EMBO/sd-character-level-ner huggingface dataset:
PhNTs enable the intercellular transfer of cytoplasmic and membrane reporters and, rarely, organelles, in culture.Figure 5a, Flow cytometry analysis of cGFP and cDsRed following co-culture of Nrl.Gfp and DsRed cells (1:1) and analyzed at 3 DIC (N = 3 independent co-cultures with a technical triplicate), 7 DIC (N = 5), 21 DIC (N = 4); One-way ANOVA non-parametric, Kruskal Wallis; b, flow cytometry analysis of DiI/DiO dual labelling of CD73+ cells following co-culture of DiI- and DiO-labelled photoreceptors (1:1) with pharmacological interventions applied at 5 DIC for 48hrs, including vehicle control (DMSO), Cytochalasin D (2 μM) and Latrunculin B (5 μM), after wash-off of Cyto D and LatrB, and a final wash control for DiI/DiO carry over; N = 4 independent co-cultures per condition; One-way ANOVA non-parametric, F-test, Kolmogorov-Smirnov; c, Representative MIP of DiI/DiO co-cultures after fixation, showing DiO+ve cell (A) and a DiI+ve cell (B) cell connected by a PhNT. Note DiI and DiO puncta in the respective acceptor cells. Blue = Dapi (nuclei), Green = DiO, Red = DiI. Scale bar = 5 µm; d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell "A" and a PhNT connecting cells "A" and "B"; Scale bar = 2 µm; e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.
Asm-mediated β1 integrin activation mediates adhesion of tumor cells in the lung in vivo.Figure 7B16F10tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [3H]thymidine, washed, and treated (+) for 10 min with 1 U/ml ASM, 10 μM C16ceramide, 10 μM S1P, 10 μM PDMP, 20 μM sphingosine kinase inhibitor SKI II, and 10 μM myriocin, or left untreated (−). The cells were then injected intravenously into wild-type or Asm-deficient mice as indicated. If indicated, 10 μg of arginine-glycine-aspartic acid (RGD) peptide or 10 μg/ml neutralizing anti-β1 integrin antibodies were added to B16F10tumor cells together with ASM for 15 min prior injection. Mice were sacrificed 30 min after tumorcell injection, the lungs carefully flushed via the right heart, and the radioactivity in the lung as a measurement for adherent tumor cells was determined. Shown is the mean ± SD of the counts per minute (cpm) in the lung lysates from three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's test for multiple comparison to determine P-values.
Pgs1 depletion does not rescue apoptotic sensitivity nor cristae structure in Opa1-deficient MEFs.Figure 6(Top) MEFs of the indicated genotypes were subjected to 4μM Actinomycin D and 10μM ABT-737 in the presence or absence of the pan-caspase inhibitor qVD. Dead cells (PI+ nuclei, orange) and total cells (NucBlue, blue) were imaged every hour for 25 hours. PI+ nuclei number divided by the total nuclei number was then quantified over time. (Bottom) Representative confocal images of (Top). Scale bar=100μm. Data represent mean ± SD of three independent experiments, (1380-2157 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant. Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs. Scale bar=200nm. Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.
Figure 2(A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student's t-test) (B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001). (C) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked. (D) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (The scale bars represent 1000 μm). (E) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e. (F) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (The scale bars represent 1000 μm).
Figure 6(A) Changes in Aβ40 relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8). (B) Changes in sAPPβ relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8). (C) Changes in sAPPα relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8). (D) Dose response of Aβ40 (mean ± SD) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing. (D) CNP520 was administered daily for 14 days; CSF was sampled via a lumbar catheter 2 hours before dosing, and 24 hours after the last dose (mean ± SD, n = 8 in treatment groups, n = 16 in placebo group).
Figure 2A. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). B. Western blot of Dyn2, Myc-hFis1, Drp1 and GAPDH in WT 293T cells collected from the experiments in (A). C. Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments. D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated. E. Western blot of Dyn2, Myc-hFis1 and GAPDH in Drp1-/- 293T cells collected from the experiments in (D). F. Percentages of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments.
Four groups of transcriptional induction upon exposure to mannitol.Figure 2A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. The first group contains TFs that reached the log2(FC) threshold 40 min after mannitol treatment (A) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the second group reached the threshold after 1 h (B) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the third group after 2 h (C) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the fourth group after 4 h (D). The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.
Figure 6(A) Scheme of a zebrafish eye. (B-B'). Cataract detection revealed abnormal lens reflection in psmc3 morpholino (MO)-mediated knockdown but not in controls (uninj, ctrl-mo). Similarly, abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 (sgRNA2). Co-injection of wt psmc3 mRNA with either psmc3-mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection. Scale bar = 50 µm. (B') Quantification of embryos with abnormal lens reflection. (C) Representative image of a zebrafish ear at 4 dpf. kc= kinocilia. (D-D') bright field images of inner ear development (lateral position). (D) Epithelial projections were fused and formed canal pillars in 4-day-old uninjected and control injected fish (ctrl-mo, sgRNA2) but not in morphants (mo) and crispants (sgRNA2+Cas9). Co-injection of wt psmc3 mRNA with psmc3-mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype. Black asterisks indicate fused pillars. Red arrowheads mark unfused projections. Scale bar = 100 µm. (D') Quantification of embryos with abnormal projection outgrowth. (E-E') An anti-acetylated tubulin antibody (green) staining revealed an abnormal amount of kinocilia in psmc3 crispants (sgRNA2+Cas9) compared to uninjected and control injected embryos (sgRNA2). Nuclei are stained in blue with DAPI. Representative images show kinocilia of the lateral cristae. Scale bar = 20 µm. (E') Quantification of embryos with an abnormal amount of kinocilia.
Figure 6(A) Heatmap of the transcriptomic dysregulation caused after treatment with lurbinectedin in five different cell lines of SCLC -DMS-53 (green rectangle), NCI-H510A (Brown rectangle) and NCI-H82 (Yellow Rectangle) with a False Discovery Rate < 0.05 (2998 common Differentially Expressed Genes, DEGs) shows that the vast majority of genes are downregulated (n=3 biological replicates). Color legends correspond to the three cell lines and ctl (basal - Pink rectangles), as well as samples treated with lurbinectedin (Red rectangles). (B). Venn Diagrams comparing DEGs with FDR<0.05 in three different cell lines after treatment with lurbinectedin shows the overlapping among the three DEG analyses. 3374 genes are commonly downregulated in the three cell lines, independently of their ASCL1 or NEUROD1 expression levels. Over-representation analysis of the common genes downregulated in the three cell lines after lurbinectedin treatment (blue table). (C) Lurbinectedin downregulates genes that have pivotal functions in the pathogenesis of SCLC as well as in the tumorigenic properties of the cells (ASCL1, NEUROD1, BCL2, INSM1, MYC, MYB, AURKA and CDK7). Data are expressed as normalized counts before (dark blue) and after (light Blue) lurbinectedin treatment. Data are presented as Mean ± SD (n=3 biological replicates).
Figure 2A. Deep coverage exome sequencing Integrative Genomics Viewer (IGV) view of CBL c.2322T>G in DNA sample that was derived from CD31 selected cells from pleural effusion fluid. The alternate allele fraction was 4% (20/493). B. Droplet digital polymerase chain reaction (ddPCR) result with 5 mutant positive droplets (circle) from a low concentration left over sample which resulted in ~0.005% fractional abundance.
Figure 6A, B Representative graphs showing the climbing ability flies carrying the UAS-(G4C2) x36 construct in neurons using the Elav-gal4 promoter upon treatment with H89 10 µM diluted with 0.1 % of DMSO and 5% sucrose in PBS. The wild-type (w1118) line was used as control. (A) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment ****P<0.0001 (G4C2) x36 vs (G4C2) x36 + H89. (B) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment. ****P<0.0001 (G4C2) x36 vs (G4C2) x36 + H89. Experiments were performed three times, N=15 adult flies were used for each genotype, sex and treatment (N tot=45). C, D Representative graphs showing climbing activity of flies co-expressing the UAS-Pka-RNAi construct (1, v330111) in combination with UAS-(G4C2) x36 in neurons using the Elav-gal4 promoter. The wild-type (w1118) line was used as control. (C) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 9th day ****P<0.0001 (G4C2) x36 vs Pka-RNAi (1);(G4C2) x36. (D) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 7th day ****P<0.0001 (G4C2) x36 vs Pka-RNAi (1);(G4C2) x36. Experiments were performed twice with different RNA interfering sequence, N=15 adult flies were used for each genotype and sex (N tot=30).
NAV3 knockdown associates with altered EGFR trafficking and disoriented chemotaxis.Figure 5MDA-MB-231 cells, pre-treated with the indicated siRNAs, were stimulated with Alexa Fluor 488-EGF (20 ng/ml) and imaged for 25 min. Frames taken at 2 and 25 min are shown (scale bars, 10 μm; see Supplementary Movie S3). Cells were stimulated with EGF (20 ng/ml; 10 min) as in (A), acid-washed and incubated at 37°C for the indicated intervals. Thereafter, cells were detached, fixed and analyzed using imaging flow cytometry (ImageStreamX). Shown are means ± SD from three experiments. MDA-MB-231 derivatives were serum-starved and stimulated with EGF for the indicated time intervals. Whole extracts were subjected to immunoblotting as indicated. Rose plots obtained with MDA-MB-231 derivatives exposed to an EGF gradient (shown as a blue and green wedge) in chemotaxis chambers. Blue lines indicate properly oriented tracks. Histograms (mean ± SEM) show quantification of persistence and chemotactic orientation from three experiments (45 cells).
Figure 8DFP treatment enhances SENP3-mediated deSUMOylation of Fis1, thereby facilitating Fis1 mitochondrial localisation that is required for mitophagy.
Figure 1(A) A scheme of the codon-tag reporter system. A codon to be tested (pink, in this case leucine) and one of the 20 sense codons (gray) are alternately repeated 20 times. This codon-tag sequence was inserted at the end of the sfGFP ORF. (B) Poly(A) tail analysis of codon tag reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (C) Quantification of the PAT assay at 6hpf in (B). (D and E) qRT-PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence (D) or presence (E) of translation-blocking GFP MO. Bar charts show the average of three independent biological replicates. Error bars show standard deviation (SD). Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.
Figure 3(a) After infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100), POLR3-dependent RNA transcripts in Ramos cells were detected by RNA-seq analysis. The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock (mock group vs. mock group), EBV-WT (EBV-WT group vs. mock group) and EBV-ΔBRLF1 (EBV-ΔBRLF1 group vs. mock group) comparisons. (b) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis. (c) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1(MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis. (d) EBER1 5'-pppRNA detection using splint-ligation. EBV-WT- or EBV-ΔBRLF1-harboring HNE1 cells were cotransfected with empty vector, BRLF1 or BRLF1 L578A, BRLF1 plus BZLF1, or BRLF1 L578A plus BZLF1 for 48 h. The total RNAs were extracted, and 5 μg of each RNA was subjected to splint-ligation with FAM-labeled probe to quantify the 5′-monophosphorylated EBER1 and total EBER1 RNA. The ligation products were separated using urea-PAGE, visualized by a fluorescent scanner and analyzed using ImageJ software. The ratio of 5'-pRNA density to total RNA density and the relative 5'-pppRNA level were calculated for three independent experiments. (e) EBV-WT-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A-expressing plasmid in the presence of GST-tagged RNA polymerase subunit POLR3F for 48 h. The cells were harvested and ChIP assay was performed with anti-GST affinity beads. The POLR3F-binding promoter fragments of BSRF1, BMLF1, EBER1, EBER2, EBNA1, 7SK, RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real-time PCR assays.
Figure 3(A) Schemes of Far1(1-150)-GFP constructs, either wild-type or mutant where NLxxxL motif is replaced with RxL motif (VKRTLF), that are used in time-lapse microscopy experiments in panel 'C'. (B) Sequence alignment showing the introduction of wild-type or mutated RxL motif to Far1. (C) Plot showing the mean±SEM fluorescence intensities of Far1(1-150)-GFP constructs with the indicated docking site. In rxl mutant, the NLxxxL motif is replaced with a mutated non-functional RxL motif (VKATAF). (D) Kinetic characterization of Far1(1-150 WT)-GFP and Far1(1-150 RxL)-GFP phosphorylation by Clb5-Cdk1. The data is from two experimental replicates. (E) Mean±SEM fluorescence intensities of wild-type or RxL-dependent Far1(1-150)-GFP in wild-type or the indicated cyclin hpm mutant strains. (F) Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A) or far1(RxL). Different concentrations of α-factor were pipetted on the paper discs. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-depedent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown.
Figure 6Representative transcriptional factor genes containing the SRSF3-dependent exons. Edges show protein-protein interaction (PPI) as annotated within the STRING database (Szklarczyk et al., 2019). Solid lines and dotted lines indicate PPIs within a cluster and over a cluster, respectively. Genes are clustered by the Markov cluster algorithm. SRSF3-responsive alternative 3′ ends of the Med1 gene. (Top panel) Disorder analysis for MED1 using PONDR VL-XT (deep blue) and IUPred2 (light blue). The scores in the ordinate indicate disordered tendencies between 0 and 1 (a score of more than 0.5 indicates disordered). (Middle panel) Protein and gene structures of MED1. The protein structure was segmented into individual exonic regions. The SRSF3-responsive last exon is indicated in pink. (Bottom panel) Sashimi plots of RNA-seqs from Srsf3- and/or Hnrnpk-silenced cells. Immunofluorescence images of MED1 in C2C12 cells treated with siCont (C), siSrsf3 (S), siHnrnpk (K), or siSrsf3+siHnrnpk (S+K). A white dotted contour outlines the nucleus. Box plots (right) show quantification of nuclear (NUC) and cytoplasmic (CYT) localization of MED1. The average intensity of one nucleus or a cytoplasmic segment is individually plotted. The intrinsically disordered region (IDR) in MED4. Disorder analysis, protein and gene structures, and Sashimi plots are shown in (B). Splicing of these genes was not affected by the siRNA treatment. Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs. Box plots show quantification of nuclear localization of MED4. Each dot represents one nucleus. Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta, including both MED1 and MED4. The number of MED1 puncta (left) and MED4 puncta (right) per nucleus in cells treated with the indicated siRNA. MED1 signals on MED4 puncta normalized to nuclear MED1 signals. MED1 signal intensity on each MED4 punctum was individually plotted.
Fig.Figure 5Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2 Insets show paired samples by donor in two different conditions. chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=4. Insets show paired samples by donor in two different conditions.