). Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h. Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM). Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal). Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h). Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h). Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h).
Figure 1A Representative IF image of NPCs stained for Nestin, FoxG1 and Pank2. B NPCs differentiated into neurons by overexpressing Ngn2 (one representative experiment is shown). Two weeks after the infection differentiated NPC were positive for neuronal markers βIII-tubulin (Tuj1), Map2, NeuN and humannuclei (hNu) and synaptic markers, the voltage-gated Na+ channels (PanNav) and the vesicular glutamate transporter 1 (VGlut1). C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β-actin antibodies (arrows). Asterisk indicates nonspecific band. Data are representative of three independent experiments. D Plots showing the total dendritic length and branching points. Data presented as mean and + SEM from at least three independent experiments. A total of 38 neurons were counted for each sample. Statistics were determined by the t-test and resulted not significant. E Representative example of co-culture containing control (green), PKAN (red) humanneurons, and E18 cortical miceneurons. Control and PKAN NPCs were infected with GFP-LV and tdT-LV expressing vectors, respectively, and differentiated for 8 weeks. Scale bars 20 μm. F Examples of electrophysiological properties of humanneurons: control individual (top); PKAN patient (bottom). Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current-clamp mode. Middle traces show Na+ and K+ currents (down- and upward-deflecting from baseline, respectively) in response to a 60 mV step from a holding voltage of -70 mV in voltage-clamp mode. Insets on the right display enlarged portions of the traces to magnify fast Na+ currents. G Histogram with percentages of recorded cells showing repetitively firing (z-test).
a) Chemical structure of allosteric inhibitors.figf3(a) Chemical structure of allosteric inhibitors. (b) Visualization of the VCP structure and the amino acids modified by the azido derivatives. Top, schematic representation of the three-dimensional hexameric structure derived from PDB code 3CF1 (left), showing the allosteric binding site (right). Blue, green and pink are used to distinguish the N, D1 and D2 domains of individual protomers in the hexameric structure, respectively. Different shades distinguish adjacent protomers. The region of binding identified by photoaffinity labeling experiments is highlighted. The right panel shows in detail the lateral tunnel formed by two D1 domains and one D2 domain of two adjacent protomers leading to the central pore of the hexamer. The position of the Lys615 (red) and Asn616 (yellow) residues in D2, derivatized by the azido probes, is highlighted. Bottom, modeled binding site of allosteric inhibitors and one example of a superimposed azide derivative (NMS-454) in close proximity to the reacting amino acid side chain (Asn616). The azido moiety was added after optimizing the orientation of the compound in the unmodified three-dimensional structure. The binding site is situated in a region connecting one nucleotide pocket (N protomer) to the next (N + 1 protomer) by means of a close interaction between the ISS motif residue Asp609 and the second region of homology (SRH) residue Arg638. Additional amino acids involved in the mutation studies are also shown.
Genetic switch to cyclin D1b promotes serum independence.Figure 5Ccnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls. Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control. Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates. Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate. Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls. Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.
Figure 4Schematic computational workflow: The SVM predictor was initially developed using a bootstrapping procedure on n=44 primary SQCLC and n=30 HNSCC cases. An independent set of n=38 tumours was used for validation, before we applied the classifier to n=51 lung tumours of unknown origin. Survival analysis was used to assess classification performance on this final data set. Classification accuracy of five different machine-learning methods used for classification of HNSCC vs. SQCLC. SVM, Support Vector Machine with a linear kernel; RPART, Classification and Regression Tree (CART); NB, naïve Bayes classifier; PAM, Partitioning Around Medoids / Nearest Shrunken Centroids; PLR, L1-Penalised Logistic Regression. All models were fitted by using the R/Bioconductor package 'caret'. Model hyperparameters were optimised by using caret's tuning option; accuracies were computed by bootstrapping with 25 repetitions, shown are mean accuracy ± standard deviation. Classification accuracy using a linear support vector machine. The figure shows the bootstrapped accuracy achieved over the number of proteins included in the computation. Feature selection was performed using recursive feature elimination. Boxplot showing the number of quantified proteins derived from 19 SQCLC, 19 HNSCC and 51 squamous-cell lung tumours of unknown origin derived from HNSCC patients. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum. Classification accuracy of the SVM-prediction model described, in an independent validation cohort of 19 SQCLC and 19 HNSCC cases. Representative H&E and immunohistochemical stainings of CK5/6 and p63 in squamous-cell lung tumours of unknown origin derived from HNSCC patients. Scale bar: 500 µm. Kaplan-Meier analysis of overall survival (OS) in which all patients with lung tumours were grouped according to the clinical classification (left, median survival SQCLC: 55 months; metHNSCC: 31 months) or proteomic classification (right, median survival SQCLC: 55 months; metHNSCC: 8 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test. Kaplan-Meier analysis of overall survival (OS) in which only the patients that were independent from the training cohort were grouped according to the clinical classification (left, median survival SQCLC: 84 months; metHNSCC: 35 months) or proteomic classification (right, median survival SQCLC: 84 months; metHNSCC: 5 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test.
Binding specificities of retrieved anti-PrP Fabs by ELISA.Figure 2(A) ELISA titration curves (OD at 450nm) of Fab3 (blue; left panel) and Fab71 (blue; right panel) against mouse recPrP23‑231 compared to their best affinity-matured versions, Fab83 and Fab101 Curves were fitted by non-linear least-squares regression analysis for statistical analysis: log(agonist) vs. response. (B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab).
Figure 1A, Representative images of DIV 3+2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 hours; 10 nM Bafilomycin A1 (BafA1) for 24 hours; 0.5 µM 17-AAG for 4 hours; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes. B, Quantification of FlucSM-EGFP foci formation in transfected neurons. N=4 biological replicates for BafA1 and corresponding control group, 3 biological replicates for all the other conditions; Two-tailed t-test. C, Left, quantification of specific luciferase activity of FlucSM-EGFP upon indicated treatments, normalized to respective vehicle-treated controls. N=3 biological replicates; One-sample t-test. Right, representative Western blot of neuronal lysates for the indicated conditions. Tubulin was used as a loading control. D, DIV 3+2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (grey) as a neuronal marker, nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig. EV1D. E, Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N=4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **p=0.0046; 4-PBA, n.s.; HTT x 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph. D
Figure 4Clustered heat map using Nearest Point algorithm and representing the weights of different features in 109 bacterial genomes, and the clustering relations between features (top dendrogram) and species (side dendrogram). Rightmost light-orange and light-blue bars are included to differentiate the two main clusters. Numbers in the right vertical axis are short references representing the names of the bacterial genomes (Dataset EV15). The right three columns represent biological features not used in the classification. The ratio of the percentage of SEPs compared to the median value are colored as blue and orange for ≤13.16% of SEPs and >13.16%, respectively. Blue and orange colors in the %GC column represent genomes with ≤38% and >38% GC content (median value=38), respectively. Genome size column separates species into small-genome bacteria (≤1.5Mb, blue) and large-genome bacteria (>1.5Mb, orange).