EMBO/sd-character-level-ner huggingface dataset:
MicroRNA-342-3p directly targets SOCS6 (A) Predicted binding sites of miR-342-3p and Socs6 3′-UTR.Figure 3(A) Predicted binding sites of miR-342-3p and Socs6 3′-UTR. (B) HEK-293T fibroblasts were co-transfected with miR-342-3p mimic and luciferase reporter construct containing wild-type or mutated UTR. After 24 hours, cells were collected for luciferase assays. The Y-axis showed relative luciferase activity. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. (C, D) MiR-342-3p mimic or inhibitor was transfected to NIH-3T3 fibroblasts. After 48 hours, cells were collected for qRT-PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) and western blotting (D, representative blots from n = 3 biological replicates are shown) to detect SOCS6 expression. (E) RAW264.7 cells were transfected with Myc-tagged Ago2 in the presence of miR-342-3p mimic or scramble for 24 hours. Immunoprecipitation was performed with Myc-antibody or IgG, and Socs6 mRNA was detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. (F-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. (I-K) B6 BMDMs were transfected with miR-342-3p inhibitor or Socs6 siRNA, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (I), cell viabilities (J), Mtb growth rates (K) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. (L) Relative expressions of miR-342-3p and Socs6 in PBMCs from healthy individuals (n=27) and TB patients (n=34) were detected by qRT-PCR. The correlation between miR-342-3p and Socs6 was analyzed by Spreaman test. Spearman rank correlation: r = -0.532, p < 0.001, n = 61.
The function of LGP2 in sensing unedited self RNA involves its canonical role as dsRNA sensor that requires RNA binding and ATP hydrolysis.Figure 4A) Schematic illustration of the domain structure of LGP2 and various point mutants and truncation mutants that are used in this study. The N-terminal domain (NTD) of LGP2 is composed of a conserved DExH/D helicase domain, subdivided into the helicase 1 (Hel1), helicase 2 (Hel2) and helicase insertion (Hel2i) domain, and a pincer motif (P). The NTD is followed by a C-terminal domain (CTD), involved in RNA binding. B) HEK293 WT or ADAR1-knockout cells (clone 1) were transiently transfected with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of LGP2. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments. C) Cells were treated as in (B). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). The dotted line indicates the juxtaposition of two non-adjacent lanes.
Figure 1(A) Gross images and H&E staining of lungs from liver-specific gene knockout mice with hydrodynamic tail vein injection of Akt and β-catenin. Scheme of the experiment was shown. Scale bars, 1mm. Data information: Sample sizes of about 30 mice per group is shown when calculating the metastasis rate or tumor burden. (B and C) Gross images and H&E staining of lungs from TFAM+/LKO or TFAMLKO mice injected with Akt/N-ras or treatment with DEN and CCl4. Scale bars, 1mm. Data information: Sample sizes of about 30 mice per group is shown when calculating the metastasis rate or tumor burden. (D) Gross images and H&E staining of lung and liver from TFAM+/LKO or TFAMLKO mice injected with Akt/β-catenin. Different time points were shown. Liver/body weight ratio and percentage of microscopic tumor area was used as the measurements of the tumor burden. Scale bars, 1mm (lung); 50μm (liver). Data information: Sample sizes of about 30 mice per group is shown when calculating the metastasis rate or tumor burden.
Study designStudy design flow diagram for DNA-seq-informed optimization of RNA-seq variant calling.Figure 1Study design flow diagram for DNA-seq-informed optimization of RNA-seq variant calling.
a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue).figf1(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample. b) Anti-VHR, anti-actin and anti-Prc1immunoblots of lysates of the same cells. (c) DNA histograms of the same cells at the indicated time points. (d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue). (e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.
A 2-nm tomographic slice of each ER–IM complex, reconstructed from a 200-nm thick section, and their 3D models are depicted in the left panels.figf4A 2-nm tomographic slice of each ER-IM complex, reconstructed from a 200-nm thick section, and their 3D models are depicted in the left panels. Red arrows indicate sites at which an edge of the IM is connected to the ER. Boxed regions are enlarged in the right panels. 3D models of the boxed regions, shown in a different orientation, depict the connection between the IM and ER (red arrows). Tomographic slices of the boxed regions show the edge of the IM (red arrowheads) is connected to the ER. Numbers indicate the depth of the tomographic slices. Scale bars, 100 nm.
Figure 1A. Experimental workflow. Single gene knockout strains from the Keio collection were grown in M9 supplemented medium at 30˚C in 96-well plates. DNA was stained with DAPI prior to imaging and nine images were taken in both phase-contrast and DAPI channels. The images were then processed with MicrobeTracker and Oufti to identify the cell and nucleoid contours. In parallel, we recorded the growth curve of each imaged strain in order to extract growth parameters. B. A SVM model was trained via visual scoring of 43,774 cells. C. Confusion matrix of the SVM model based on a large validation dataset (102,137 cells), illustrating the distribution of the SVM classifier output in comparison to the visual classification. D. Comparison of the average cell length of 178 strains obtained from two independent 96-well cultures of the 176 most phenotypically remarkable Keio strains and 2 WT replicates.
Fig.Figure 8Plaque burden in ligated carotid arteries from ApoE-/- (-/-) and ApoEYF (YF) mice fed a high cholesterol diet for 21 days; scale bars: 2 mm. Intimal and medial thickness was determined at four specific locations (proximal, middle 1, middle 2, distal) in cryo-sectioned arteries stained with Oil Red O; scale bars: 200 µm. n=7 mice per group (Unpaired Student's t test). Oil Red O staining of plaques in aortae from mice fed a Western diet for four (B) or six months (C); scale bars: 3 mm. n=6 (B) or n=12 (C) mice per group (Unpaired Student's t test).
Figure 4A. Yb bodies (Yb in green) disappeared after 1,6-hexanediol treatment of OSCs, as did P bodies (Dcp1 in red). Nuclei are shown in blue. Scale bar: 5 μm. B. Snapshots of live imaging of GFP-Yb expressed in OSCs Fusion- and fission-like dynamics of Yb bodies were observed. Saturated pixels are shown in red. Scale bar: 0.5 μm. C. Yb Y23A and F129A mutants (green) impaired Yb body formation. Nuclei are shown in blue. Scale bar: 5 μm. D. Yb Y23A and F129A mutants failed to produce flam-piRNAs. miR-310 was used as a loading control. E. Mapping of flam- and tj-piRNAs produced in the flamKG mutant and heterozygote ovaries. F. Summary of the functionality of each domain of Yb. Production of non-transposon-repressing piRNAs requires RNA helicase and eTud domains of Yb. Yb body formation via LLPS requires all three domains of Yb, but Armi, SoYb, and Vret are dispensable. Production of transposon-repressing piRNAs (flam piRNAs) requires all three domains of Yb and also the Yb body components, Armi, SoYb, and Vret. The Hel-C domain is responsible for Yb−Yb interaction (self-assembly of Yb). The RNA helicase and eTud domains are required for Yb−RNA binding. The eTud domain is also required for Armi binding. Yb binds the SoYb−Vret heterodimer Armi-dependently.
CEP55 regulates human breast cancer cell survival.Figure 1Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines (n=25) Tubulin served as loading controls. Immunoblot analysis of CEP55 expression in progression series of MCF10A lines (MCF10AT and MCF10CA), respectively. COX-IV served as loading controls. A panel of selected breast cancer and near normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean± SEM of three independent experiments. Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA-MB-231 cells. Two isogenic lines (sh#2 and sh#8) were obtained using two different shRNA sequences as described in method section. COX-IV served as loading control. Effect of CEP55 knockdown on cell proliferation in MDA-MB-231 cells assessed using the IncuCyte ZOOM® live cell imager (phase-only processing module). The percentage of cell confluence was determined using an IncuCyte mask analyzer. Graph represents the mean±SEM of three independent experiments. Representative images of colony forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA-MB-231 cells. Six-week-old female NOD/SCID cohorts of mice were injected in the 4th inguinal mammary fat-pad with the control and CEP55 knockdown cells. Growth rate (area, mm2) of the tumors was measured using digital calliper. Differences in growth were determined using Student's t test. Graph represents the mean tumor area±SEM, n=6 mice/group. Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag-CEP55 expression construct and two isogenic lines were obtained (CEP55-#16 and CEP55-#17) with its respective empty vector (EV) transfected parental cells. COX-IV served as a loading control. Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM® live cell imager as described in panel E. Graph represents the mean±SEM of two independent experiments. Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector (EV) and CEP55-overexpressing (CEP55#17 and CEP55#18) lines. Representative images of single plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: Cytokeratin-19; Green: F-actin stained by Phallodin and Blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH.
/3 at K11 limits the formation of SUMO chains (a) Structure of SUMO2.Figure 1(a) Structure of SUMO2. Lysine residues within the flexible N-terminus (K5,7,11) are marked in magenta, lysine residue 21 is colored in teal and lysine residues involved in SUMO/SIM interactions (K33,K35,K42,K45) are colored in green. (PDB: 2N1W). The red circle indicates K11. Ac. indicates the acetylation at K11. (b) Comparison of the amino acid sequences for SUMO1/2 and 3 and the yeast variant Smt3. Lysine residues are marked in blue. SUMO consensus motifs are marked in red. SUMO2/3 and Smt3 display a conserved consensus motif, whereas SUMO1 shows an inverted consensus motif. (c) Substrate independent in vitro SUMOylation assay with SUMO2WT, SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies. (d) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451-N. Reaction products were analyzed by immunoblotting with the indicated antibodies. Note that - when compared to (c) assays were done at reduced concentrations of E1 and E2 (e) In vitro SUMOylation assay using a fixed amount of SUMO2WT (220 ng) with increasing concentrations of SUMO2K11Ac. Ratios ranged from 3:1 (unmodified/K11Ac) to 1:2 (unmodified/K11Ac) (f) In vitro SUMOylation assay, using SUMO2WT or SUMO2K11Ac, with p53 as a substrate. p53 was generated by in vitro translation/transcription in a rabbit reticulocyte lysate system. SUMO chain formation on p53 was analyzed by immunoblotting with α-p53 antibodies.
Figure 8A Visualization of interaction between MAPKKK5K375M and MKK1 / MKK2 / MKK4 / MKK5 by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction. Scale bar=10µm. B MAPKKK5-KD phosphorylates MKK4 and MKK5 in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. C MAPKKK5-KD does not phosphorylate a T224A/S230A mutant of MKK4 or a T215A/S221A mutant of MKK5. The above experiments were performed three times with similar results.
Figure 5(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment. RT plus anti-PD-L1 (B) reverses T-cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test). RT plus anti-PD-L1 (C) increases CD8:Treg ratio. Bar graphs show ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
Figure 3Hela-hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS-CoV-2, GX/P2V/2017 and GD/1/2019, respectively. Green fluorescence indicates Hela-hACE2 cells infected with pseudovirus. Untransfected HeLa cell is used as negative control. The scale bar indicates 400 μm. Statistic for the infectivity of the three pseudoviruses. Data represent the results of five replicates. All data are presented as mean ± SD. ***p < 0.001 (Student's t-test). ns, no significant differences.
Figure 7A, Topological overlap matrix (TOM) plot of coexpression network created from gene expression profiling of all samples across both timepoints (n = 42 patients × 2 timepoints × 2 technical replicates = 168 samples). At top, dendrogram of the hierarchical clustering of the matrix that undergoes a dynamic tree-cut operation to form 92 gene coxpression network modules (coEMs), depicted by the colored bars on the edges of the TOM plot. Color assignment is arbitrary. Three coEMs are highlighted (boxes). B, enrichment of five subsets of the DET signatures for CHIKV infection phase and viral titer among each of the 92 coEMs (X axis), showing the fractional overlap of the module with the DET signature (Y axis). Negative log10-scaled q values (Benjamini-Hochberg adjusted P values; Fisher's exact test) are depicted as red asterisks on the secondary Y axis (right hand side), clipped to a maximum of 10. The only modules with overlaps in any of the subsets that exceed FDR < 0.05 are turquoise and sienna. The horizontal line indicates a threshold of FDR < 0.05. The three highlighted coEMs from A are again labeled. , correlations between coEM eigengenes (colored bars on each axis) and the clinical variables (text labels; Ab, anti-CHIKV antibody). The three coEMs in A are again highlighted. There is a strong inverse relationship between turquoise + convalescent vs. greenyellow + sienna (black boxes). D, multiscale network of cell sub-community and coEM eigengenes depicted with a force-directed layout and edge bundling. Gold nodes, cell sub-communities; gray nodes, coEM eigengenes; large diamonds, clinical variables; red edges, positive correlations; blue edges, negative correlations. Edges are filtered to Pearson correlations significant at P < 0.001, and thickness corresponds to the square of the correlations. A cluster of sub-communities and coEMs associated with convalescence (dashed gray box) and a corresponding cluster of sub-communities and coEMs associated with acute infection (solid black box) surround the "convalescent" node. A positive correlation between convalescent anti-CHIKV antibody (Ab) titer and CD14+CD16+ monocyte sub-community 1 frequency (shown previously in Appendix Figs S3A and S3B) is also visible (asterisk and arrow).
Figure 1A Disruption of lysosomes with GPN abolished LysoTracker staining (red) in GFP-expressing hippocampal neurons. Scale bar, 20 µm. B Upper panels: Kymographs of LysoTracker-labeled lysosomes in the proximal dendrites of neurons infected with scrambled shRNA (shSc), LAMTOR1 shRNA (shLAMTOR1), or LAMTOR1 shRNA and RNAi-resistant LAMTOR1 (rLAMTOR1). Stationary, anterograde, and retrograde traces generated using the KymoGraphClear plugin for ImageJ were coded blue, red, and green respectively. Lower panels: Tracks of mobile lysosomes. Scale bar, 5 µm. C Quantitative analysis of lysosomal movement from kymographs. Results were expressed as percent of total lysosomes (N = 44, 49, and 16 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1 respectively from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 n.s., not significant. D, E Quantification of track speed (D) and displacement length (E) of lysosomes with low (0-1000 arbitrary unit) and high (>1000) intensity of LysoTracker (dashed line, median; thin dashed line, quartiles; same length dendrites for each group from 3 independent experiments were analyzed). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Dunnett's post-test *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 n.s., not significant. F, FRAP analysis of dendritic lysosome movement. F Representative images of neurons at 0 and 4 min after photobleaching. Photobleached regions are indicated by the cyan and red lines, and lysosomes traveling anterogradely (crossing cyan line first) or retrogradely (crossing red line first) are labelled cyan and red respectively. G FRAP analysis of dendritic lysosome movement. Quantitative analysis for F (N = 7, 7, and 9 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Dunnett's post-test *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 ; n.s., not significant. H Left: Kymographs of Magic Red cathepsin B substrate-labeled degradative lysosomes in the proximal dendrites of neurons infected with shSc and shLAMTOR1. Scale bar, 5 µm. Right: Quantitative analysis of vesicular movement from kymographs (N = 16 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test
Figure 7(A) Schematic illustration of barasertib treatment protocol. Control and TGFα mice were treated with either vehicle or barasertib (40mg/kg; twice a day) for last 3 wks while they were fed with Dox containing food for total of 6 wks. (B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group). (C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group). (D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm. (E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).
Blockage of Dll4 increases the number of cells in IAHCs (A)Whole mount images of the aorta endothelium stained for CD31 (white in image, black in label) and Kit (IAHC, cyan) upon IgG (left) or αDll4 antibody treatment (right).Figure 4(A) Whole mount images of the aorta endothelium stained for CD31 (white in image, black in label) and Kit (IAHC, cyan) upon IgG (left) or αDll4 antibody treatment (right). Multistack reconstruction of confocal images. Red arrows indicate IAHCs on the ventral part of the aortic wall. Asterisks indicate aspecific autofluorescence. Scale bar: 50μm. Representative IAHCs of IgG and αDll4 treated embryos. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Note the increased IAHC size in the right panel. Scale bars: 50μm (overview), and 10μm (magnification). Frequency of IAHC categories based on the number of Kit+ cells forming a cluster. Quantification of confocal images. Bars represent the mean±SE (n=6) Number of Kit+ cells per IAHC increases following αDll4 antibody treatment. Each dot represents one cluster. Quantification on confocal images. Statistical analysis: Mann-Whitney U test. ****p< 0.0001. Total number of IAHCs per 100μm. Quantification of confocal images. Mean±SE. Statistical analysis: t-test. ns p> 0.05; (n=6). (F) Quantification of the number of Kit+ cells per 100 μm using confocal images. Bars represent the mean±SE. Statistical analysis: t-test. ns p> 0.05; ***p< 0.001 (n=6).
Desmogelin-2 palmitoylation by DHHC5/Golga7b regulates its plasma membrane localisation and incorporation into desmosomes.Figure 6ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5. Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D. Immunoblots and quantification of Dsg2 pulled-down in cell surface biotinylation assays in nt siRNA, DHHC5 siRNA or Golga7b siRNA treated conditions. Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input. A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA. *= Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.