EMBO/sd-character-level-ner huggingface dataset:
a) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation.f2(a) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation. A HeLa cell labelled with Lysotracker Red and AlPcS2a was 635-nm illuminated within the white square region (left panels). Lysotracker fluorescence within the illuminated region was immediately lost after light illumination (middle panels), indicating the successful triggering of LMP. Right panels: magnified view of Lysotracker fluorescence within the 635-nm illuminated region pre- and post light activation. (b) 635 nm illumination did not lead to complete lysosome rupture. A HeLa cell labelled with FITC-dextran (molecular weight=10,000) and AlPcS2a was 635-nm illuminated within the white square region (left panels). FITC-dextran remained within illuminated lysosomes (an increase in FITC fluorescence was observed, in accordance with an increase in pH in damaged lysosomes; middle panels). Right panels: magnified view of FITC-dextran fluorescence within the 635-nm illuminated region pre- and post light activation. (c) Light activation did not affect other cellular structures, such as mitochondria. A HeLa cell labelled with TMRE and AlPcS2a was 635-nm illuminated within the white square region (left panels). Illumination did not affect TMRE fluorescence, indicating that mitochondria within the same region were not affected/impaired. Right panels: magnified view of TMRE fluorescence within the 635-nm illuminated region pre- and post light activation. Scale bars, 10 μm.
Figure 9A HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies. HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH, GFP and CENP-C. Example images of anaphase cells are shown (B). The percentage of anaphases with PICH was determined in at least 152 cells in each condition. Means and S.D.s from three independent experiments are shown (C). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm. HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells Lysates of nocodazole-arrested mitotic cells were immunoblotted (D). * represents non-specific bands. E, F HeLa cells transiently expressing CB-GFP or CB-TOP2A-GFP were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (B). Example images of anaphase cells are shown (E). The percentage of anaphases with PICH was determined in at least 142 cells in each condition. Means and S.D.s from three independent experiments are shown (F). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm. HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. Example images of anaphase cells are shown (G). Arrows point to the UFBs. Scale bars, 10 μm. HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. The percentage of anaphases with PICH was determined in at least 174 cells in each condition. Means and S.D.s from three independent experiments are shown (H). Data information: Means and error bars representing S.D. are shown unpaired t test). HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then Lysates of asynchronous cells were immunoblotted (I).
Figure 6(A) C9ORF72 in complex with SMCR8 and WDR41 acts as a GDP/GTP exchange factor for RAB39b GTPase, which interacts with the P62 autophagy receptor. Phosphorylation of SMCR8 by TBK1 potentially promotes C9ORF72 GEF activity and enhances autophagy turnover of proteins such as TDP-43 or ATXN2 with intermediate polyQ size. In absence of C9ORF72, autophagy clearance of these proteins is reduced and TDP-43, P62 or ATXN2 with intermediate length of polyQ accumulate into cytoplasmic aggregates. (B) Autophagy receptors such as P62 or OPTN act as hubs to gather Rab GTPases with their GEF effectors and kinase regulators to initiate autophagy precisely at the site of damaged organelles, protein aggregates or intracellular pathogens.
USP17 is necessary for peripheral trafficking of early endosomes.Figure 5A HeLa cells were transfected with constructs for EEA1-GFP and RAB5-GFP in addition to non-targeting scrambled (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 72 hours post transfection the cells were stained with DAPI. The scale bar represents 25μm. B The distribution of at least 400 EEA1 or RAB5 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups. E
Role of neutrophils in fracture healing.Figure 2Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model. Compared to control and number of blood neutrophils at 3 h ***P = 0.0006 and at 6 h *P = 0.042, by one-way ANOVA with Dunnett correction. Depletion of neutrophils using anti-Ly6G. Intravenous anti-Ly6G treatment led to depletion of neutrophils in the systemic circulation (left) as well the local peri-fracture soft tissues (right). Left: counts of neutrophils in the blood harvested by cardiac puncture at 3 h post-injury. Data are presented as mean ± SEM (**P = 0.0074 using unpaired one-tailed t-test). Right: counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti-Ly6G antibody versus IgG control at day 1 and day 7 post-fracture. At day 1, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment: ****P = 0.0001 for both Ly6G and NE, by unpaired two-tailed t-test. At day 7, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups: ****P = 0.0001 for Ly6G and *P = 0.020 for NE, by unpaired two-tailed t-test. Number of neutrophils at day 1 versus day 7 in control groups, *P = 0.037 for Ly6G and ***P = 0.0006 for NE, by unpaired two-tailed t-test. Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti-Ly6G and anti-neutrophil elastase primary antibodies at day 1 post-fracture following treatment with anti-Ly6G antibody or IgG control. Scale bar, 100 μm. Depletion of neutrophils using anti-Ly6G led to impaired fracture healing. Representative section stained with Masson's trichrome at day 14 comparing fracture healing in mouse treated with isotype control (top) versus anti-Ly6G (bottom). Black and white images in the right column are identical to the color images in the left column; they have been labeled to clearly demonstrate the anatomical structures. Control section shows advanced mineralized callus, while treatment section shows a large immature unmineralized callus. Muscle fibers: red; collagen, bone, and mineralized callus: green. Scale bar, 1 mm. Anti-Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro-CT images. Anti-Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control. Data are presented as mean ± SEM. ***P = 0.0006 by unpaired two-sided t-test. Scale Bar, 2 mm.
Antisense oligonucleotides against the GA-rich region specifically interfere with localization of Rab13 RNA.Figure 3A. Schematic showing positions along the mouse Rab13 3'UTR targeted by the indicated PMOs. PMOs #1, 2 and 3 target the RGAAGRR motifs or the adjacent GA-rich region. PMOs #6 and #7 target the Rab13 3'UTR outside of the GA-rich region. The control PMO targets an intronic sequence of human β-globin. Red rectangles and text indicate the location of the GA-rich motifs. B. FISH images and corresponding PDI measurements of mouse fibroblast cells treated with the indicated PMOs. Cyb5r3 is an APC-dependent RNA also enriched at protrusions. Arrows: peripheral Rab13 RNA. Arrowheads: perinuclear Rab13 RNA. Boxed regions are magnified in the insets. Note that Rab13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region. Scale bars: 10 μm. (4 μm in insets). ****: p<0.0001 by analysis of variance with Dunnett's multiple comparisons test. N=40-90 cells observed in 3-6 independent experiments. Bars: mean ± 95% CI. C. Protrusion (Ps) and cell body (CB) fractions were isolated from cells treated with control-PMO or Rab13-PMO #2. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n=3; bars: mean ± s.e.m.). Note that only the distribution of Rab13 RNA is affected. **: p=0.01 by two-way ANOVA with Bonferroni's multiple comparisons test against the corresponding control. D. Levels of the indicated RNAs were determined using nanoString analysis from control- or Rab13 PMO #2-treated cells. (n=4; bars: mean ± s.e.m.). No significant differences were detected by two-way ANOVA against the corresponding controls.
Figure 6A) Real time image-based killing of A549 cells by NK cells isolated from two individual healthy donors at an E:T ratio of 10:1. B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-. C) Representative image of NK cell infiltration (red) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells, scale bar: 400 μm. D, E) Incucyte quantification of D) total and E) tumor-infiltrating CD25+/CD54+ and CD25-/CD54- NK cells. RCU, Red Calibrated Unit, (n=4, biological replicates). F, G) Flow cytometry quantification of (F) tumor-infiltrating and (G) non-infiltrating NK cells. Results are depicted as total NK cells among CD45 positive live cells. (n=4, biological replicates). H) 48 hours dpi zebrafish injected with A549, A549+ CD25+/CD54+, or A549+ CD25-/CD54- NK cells after 24 hours. Fluorescent image was taken by Leica epi-fluorescent stereomicroscope. Scale bar: 500 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. I) 3D view of tumor injection area 24 hours posts tumor inoculation (left). Scale bar=50 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. Magnified view for tumor area (right). The A549-td tumor cells are annotated with a purple translucent mask and NK cells are color-coded based on the distance to the tumor area (near-far: blue-red). Scale bar=10 µm. J) The distance between NK cells and tumor area in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). K) Frequency of tumor infiltrating NK cells in in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). L, M) Quantification of (L) tumor cell and (M) NK cell counts 24 hours after injection of NK cells by Imaris (n=5, biological replicates). N) Frequency of tissue-infiltrating total NK cells (CD56+/CD3-) (left) and CD54 positive NK cells (right) among CD45 positive live cells (n=10, biological replicates).
Figure 6The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ΔΨCr-IBM is decreased. Scale bar = 500 nm. N = 3 independent experiments. Quantification of A, showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments. The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10- and Mic60-deficient cells. Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models. Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ΔΨCr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (see Figure 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed n Mic10-KO cells, indicating decreased ΔΨCr-IBM. Scale bar = 500 nm. N = 3 independent experiments. Quantification of C, showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments. Quantification of C, showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr-IBM. Note: quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.
Figure 4A Principal component (PC) analysis of DEGs in OC43p (red) and OC43p-SCR (blue) groups (n=3/group). B Hierarchical clustering plot of 548 significant upregulated DEGs (Log2FC>1, Padj<0.1, n=3/group). Heatmap color scheme corresponds to logarithms of variance stabilized counts. C Hierarchical clustering plot of X-linked DEGs upregulated DEGs (Log2FC>1, P<0.05, n=3/group).
Change of Apg8p localization in Δpep4 cells after shift to starvation.Figure 6Change of Apg8p localization in Δpep4 cells after shift to starvation. The Δapg8Δpep4 cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in Fig. 5. A-E, FITC staining of 3 × HA-Apg8p. F-J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.
Figure 5The ratio of Bacteroidetes and Firmicutes, the two dominant phyla in intestinal flora, decreases significantly in high-fat diet (HFD)-fed mice compared to Regular-chow Diet (RD)-fed mice, with a relative increase of Firmicutes in HFD-fed mice and decrease of Bacteroidetes. This dysbiosis is accompanied by heightened intestinal permeability, which allows increased translocation of pathogen associated molecular patterns (PAMPs) (endotoxemia). Recognition of these microbe-specific molecules by pattern recognitions receptors (PRRs) present on innate immune cells triggers synthesis and excretion of cytokines causing chronic systemic low-grade inflammation. Ultimately, this exacerbates CNV.
Figure 2(A) Representative tissue immunostaining of human melanoma, colon carcinoma and colon adenoma; confocal images from serial sections (scale bars: 50μm tissues). Arrowheads point to CD31+/p-EphrinB+; CD31+/p-SHP2+; CD31+/p-Tie2+ and CD31+/Ang2+ endothelial cells. (B) Gene expression in tumor (black bar) and control (white bar); top panel: melanoma (GSE7553, primary and metastatic; tumor n=54; control skin n=5); bottom panel: colon cancer (TCGA, primary and metastatic; tumor n=269; control n=41. **P<0.01, ****P<0.0001; P values from Mann Whitney U-test. (C) Impact of high and low (75% quartile) mRNA levels on the survival probability of patients with primary melanoma; Kaplan-Meier analysis using TCGA primary melanoma datasets (n=103); P values from Mann Whitney U-test. (D) Impact of high and low (median) mRNA levels on the survival probability of patients with colon cancer; Kaplan-Meier analysis using TCGA colon cancer dataset (n=262). (E) Proliferation of endothelial (HUVEC, BMEC and HDMED) and tumor (B16F10, LLC1 and 4T1) cells in three-day cultures supplemented with SHP099. Representative experiments of 3-8; error bars: ± S.D.; 5-10 replicate cultures/experiment. (F) SHP2 depletion in BMEC and B16F10 by 6 distinct shRNAs (#1-#6); pLKO: vector; none: no infection. Immunoblotting with SHP2 antibody followed by reblotting for β-actin. (G) SHP2 shRNAs #1, #2, #3 reduce BMEC, not B16F10 cell proliferation; representative of 3 experiments; error bars: ± S.D.; 5 replicate cultures/experiment. (H) SHP099 (5μM HUVEC and HDMEC; 20μM BMEC) minimally alters endothelial cell cycle distribution measured by flow cytometry; 3-day culture; 3 independent experiments; error bars: ± S.D. (I) Non-viable endothelial cells identified by flow cytometry (DRAQ5+/DAPI+) after 3-day culture with or without SHP099 (5μM HUVEC and HDMEC; 20μM BMEC); quantification of 3 independent experiments; error bars: ± S.D. P values from two-tailed Student's t-test; ***P<0.001;
Figure 5A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines. B Isotope profiling of [13C6,15N3]-labeled L-histidine measured by LC-MS/MS in Tsc2-deficient and wild-type MEFs grown in DMEM 10% FBS without unlabeled L-histidine. Labeled-measured metabolites are depicted in the X-axis. The significant differences correspond to two-sided t-test (*P = 0.032 and **P = 0.008; replicates/condition n = 5). Average values are indicated with lilac-colored lines. C Fold-change variation of histamine and MIAA levels measured by LC-MS/MS assays in rapamycin-exposed (20 nM, 16 hours) Tsc2-deficient MEFs relative to DMSO-exposed for the same period. The significant difference corresponds to one-sided t-test (P = 0.012; replicates/condition n = 4 and independent experiments n = 2). Average values are indicated with lilac-colored lines. D Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal setting E Representative images of immunohistochemical detection of HRH1 in LAM lung lesions. F Evaluation of cell viability inhibition by loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD. G Percentages of viability of Tsc2-dificient MEFs exposed to increasing concentrations of histamine (X-axis) in DMEM 0.5% FBS without L-histidine for 72 hours. The asterisk indicates significant difference with one-sided t-test (P = 0.040; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD. H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5% FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10% FBS, P = 0.017; 0.5% FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD. I Western blot results of phospho-Tyr783 PLCγ1 and loading control in Tsc2-deficient MEFs grown in DMEM 0.5% FBS without L-histidine and exposed to histamine (1 μM) for 10 minutes or 1 hour. The signals of phospho-Tyr783 PLCγ1 relative to loading control or non-phospho PLCγ1, and to basal setting (noted as 1(ref)) are indicated.
Figure 4NLRP3 oligomerisation analysed by native PAGE. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 min.). Digitonin-solubilised cell lysates were analysed by native-PAGE or SDS-PAGE and NLRP3 western-blotting. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120 kDa). β-actin is shown as a loading control. Blots are representative of 4 independent murine donors. NLRP3 ubiquitination analysed by western blot. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 mins.). Endogenous NLRP3 was immunoprecipitated (IP) with anti-NLRP3 and probed with either anti-ubiquitin (Poly-Ub), anti-K63-ubiquitin (Poly-K63-Ub) or anti-NLRP3. As a negative control, cell lysate made from LPS-primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120kDa). β-actin is shown as a loading control. Blots are representative of 2 independent murine donors for 2.5µM P22077 and 4 independent murine donors for 10 µM.
Figure 6(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown. (B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates