EMBO/sd-character-level-ner huggingface dataset:
Figure 6A Heat map showing mRNA expression in peritoneal macrophages derived from wild-type and M-Shp2-/- mice after IMQ treatment based on RNA sequencing (n=3/group). Colors represent high (red) and low (blue) intensity. Genes labeled in green indicate they are in the NF-κB signaling. B Volcano plot image of upregulated (in red) and down regulated (in green) genes (M-Shp2-/- mice compared to wild-type mice) in peritoneal macrophages from wild-type and M-Shp2-/- mice after IMQ treatment (n=3/group). C Expression levels of representative psoriasis-related genes decreased in the IMQ model of M-Shp2-/- mice compared to wild-type mice (n=3/group). Data information: Data are represented mean ± SEM. P values are determined by Tukey multiple comparison test (C). **P<0.01, ns, not significant. D BMDMs and PMs derived from wild-type and M-Shp2-/- mice were stimulated by IMQ (10 µg/ml) for indicated times. Whole cell lysates were subjected to Western blotting. E Representative p-p65 staining of skin sections from mouse. Scale bar: 100 μm.
Figure 1We utilized exome sequencing data from 24 MSI CRCs and corresponding normals and whole genome sequencing data from 12 MSI CRCs and corresponding normals. The tumor data was filtered against the corresponding normals as well as outside controls to remove germline variants. Only the areas targeted by the exome sequencing kit were included in the analysis. Insertions and deletions were then filtered out, leaving the somatic missense, nonsense, synonymous and non-coding mutations in the region targeted by the exome sequencing kit. On this set of variants a) hot spot analysis was performed, and b) MutSigCV was utilized, resulting in a ranking of genes Genes with mutations in one or two tumors only were excluded. The top 73 genes were validated by MiSeq sequencing in a validation set of 93 additional MSI CRCs. The data was filtered against > 60,000 outside controls to remove germline variants. Insertions and deletions were left out, resulting in a set of somatic missense, nonsense, and synonymous changes with MAF < 5 x 10-5. On this set of variants, OncodriveFML was utilized. Genes with mutations in only one tumor were excluded, resulting in a ranking of 57 candidate driver genes. Further functional studied were carried out for SMARCB1 and STK38L
Figure 7(A) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level (left). The cells were incubated with NAC (1 mM) and then stained with MitoSOX or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX (right) was re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (B) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3 (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose. (C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox). ( Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (D) Min6 GK or KD cells infected with a lentivirus expressing a cDNA of full-length Ndufs3 or p22phox were selected and tested for the level of Pdia4 and Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), Pdia4 and p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
a,b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively.figf7(a,b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively. Data shown are an average of at least three experiments ± s.d. (*P 0.05). (c) Mechanism by which C18-ceramide (Cer) induces lethal autophagy involves, at least in part, the lipidation of LC3B, forming LC3B-II (LC3-PE), and interaction of LC3B-II and ceramide on mitochondrial membranes upon Drp1-mediated mitochondrial fission, which targets autophagolysosomes to mitochondria for lethal mitophagy.
Figure 5Northern blot experiments assessing the half-lives of GlmY* and GlmZ under normal growth and GlcN6P starvation conditions. Bacterial cultures were either treated with 100 μg/ml Nva-FMDP or H2O (mock). Transcription was stopped by rifampicin addition when cultures attained OD600 = 1.0 and samples were removed at indicated times for Northern analysis. A Analysis of GlmY* and GlmZ decay in the wild type strain Z8. B Analysis of GlmY* and GlmZ decay in the ∆rapZ mutant Z28. C Semi-logarithmic plots of GlmY* and full-length GlmZ decay for half-life determination.
CRISPR-based detection method for novel coronavirus.Figure 1.CRISPR-based detection method for novel coronavirus. General scheme of CRISPR detection procedure. In the two strategies all the process takes less than 60 min.
Figure 7A. MTIF2-dependent reading of initiation codons AUG, AUA or AUU in the P site of the ribosome by unmodified (unmod.) or C34-modified [14C]Met-tRNAMet. Binding was determined by nitrocellulose filtration and [14C]Met-tRNAMet retrieved on the membrane was quantified by scintillation counting. Binding in the absence of ribosomes (no 70S) or mRNA (no mRNA) served as controls. Data from three independent experiments are presented with error bars indicating ± SEM. The statistical significance of the results was analysed by T-test and is indicated by the asterisks in the graph. B. TUFM-dependent recognition of A site codons during elongation. Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in (A).
Figure 7A) Ptc is mainly secreted in small EVs (P100K) from Cl8 cells. B) Large EVs (lEVs), small EVs (sEVs) and very small EVs (vsEVs) were analysed in triplicate by Western blotting. sEVs are enriched in Ptc and in the EV-associated proteins Syntaxin1 (Synt1A), Late bloomer (Lbm) and Hsp70. C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation. D) Fractionation of sEVs by Size Exclusion Chromatography: Ptc perfectly co-fractioned with Synt1A, Lbm and Hsp70. In agreement with the density gradient analysis, unprocessed form of co-receptor Ihog does not co-fraction with Ptc in the same subpopulation of sEVs. E) Effect on EV-associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α-Snap, the tetraspanin Tsp96F and the ESCRT components Hrs, Tsg101, Shrub and Vps4. Hsp70 in cells and EVs is detected by different exposition time.
Figure 3A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image. B ASO improves exon 19 inclusion in a dose-responsive manner. RT-PCR analysis of RNA from mouse cells transfected with increasing doses of ASO-15. The percent inclusion of exon 19 is shown below the gel image. The half-maximal effective concentration (EC50) was calculated using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA) after fitting the data using nonlinear regression with normalized response and variable slope. C Sequence and ApoER2 target region of most active mouse ASOs from A. Exonic and intronic sequences are in capital and lower-case letters, respectively. D RT-PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs. C refers to a non-specific control ASO.E Quantitation of ApoER2 exon 19 inclusion from D (mean s.e.m., one-way ANOVA with Bonferroni multiple comparison test.
Shp2 activates the expression of Skp2, Aurka, and Dll1/Hey1 genes to suppress senescence in PyMT tumor cells Heatmaps for cell cycle, DNA replication, and p53 target genes defined by gene ontology terms in control, Shp2 ko, and Shp2‐inhibited PyMT spheres at day 7.Figure 2Heatmaps for cell cycle, DNA replication, and p53 target genes defined by gene ontology terms in control, Shp2 ko, and Shp2‐inhibited PyMT spheres at day 7. qRT-PCR analysis of mRNA levels of Skp2, Aurka, Dll1, and Hey1 in control, Shp2 ko, and Shp2‐inhibited cells. Error bars represent SEM (n = 3). SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNA. Scale bar, 100 μm.Quantification of the numbers of senescent cells shown in (C). Error bars represent SEM (n = 3). **P 0.01; ***P 0.001.Quantification of the numbers of spheres formed by cells shown in (C). Error bars represent SEM (n = 3). *P 0.05; **P 0.01; and ***P 0.001. Statistical significance was assessed by Student's unpaired t‐test. Scheme showing an overview of Shp2‐dependent signaling systems and target genes, which are involved in senescence and cell cycle programs in PyMT tumorcells.
Figure 2endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (A) bEnd.5 cells were incubated with antigen-stimulated mouse T cells for 0-15 min. endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (B) luEnd cells devoid of PECAM-1 (KO) or re-transfected with PECAM-1 (RT) were incubated with T cells for 30 min. endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min. endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (D) bEnd.5 cells were pre-treated with anti-PECAM-1 (Mec13.3) or anti-endomucin antibodies for 30 min prior to incubation with T cells for 30 min. endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above) and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (E) HUVEC, siRNA depleted for endogenous PECAM-1, were transduced with wild-type PECAM-1 (PECAM-1 WT) or Y663,686F PECAM-1 double mutant (PECAM-1 YF) and incubated with HL60-derived neutrophils for 20 min. On the right, immunoblot analysis of lysates from siRNA treated cells before PECAM-1 re-expression. F Transmigration of human neutrophils towards IL-8 through TNFα-stimulated HUVEC expressing PECAM-1 WT or PECAM-1-Y663,686F. The transmigration rate is presented relative to that of PECAM-1 WT expressing cells, set as 100%.
Figure 7Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration. (A) RT-qPCR analysis for key EMT markers in embryonic stem cells (ES), neuronal progenitors (NP) and terminally differentiated neurons (TN). mRNA levels were measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates. (B) Western blot analyzing total JNK and phospho-JNK (p-JNK) during neuronal differentiation of stem cells. β-actin serve as a loading control. (C) RNA levels of Pcbp4, Prkrir and Tsc22d1 during neuronal differentiation as shown in A, measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates. (D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours. (E) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in (A), measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates. (F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex. (G) RNA levels of genes shown in Figure (E), represented by average normalized tag counts derived from RNA-seq data of TGF-β-induced EMT in NMuMG cells and plotted on the y-axis. Error bars represent SEM from three biological replicates. (H) Graphical representation of In Utero Electroporation (IUE) performed at the embryonic stage 12.5 (E12.5) during murine development. E12.5 mouse embryos were electroporated with GFP-tagged constructs either with an established non-target control (NTC) shRNA or a validated shRNA against Tsc22d1. Four days later (E16.5), embryos were sacrificed and brains were fixed for immunofluorescence analysis with antibodies against GFP and Tbr1 or Satb2 (early neuronal markers). (I) A representative image from immunofluorescence analysis performed with anti-GFP and anti-Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain. The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining. y axis represent percentage of cells above or below the Tbr1 stain region. Error bars represent SEM from 3 independent biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.
Regulation of DHHC5 localisation by palmitoylation of Golga7b and the C-terminus of DHHC5.Figure 2Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5. Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and the catalytic mutant DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Quantification of the plasma membrane signal of DHHC5 in each of the images (A-F), the graph displays mean +/- s.e.m. ****=p<0.001, n.s. = not significant, unpaired t-test. N=35, DHHC5 + WT Golga7b. N=41, DHHC5 + mutant Golga7b. N=35, DHHS5 + WT Golga7b. N=31, DHHS5 + Golga7b mutant. N=29, C-terminal mutant + WT Golga7b. N=30, C-terminal mutant + mutant Golga7b. Co expression of palmitoylation-deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane. The majority of the DHHC5 C-terminal palmitoylation-deficient mutant is localised to the plasma membrane in a Golga7b independent manner. All N numbers refer to the number of cells imaged and quantified, all experiments repeated 4 times. Data information: All images show ectopic protein expression only. All scale bars 10µm.
Figure 5IB analyses to determine the interaction between β-catenin and BBR peptide by in vitro pull-down assays using purified His-β-catenin and GST-BBR proteins IB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG-β-catenin and GFP-BBR or -Mock in the presence of proteasome inhibitor, ALLN, for 24 hr Effects of BBR overexpression on KYA1797K-induced RAS degradation. I analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr Effects of BBR overexpression on KYA1797K-induced RAS degradation ICC (D; Scale bars, 10 µm) analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr MT-HCT116 cells were cotransfected with GFP-Mock or -BBR and FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr, and WCLs were immunoprecipitated with pan-RAS antibody IB analyses of IP with β-catenin antibody in MT-HCT116 cells transfected with GFP-BBR-WT, -2E, or GFP-Mock in the presence of ALLN for 24 hr IB analyses of MT-HCT116 cells transfected with GFP-Mock, -BBR-WT, or -BBR-2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hr MT-HCT116 cells were transfected with GFP-Mock, -BBR-WT, or -BBR-2E together with FLAG-Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses
Figure 4E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT or variant were grown in 2% arabinose supplemented LB in the absence of drug and harvested at mid-exponential phase. Membrane samples were prepared from three individual colonies grown independently (Figures S5 and S6). Compared to wild-type, strains expressing tet(B)L9F consistently have higher in vivo protein levels where as strains expressing tet(B)V339C and tet(B)G366C have lower in vivo protein levels as detected by Western Blot analysis. All four variants located in the interdomain loop exhibited in vivo protein levels similar to wild-type.
Figure 5(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone. (B) Experimental scheme of detecting WH2 occupancy by FRET. (C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve. (D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).
Figure 6A, B NB-3-Fc-coated bead aggregation assay (A). BSA-coated beads as a negative control (B). C Co-immunoprecipitation of NB-3-Myc with NB-3-HA from co-transfected COS1 cells. D, E Cis interactions of NB-3-Myc with NB-3-HA in COS1 cells. (E) High-magnification image of the boxed area in (D). Cells were co-stained for Myc (red), HA (green), and DAPI (blue). The arrowheads indicate cell surface co-localization of NB-3-Myc and NB-3-HA. F, G Trans interactions between NB-3 expressed in COS1 cells and treated NB-3-Fc. Cells were co-stained for Myc (red), Fc or NB-3-Fc (green), and DAPI (blue). The arrows indicate co-localization of NB-3-Myc and NB-3-Fc at the cell membrane (G). H Cell membrane co-localization of NB-3-Myc and NB-3-HA separately expressed in 293T and COS1 cells. Cells were co-stained for Myc (red), HA (green), and DAPI (blue). I, J NB-3 expression in cultured pyramidal neurons and astrocytes. Pyramidal neurons were co-stained for NB-3, Ctip2 and Tuj1 (I). Astrocytes were co-stained for NB-3, GFAP, and DAPI (J). K Western blot analysis and quantification of NB-3 in cultured pyramidal neurons and astrocytes. NB-3 expression levels were normalized by b-tubulin and expressed relative to that of neurons. ***p < 0.001; one sample t-test. Data were analyzed from 3 independent experiments. L-Q NB-3+/+ (L and M) and NB-3−/− (O and P) pyramidal neurons were co-cultured with astrocytes that did not (L and O) or did (M and P) express NB-3, and immunostained for Tuj1, NB-3, and GFAP (L, M, O and P). NB-3+/+ pyramidal neurons (N) and NB-3−/−pyramidal neurons that overexpressed NB-3ΔECD-Myc (Q) were co-cultured with astrocytes that overexpressed NB-3ΔECD-Myc, and immunostained for Tuj1, Myc, and GFAP (N and Q).R The length of neurites on astrocytes relative to total neurite length. n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified.