EMBO/sd-character-level-ner huggingface dataset:
(c) Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. (d) Morphometric analysis of mitochondrial shape. Experiments were carried out as in c. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment).
(E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.
C | Representative flow cytometric analysis of HEL-binding in reconstituted Ramos cells after staining with fluorescently-labeled HEL. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).
K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).
(h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson's correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.
Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).
(A) A schematic of IFITM3 protein is shown, with gray boxes indicating transmembrane domains and colored bars indicating residues targeted for post-translational modification: ubiquitinated lysines (blue), palmitoylated cysteines (purple), and a phosphorylated tyrosine (red). Residues 17-24 are shown in detail to highlight two adjacent, overlapping motifs that recruit NEDD4 (PPNY) and the AP-2 complex (YEML). The IFITM3 Δ1-21 variant initiates translation at an internal methionine residue (green) and thus lacks these motifs.
(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.
(C) The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a KD of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line).
C. Number of CD41+ megakaryocytes derived from isolated CD34+ progenitors from myelofibrosis patients. Number of CD41/CD61+ cells counted on day 12 using trypan blue exclusion (n=1 technical replicate). D. Summary of fold change reduction of CD41/CD61+ megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.
(A) Depiction of the three main signaling branches of VAV1 in T cells. Additional binding partners of the CSH3 domain that will be studied in this work are also shown. Signaling crosstalk between the NFAT route and parallel TCR-triggered pathways are also depicted. DAG, diacylglycerol; RAS-GRP1, Ras GDP releasing protein 1; cNFAT, cytosolic NFAT; nNFAT, nuclear NFAT. Rest of abbreviations have been introduced in the main text.
Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Representative pictures are shown in (A).
(B) Immunoblot analysis of endogenous PLZF protein levels in HEK293T cells or CRBN-/- HEK293T cells treated with DMSO or lenalidomide (Len) for 24 h.
(D) Cellular localization of recombinant FAM210A in human AC16 CM cells. FAM210A-3xFlag expression plasmid was transfected into AC16 cells for 48 hrs. Scale bar: 40 μm.
G. CD4+ T cells were stimulated for 96 h with anti-CD3, anti-CD28 and anti-CD49d in the presence or absence of ATM inhibitor (KU, upper panel) or GSNOR inhibitor (N6022, bottom panel). Cell death was assessed cytofluorometrically upon staining with Annexin V (AnV) and PI. Values are expressed as fold change of AnV+/PI+ cells relative to control (CD4+ without inhibitor, vehicle, arbitrarily set to 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 independent experiments. *p<0.05; ***p<0.001.
(C) The number of occurances where each drug within the library is in the top 20 ranking interactions for each target is tabulated for drugs that cause siginificant (p-values < 0.05) changes in at least 5 metabolites.
G: IL-6 protein quantification in AAVS1KO and TLR7KO pDCs after SARS-CoV-2 exposure (1 MOI) at indicated time points. Data information: Bars represent mean values and equal symbols represent the donors used throughout the experiments (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to their respective mock treated conditions, or by unpaired non-parametric T test when comparing matched conditions between different KOs. *
D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.
MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D (solid bars, MISTRG; hatched bars, MISTRG6),
Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.
HCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays.
E Top panel represents overlap between genes within 20 kb of the bicalutamide‐responsive AR locations and enzalutamide‐responsive AR locations. Bottom panel indicates microarray analysis of genes around enzalutamide‐responsive AR locations following treatment with DHT or enzalutamide in LNCaP cells.
A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Scale bar, 25 µm. B Quantification of LC3 signal morphology of images shown in (A). Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei. 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=4 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).
(D-H) Western blot analysis of THP-1 cells untreated or treated with (D) 5'pppRNA ( 1 μg/ml) for 3 h, 6 h, 12 h, 18 h and 24 h (E) Poly I:C (1 μg/ml) for 3 h, 6 h, 9 h, 12 h and 24 h (F) Poly dA:dT (1 μg/ml) for 3 h, 6 h, 9 h,12 h and 24 h (G) cGAMP (1 μg/ml) for 6 h, 12 h, 18 h, and 24 h (H) dsDNA (1 μg/ml) for 3 h, 6 h, and 12 h and probed with antibodies as indicated.
E. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).
Representative traces of cell-attached recordings performed in a WT (blue) and a KO+A-mWwox (purple) adult mice (6 months old).
A. Schematic of CLASH protocol for purification of RNA-RNA interactions. RNAs were UV-crosslinked to RNase E-HTF in vivo and purified using M2 anti-FLAG resin. RNAs were trimmed using RNase A/T1 and further purified under denaturing conditions. RNA linkers were ligated to the immobilized RNA-RNase E complexes. Duplexed RNAs may be ligated into a single contiguous molecule (left, CLASH) that gives information on RNA-RNA interaction occurring on RNase E. The remaining single RNAs reveal the site of RNase E binding within the transcriptome. Linker ligated RNA-RNase E complexes were size selected by SDS-PAGE and RNAs recovered for library preparation and sequencing.
D. Immunostaining of SCNT embryo for H3K27me3 and HA epitope tag after injection of different mRNA as indicated. Representative images from ≥ 187 embryos analyzed in four independent micromanipulations for each condition are shown. Scale bar, 20 μm
C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP.
D Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP, and 6Myc-Atg14 were immunoprecipitated with an anti-Myc antibody and Protein G PLUS-Agarose. The bound proteins were analyzed by immunoblotting.
(A) CD45.2 frequency among GCs induced by eOD-GT8 60 mer at different time points. Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD-GT8 60mer. On days 8, 15 and 36, the splenic B cells were isolated to measure the CD45.2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS. Gated plots represent the CD45.2 frequency among GC B cells. (B) Quantification of CD45.2 frequency among GCs at three time points. X-axis represents adoptively transferred mice groups with different precursor frequency (1 in 104, 2 in 105, 3 in 106) at three time point, y axis represents CD45.2 frequency among GC. Each circle represents one mouse. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. Bars indicate mean ± SD from mice in each group.
Proximity ligation assay for p53 with Tom20 in indicated untreated (Mock) or Doxo-treated (0.1 µg/ml, 24 hours) H1299 cells following tet-induction. Shown are representative cells with PLA signals (red) counterstained with DAPI (blue). Quantification of proximity ligation assays for p53EE with Tom20, Bcl-2, Bcl-xL and Bak in H1299 cells. Doxo treatment (0.1 µg/ml, 24 hours) as indicated. Plotted is the average number of PLA signals per cell for 10 fields of view.
B. Generation of double-stranded fragments S1 and S2 in SRS2 and srs2Δ during a time-course experiment. Also see Figure S2B-C for blot images. Red two-ended arrow indicates time delay of ~ 0.5 h in fragment S1/S2 formation in srs2 mutants relative to SRS2 cells. Average ± SD (n=4) is shown for each time point. Strains used: NK4691-4693; NK4805-4808.
(B) Sequences of the TMD regions used for cloning and the immediate flanking residues are represented. The regions defined as TMDs were determined using TOPCONS (Tsirigos et al, 2015) and are in bold/underlined. Red residues indicate mutations made in the Xport-A TMD to increase its hydrophobicity. To the right of each sequence is the "transmembrane tendency" score of Zhao & London
(J) The graph depicts the percentage of WNV (MOI 0.3, 16 h) infected control and IRGM knockdown Huh7 cells stained with 4G2 antibody analyzed by flow cytometry
A Effects of expression of either FXR‐WT or FXR‐K277R on the mRNA levels of the indicated genes in hepatocytes isolated from FXR‐KO mice. Values are presented as mean ± SEM (n = 6); two‐tailed Student's t‐test, **P 0.005, NS, not significant.
Alignment of micV sequences, including the promoter regions, from various Vibrio species. The -35 box, -10 box, the TSS, the highly conserved potential seed region, and the rho-independent terminator are indicated. Lower part: Consensus motif of E. coli σE-dependent promoters.
(e) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h.
DNA damage, e.g. induced by chemical mutagens, is followed by the activation of the checkpoint kinase ATM (Ataxia Telangiectasia Mutated) that activates the transcription factor SOG1 (Suppressor of Gamma Response 1). On the one hand, SOG1 represses, directly or indirectly, the expression CDKB2 and possible other CDKs as the major driving force of mitosis to allow a cell time for DNA repair. On the other hand, SOG1 directly binds to the promoter of CYCB1;1 and activates its expression. CYCB1;1 builds an active complex together with CDKB1. This complex phosphorylates the DNA binding protein RAD51 (Radiation Sensitive 1) that gets recruited to the DNA damage site. This cascade is required for HR in S and G2 phases of the cell cycle in meristematic cells. To trigger this response, the action of all four B1-type cyclins is necessary, possibly by providing a threshold of mitotic CDK activity that then gets amplified through SOG1-dependent stimulation of CYCB1;1 expression.
Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.
C. Protein sequence alignment of E. coli GlpG, human RHBDL4, S. pombe Rbd2, and S. cerevisiae Rbd2. Alignments were generated using Clustal Omega server (http://www.ebi.ac.uk/Tools/msa/clustalo). Predicted transmembrane domains (TM) and shared sequence motifs were used for alignment. Positions of functionally important, conserved rhomboid amino acid residues (including predicted catalytic dyad serine and histidine) are shaded in red. Residues boxed in green were identified as required for Sre1 activation from a genetic selection and were analyzed in Figures 2E and F. The starting residue numbers of the indicated sequences within their full-length proteins are given. Asterisks mark residues identical in all sequences and colons denote conservative substitutions.
E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.
(H) The blotting band intensities of LC3-I, LC3-II and β-actin were quantified (n = 6), and the relative ratios between LC3-II/actin and LC3-I/actin are shown in the bar graph; the number (n) of animals is for each genotype.
J. Insulin (ITT) and glucose (GTT) tolerance tests after 60 days of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.
B Immunoblot analysis for O-GlcNAcylation of ATX1 by SEC using anti-CTD110.6 antibody. HA-tagged ATX1 was expressed alone or together with Flag-tagged SEC in tobacco leaves. Nuclear proteins were extracted from tobacco mesophyll cells, and expression of FLAG-SEC and ATX1-HA was confirmed by western blotting.
The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of ATP (n = 3) (H)
(A) MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.
(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.
(A) Heat map showing RPKM (Reads Per Kilobase per million of mapped reads) mean values of Mmps from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for 24 h or left untreated (NT).
(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.
A) SEZ6KO and WT neurons were biotinylated with Sulfo-NHS-Biotin and surface proteins were enriched by streptavidin bead pull-down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma & Marques, 2013), therefore the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2 and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3 / SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± S.E.M., at least 10 replicates in 4 independent biological experiments, Mann-Whitney ****p-value<0.005).
44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. H TruCulture IFNγ, IL4, IL6, IL8, IL12 subunit p40, IL23 and TNFα protein quantification of LPS-stimulated whole blood samples, n=11/12/11/10/11/10/11 individual human subjects. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
B USP26 modulates the ubiquitination of TEX11. pRK-FLAG-Usp26, pEGFP-Tex11, pCMV-HA-Ub were transfected into HEK293T cells. At 24h after transfection, cells were collected for immunoprecipitation (IP) with anti-GFP antibody and analyzed with anti-HA and anti-GFP antibodies, respectively.
(G) HCT116 XIAP KO cells were treated with the indicated amounts of Nutlin for 8 h. Cell lysates were analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates.
(D) Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean +/- SEM. P values indicate significance measured by Fishers exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph).
Representative experiment of human epidermal cells analysed by flow cytometry for the expression of langerin, CD1a and S100A9 (Skin samples from n=2 donors). The enriched CD1a+/langerin+ population representing human eLC is shown (black circle).
Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti-TAPBPL or isotype Ab.
Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).
(e) During a 72-h serum starvation, NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were subsequently re-stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.
(B) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-OFD1 and anti-PCM1 antibodies. The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock_growing, n=150 cells; Mock_SS, n=149 cells; MyoVI KD_growing, n=149 cells; MyoVI KD_SS, n=150 cells, from three independent experiments. ns, not significant; * P<0,05; **** P<0,0001 by Kruskal-Wallis test.
(B) Wing size variation in absolute centroid size (CS) and relative CS (adjusted for body size).
(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3)
b, Quantification of GFP-LC3 dots in neonatal tissues. Cryosections were prepared from tissues isolated at the indicated times. The ratio of the total area of GFP-LC3 dots to the total cellular area is shown as a percentage. Values represent mean ± s.d. of three mice. Diaph., diaphragm; Panc., pancreas; Muscle; gastrocnemius muscle.
Il1b and Tnf mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments.
D. HopAO1 dephosphorylates LORE Y600 in N. benthamiana. LORE-FLAG was transiently co-expressed with GUS-T7, HopAO1-T7 or HopAO1CS-T7 in N. benthamiana leaves mediated by Agrobacteria. The protein was immunoprecipitated and probed by anti-pY600 antibody. The experiment was repeated at least three times with similar results.
PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D) Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM).
Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9.
Representative images of H&E-stained lung sections from KrasG12D recipient mice reconstituted with KrasG12D (KrasKras) or KrasG12D:Adam17ex/ex bone marrow (KrasKras:ex/ex). Scale bar, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated bone marrow chimeras (n = 6 mice per group).