EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) WB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS-IIIA brain samples at indicated ages. WB quantitation was shown. (B) α-Synuclein was immunoblotted in WT and MPS-IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.) and highly insoluble (H. Insol.) forms correspond to the protein solubilized respectively in Triton-X100, SDS 10% and 8M UREA. (C) Co-immunofluorescence analysis of α-synuclein with SMI-32 in WT and MPS-IIIA hippocampal neurons (DIV14). α-Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of α-synuclein synaptic puncta was calculated from 30 different enlarged images c. (D) Confocal analysis of α-synuclein (green) and LAMP1 (red) in WT and MPS-IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group). (E) CSPα was immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in (B). (F) Co-immunofluorescence analysis of CSPα and SMI-32 in DIV14 hippocampal neurons. CSPα- synaptic puncta were quantified as in (C). (G) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated. (H) Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents (beta-mercaptoethanol or dithiothreitol). (I) The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys-48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS-IIIA brain samples at indicated ages by WB analysis. WB quantitation was shown. (L) Proteasome activity was evaluated by measuring the chymotrypsin-like activity in WT and MPS-IIIA mouse brain samples at different ages. Proteasome activity was expressed as % of WT activity
Combining perioperative Vasculotide treatment with adjuvant sunitinib treatment after surgical resection of LM2-4luc primary tumors.Figure 6A Kaplan-Meier survival curves. Number of days gained in median survival due to treatments is listed in brackets. Yellow arrows mark the timepoints at which the bioluminescent images were taken. B Bioluminescent images of tumor burden.C-D Total bioluminescent fluxes, at 35 days post-implantation (while the 'SU_3 wk' group was still receiving 60 mg/kg/day sunitinib treatment), were separately quantified from the thoracic (C) and abdominal (D) regions. Geometric means and P values from Mann-Whitney tests are depicted in (C and D). Percentages listed below the x-axes in panels (C and D) refer to treatment-associated changes in the geometric mean bioluminescence, relative to the vehicle group.
Treatment with AB-Tie1-39 inhibits metastasis in the 4T1 breast cancer model.Figure 2(A) Following a neoadjuvant treatment regimen with IgG or AB-Tie1-39, mice were monitored by whole-body bioluminescence imaging. Shown are the images acquired two weeks after primary tumor resection. (B) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging (mean ± SD, nIgG = 8, nAB-Tie1-39 = 9 mice). *, P<0.05; **, P<0.01 (two-tailed Mann-Whitney U test). (C) Experimental outline of spontaneous metastatic breast (4T1) cancer model treated with IgG or AB-Tie1-39 in a perioperative setting. Therapy was initiated once tumors had reached an average size of 150 mm3. (D) Kaplan-Meier graph comparing percent survival between mice treated with either IgG or AB-Tie1-39 (nIgG = 10, nAB-Tie1-39 = 9 mice). **, P<0.01 (Log-rank (Mantel-Cox) test).
Sequence and structure of YMR159c.Figure 1(A) Amino acid sequence of YMR159c. (B) Structural analysis of YMR159c. The amino acid sequence was analyzed using the COILS program in the 28 residue window setting (obtained from http://www.isrec.isb‐sib.ch/software/coils_form.html) ( Lupas et al., 1991). A putative coiled‐coil region (probability of 1.0) was shown.
Fig.Figure 2A. Overlap between Myc ChIP-seq peaks. For each time-point indicated at the bottom, the percentage of peaks overlapping over ≥1 bp with any other time-point (Reference sample) is reported. Peaks showed consistent distributions along the time-course, almost all those called in untreated cells being included in LPS-stimulated samples B, C. Distribution of Myc at promoters (B) and distal sited (C) in LPS-treated B-cells (0, 2, 4 8h) and in B-cells in vivo, in either control c-mycwt/wt (C), pre-tumoral Eµ-myc mice (P) or tumors (T) 4[]. Each row represents a promoter-associated Myc peak within a 6 kb genomic interval (centered on the midpoint of the peak). The heatmap includes every annotated promoter in chromosome 1 (as representative of the whole genome) that was called as Myc-associated by ChIP-seq in at least one sample. For the same intervals, we also show the distribution of H3K4me1, H3K4me3, and H3K27ac in the in vivo control sample, and gene annotations (exons in red, introns in pink; + sense, - antisense strand). D, E. Numbers of Myc peaks at promoters (-2kb, +1kb) (D) and distal sites (E) at different time points after LPS stimulation. The shadings mark the subsets of peaks associated with H3K4me3 (D) or H3K4me1 (E), with or without H3K27ac. F. Percentage of active promoters (positive for H3K4me3 and H3K27ac) or distal sites (H3K4me1 and H3K27ac) that overlap with at least a Myc peak at the different time points after LPS stimulation (0, 2, 4, 8h) or in vivo (C, P, T1, T2, T3). G. Quantitative heatmap showing Myc signal intensities (read counts normalized by library size) at each bound region in chromosome 1, ranked based on the signal in the control sample (0h LPS). H. Percentage of Myc peaks at promoters (top) or distal sites (bottom) that contain either canonical (CACGTG) or variant E-boxes (CACGCG, CATGCG, CACGAG, CATGTG) 83637[,,] in a 200 bp window centered on the peak summit. I. Myc enrichment by ChIP-seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs. In the boxplot, the central tendency marks the median, the box ranges mark the upper and lower quartiles, and the whiskers extend to the upper and lower extreme datapoints which are no more than 1.5 times the interquartile range from the box. The Myc ChIP-seq time-course was performed on a unique pool of cells, obtained from > 10 mice.
a) Fluorescence microscopy of E.figf3(a) Fluorescence microscopy of E. coli-containing phagosomes in primary macrophages transfected with RFP-conjugated LAMP-1, showing colocalization with E. coli-eGFP or S. aureus-eGFP. Right, quantification of LAMP-1+ phagosomes in the microscopy at left. (b) Immunoblot analysis of LAMP-1 in phagosomes isolated by sucrose-gradient flotation from RAW264.7 macrophages after phagocytosis by Slamf1- or mock-transfected RAW264.7 macrophages of beads coated with E. coli outer membrane extract. WCL, whole-cell lysate. Right, quantification of LAMP-1 in the immunoblot at left. (c) Localization of MHC class II-eGFP (MHCII GFP) in phagosomes of primary macrophages from Slamf1+/+ or Slamf1−/− MHC class II-eGFP (B6) mice and 3-μm beads coated with E. coli outer membrane extract. (d) Entry of 3-μm beads coated with E. coli outer membrane extract into lysosomes loaded with Texas red-dextran. Right (c,d), quantification of fluorescence in the microscopy at left. Numbers in bottom right corners (a,c,d) indicate time (in min). Original magnification (a-c), ×60. DIC, differential interference contrast. Data are representative of three combined experiments (a) or at least three independent experiments (b-d) with at least 100 beads or 80 bacteria in each (error bars, s.d.).
Generation of a complete Fus-/- loss of function mouse model.Figure 3(A) Schematic representation of the Fus gene locus (upper panel). Lower panels depict exons 1-3 in the wild type allele (left) and loss of function allele (right). Arrow: translational start site; SA: splice acceptor; βgeo: β-galactosidase/neomycin phosphotransferase fusion gene; pA: polyA. (B) Representative immunoblot for FUS on protein extracts of E18.5 brain. Histone 3 is used as loading control.(C) Quantification of FUS protein levels from immunoblots . (**) p<0.01. (D) Quantitative real-time PCR for Fus transcript in Fus+/+ and Fus-/- mice. (**) p<0.01. (E) Immunostaining for the neuronal marker NeuN and FUS on the spinal cordventral horn of E18.5Fus+/+ and Fus-/- mice. (F-G) Body weight (F) and length (G) of Fus+/+, Fus+/- and Fus-/- pups at birth; N=14 Fus+/+, N=36 Fus+/- and N=13 Fus-/- for body weight; N=6 per genotype for body length; (**) p<0.01 vs Fus+/+, (##) p<0.01 vs Fus+/- ; One way ANOVA followed by Tukey post hoc test.
a–c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide–specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c).figf7(a-c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide-specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c). (d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC. (f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO. (g) Confocal microscopy analysis of BMDCs stained for mitochondria (red) and LC3 (green) for colocalization as an indicator of mitophagy. Original magnification, ×400; inset (red boxed region) was digitally enlarged ×10. *P 0.01 (one-way ANOVA with Tukey's post-hoc test (d) and unpaired two-sided Student's t-test (g)). Data are representative of nine experiments (a), three experiments (b,d-g) or six experiments (c) (mean ± s.d.; n = 2-3 replicates per experiment).
Figure 7B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections. B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (B-D) Mice were sacrificed on day 20 and tumors were counted. Data is presented as total/mouse (B), N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112). B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. Mice were sacrificed on day 20 and tumors were counted. (B), lung tumors (C) and metastatic tumors (all tumors found outside of the lungs; D). N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112). B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. WT lung tumors were 5.407 ± 1.843% CD45+, while EGR4KO mice were 12.445 ± 2.239% CD45+. WT spleens were 83.92 ± 3.344% CD45+, while EGR4KO mice were 95.13 ± 1.856% CD45+. Relative distributions of T and myeloid cells within the CD45+ populations were determined by flow cytometric analysis. (E,F) Distributions of T and myeloid cells found within lung tumors (E) vs. the spleen (F). B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. (G) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and F
Muscle biopsies of DMD treated patients.Figure 2(A) Haematoxilin and Eosin staining of muscle biopsies from Pt 01, Pt 02 and Pt 03 performed 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. (B) Haematoxilin and Eosin staining of muscle biopsies from Pt 05 and Pt 06 performed one month before the first MAB infusion and 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. No evident differences were observed between biopsy performed after and before treatment. (C) Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion. The staining was performed in tibialis anterior muscle for Pt 01, Pt 02 and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06. A higher number of positive (brownish staining) fibers (representing regenerating fibers) were observed in Pt 02, Pt 05 and Pt 06.
Figure 1A, Representative immunofluorescence staining of normal, tumor, and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130 (magenta); DAPI (blue). Right: human MYC oncoprotein (green) is expressed only in tumor cells, CDH1 (red). Scale bar: 25 μm. Data information: Number of replicates corresponds to individual mice used to derive organoids. B, C, Principal component (PC) analyses of normal (blue), tumor (orange) and residual (green) cells based on (B) RNA-sequencing data (normal, n=8; tumor and residual, n=4 each) and (C) lipidomics data (n=4). Data information: Number of replicates corresponds to individual mice used to derive organoids. Centroids represent the mean and concentration ellipses represent one standard deviation (level=0.68) of an estimated t-distribution based on the first two principal components. D, Heat map of untargeted metabolomics results showing the clustering of the normal - wild type (dark blue), normal - uninduced (light blue), tumor (yellow) and residual (green) populations and the most altered metabolic pathways (n=4). Hierarchical clustering was based on the complete linkage method using the Euclidean distance metric. Significance is defined as a false discovery rate < 0.05 versus the normal population, calculated using unpaired two-sided t-tests and adjusted for multiple hypothesis testing according to Storey's and Tibshirani's method PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; AA, amino acids. Data information: Number of replicates corresponds to individual mice used to derive organoids. E, Principal component analysis of normal and WT (blue), tumor (orange), and residual (green) populations based on intracellular metabolomics targeted at central carbon metabolism (n=8 each; WT n=2). Data information Number of replicates corresponds to individual mice used to derive organoids. Centroids represent the mean and concentration ellipses represent one standard deviation (level=0.68) of an estimated t-distribution based on the first two principal components. F, Selection of the most profoundly altered metabolites in extracellular spent growth medium from normal (normal and WT, n=4 and n=2, respectively), tumor (n=4), and residual (n=4) populations, based on metabolomics analysis targeted at central carbon metabolites. Values represent metabolite abundance levels as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Statistics were calculated using the limma package in R (Ritchie et al., 2015). Significant results (marked with *) correspond to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual or tumor compared to normal). ns, not significant. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. Data information: Number of replicates corresponds to individual mice used to derive organoids.
Figure 3A Schematic of a two-state promoter model for transcriptional bursting. Burst frequency (ka) and burst size (b = kt/ki) were fit to combined transcript distributions measured by smFISH. B-C Burst size (B) and burst frequency (C) parameter fits from the two-state model in the basal state and after treatment with 20 ng/mL TNF for 1, 2 or 4 hours. Error bars indicate bootstrapped 95% CIs. Significant differences indicated by non-overlapping CIs. The fit for Nfkbia (1h TNF) was unstable and thus is not reported
Structure and characterization of liposomal NPs.Figure 2(A) Schematic presentation of liposomal NP layers and composition. (B) Characterization of the diameter and zeta potential (Z-potential) with the addition of each layer. Graph below presenting NP size distribution. (C) Imaging of fluorescently-labeled NPs using confocal microscopy. The data shown are representative of three independent experiments.
Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells.Figure 1Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25-35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.) (B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25-35, mean ± SEM, Student's two-tailed t-Test). (C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30-90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.) (D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm. (E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.
Figure 10In our hypothetical model, Rab10 is initially recruited to PI(3,4) P2 /PI(3,4,5) P3 positive membrane ruffles at the early stage of macropinosome formation. The factors that underlie Rab10-patched recruitment to these initial membranes are not known. During the process of full ruffle closure, PI(3,4,5) P3 is concentrated on macropinosomes, with Rab10 now recruited at much higher levels to facilitate early trafficking of macropinosomes. Now distinct form the plasma membrane, without LRRK2 phosphorylation, Rab10 binds EHBP1L1 to facilitate the formation of tubular endosomes which mediate fast recycling of surface receptors. Our antibody staining demonstrates that virtually all macropinosomes (defined by 70 kDa dextran positivity) in the cells studied here included the GPCR CCR5 and integrins (CD11b). The fate of recycling-tubular endosomes is likely dictated by EHBP1L1 effectors and the local microcytoskeleton network. On the other hand, if LRRK2 phosphorylation of Rab10 occurs on Thr73, EHBP1L1 interaction is weakened and instead RILPL2 interaction is promoted. The net effect may be to inhibit EHBP1L1-mediated fast recycling of CCR5 to retain an endosome-localized activated receptor complex. In vesicle maturation, the former macropinosomes assume early and then late endosome markers. These endosomes, if loaded with CCL5-bound CCR5, appear to potently activate Akt signaling pathways using these PI(3,4,5) P3 enriched vesicles as a platform. Through this signaling axis, critical pathways that mediate chemotaxis, as well as other possible pathways not studied here including cell survival in tissues and respiratory bursts, may be promoted (through LRRK2 phosphorylation of Rab10).
– Tau protein is involved in the effect of EE on GABAergic innervation.Figure 7A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD-65 antibody (red), and high-power magnifications of the DG molecular layer showing GAD-65+ terminals in WT CH, Tau-/- CH, WT EE and Tau-/- EE mice. Cell nuclei were labeled with DAPI (blue). White scale bar 300 µm. Red scale bar 50 µm. Green scale bar 5 µm.B-C Quantification of the density (B) and area (C) of GAD-65+ terminals in the molecular layer of mice in each experimental condition (n= 10 mice WT CH, n= 8 miceTau-/- CH, n= 6 mice WT EE, n= 10 miceTau-/- EE).
a) Identification of biomarkers modulated upon VCP silencing.figf1(a) Identification of biomarkers modulated upon VCP silencing. HeLa, HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h. Protein cell extracts were resolved by SDS-PAGE, and filters were probed with the indicated antibodies. The extent of biomarker modulation correlated with the efficiency of silencing (oligo 4 versus pool). VCP silencing determined stabilization of cyclin E; induction of the endoplasmic reticulum chaperone GRP78, CHOP transcription factor and its downstream target GADD-34; conversion of the autophagy regulator ATG8/light chain 3 (LC3B) from the free to the lipidated form; activation of the effector caspase-3; and cleavage of the caspase target PARP-1. A graphical representation of the cell cycle distribution (percentage of cells) obtained from the cell cycle analysis (Supplementary Fig. 3b) is reported at the bottom of each sample. C, control; NT, nontargeting oligo; P, oligonucleotide pool. (b) Effect of VCP silencing on cell growth. HeLa, HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter (data represent mean values of two experiments). (c) Combination of VCP siRNA with standard-of-care therapeutics. HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose-response analysis with a variety of chemotherapeutic agents.
Figure 6Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs and treated or not with EFV. Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP, as a percentage of the sh-scr baseline (n=20 images per condition from 2 separate experiments, 2-way ANOVA, ***psh-scr < 0.001, **psh-scr+EFV < 0.01, ***psh-NPC1+EFV < 0.001).