EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB-360. B. Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB-360. C. KEGG enrichment pathway analysis of the significantly regulated proteins upon BACE inhibition in both IGRs. D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD). F. Quantitation, by immunofluorescence analysis, of YAP in different cellular compartments (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). Images were quantified by subdividing cells into mostly cytosolic YAP (Cytosol), mostly nuclear YAP (Nuclear), or equal distribution (Total) from three biological replicates. G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). H. CTGF mRNA level measured by real-time PCR in IGR39 treated with IGR39 conditioned medium (CTRL) or with IGR37 conditioned medium (CM), N=4 biological replicates. T-test analysis, *** p-value <0.001, data are mean ±SD. I. CTGF mRNA level measured by real-time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis: *** p-value <0.001, data are mean ±SD. J. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.
Figure 2A. Immunoblotting and densitometry quantification of Rab22a in JAWS-IIDCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments. B. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A. Data show mean ± SEM of triplicate values and are representative of two independent experiments. ** P = 0.0047. C. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 µm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments. D. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003. E. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types. F. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min. G. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20 and 40 min.
C38 and IB3-1 cells transfected with GFP–LC3 plasmid in nutrient-rich or starvation medium.figf1C38 and IB3-1 cells transfected with GFP-LC3 plasmid in nutrient-rich or starvation medium. (a) Top: confocal microscopy of GFP. N, nucleus. Scale bar, 10 μm. Bottom: percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. (n = 5). Asterisk, P 0.001 versus C38 cells; analysis of variance (ANOVA). (b) Left: electron microscopy of starved C38 and IB3-1 cells. The right-hand images are higher magnifications showing areas enriched in autophagosomes in C38 cells (arrows) and perinuclear mitochondria in IB3-1 cells. N, nucleus. Scale bar, 250 nm. Right: numbers of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus C38 cells; ANOVA. (c) Fluorescence-activated cell sorting (FACS) analysis after staining with fluorescent dye DiOC6 in starved C38 and IB3-1 cells. (d, e) Immunoblot of LC3 in starved C38 and IB3-1 cells (d) and IB3-1 cells (e) after incubation with E64d and pepstatin A. (f, g) Immunoblots (IB) of polyubiquitin (f) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (f, g) Immunoblots (IB) of p62 (g) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (h) Immunoblot of LC3 and p62 in control (n = 10) and CF (n = 10) human nasal mucosa. (i) Number of LC3-positive dots (per 104 mm−2 of mucosa) in control (n = 10) and CF (n = 10) human nasal mucosa. Means ± s.d. Asterisk, P 0.01 versus controls; ANOVA. (j) Confocal microscopy of p62 in control and CF human nasal mucosa. Representative images from ten patients with CF. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 10 μm. (k) Number of LC3-positive dots in lung tissues from wild-type (WT) and CftrF508del mice. Means ± s.d. for lung tissues from ten mice per group. Asterisk, P 0.001 versus WT mice; ANOVA. (l) Confocal microscopy of p62 in WT and CftrF508del homozygous mice. Representative staining from at least five airway epithelial areas per mouse (n = 10 mice per group). L, lumen. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 5A Recombinant Gadd45a protein and its G39A mutant were added to the histones-oligo DNA mixture. The mixtures were separated by 4% non-denaturing PAGE and stained with ethidium bromide (EB). The oligo DNA probes were amplified from the Oct4 promoter. B Recombinant wild-type and G39A Gadd45a were added to the mixtures of oligo DNA and the recombinant H2A/H2B heterodimer or H3/H4 tetramer. The mixtures were separated by 4% non-denaturing PAGE and stained with EB. C Co-immunoprecipitation of wild-type Gadd45a, G39A Gadd45a and H3 shows that the interaction between Gadd45a and H3 is dependent on G39 residue. D Two step crosslinking method identified Gadd45a could interact with chromatin in the promoter regions of Oct4 and Nanog. Cells were crosslinked with DSG before ChIP-PCR analysis. (**p≤0.01; ***p≤0.001; n=3) E The chromatin compaction of the indicated regions was detected by nuclease accessibility assay. Genomic DNA was purified from MEFs infected with Flag alone and SKO plus Flag, wild-type Gadd45a or G39A Gadd45a on day 8. (*p≤0.05; **p≤0.01; n=3) F ChIP-PCR analysis of the binding of Oct4, Sox2, and Klf4 to their targets individually in MEFs infected with SKO plus Flag, wild-type Gadd45a or G39A Gadd45a on day 8. (***p≤0.001; n=3)
Figure 6(A) Schematic representation of the experimental procedure. Catheters pre-colonized with S. aureus were allowed to form biofilms in an in vivo context by subcutaneous implantation in mice. 24 h post-implantation mice were treated with a single subcutaneous injection of different mycoplasma strains and the effectiveness of each treatment was followed by positron tomography with [18F]-FDG-MicroPET. (B) Representative images of longitudinal slices of [18F]-FDG-MicroPET uptake in mice carrying implanted catheters (red arrows) at day 1 or day 4 of the treatments. Micro-PET images have been superimposed with CT-3D images used as anatomical reference. Brain location is highlighted (b). (C, D) Plots showing the SUV60 variation (%) between day 1 and day 4 of the different treatments. Each circle represents the SUV60 variation obtained for individual animals (n≥4), whereas mean ± s.d. is represented with lines inside each group. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post-treatment. Results from one-sided ANOVA followed by Fisher's PLSD test are shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.
Figure 7Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Taf1-FRB.
a, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times.figf3(a, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex (a), Beclin 1 (b) and VDAC (loading control) was analysed by Western blot analysis. (c) Inhibition of etoposide-induced autophagy by silencing of Beclin 1. Bax/Bak−/− MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy (×8,500). d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were transfected with GFP-LC3 together with the indicated siRNAs, were incubated without (NT) or with 20 μM etoposide for 24 h, and then examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4). (e, f) Inhibition of etoposide- and staurosporine-induced cell death by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide (e, f) or 1 μM staurosporine (f) for the indicated time. Cell viability was measured by CTB assay (e) and clonogenicity assay (f). n = 4.
Figure 4(A) Schematic of the PIDD1 domain structure and of the different PIDD1 species generated by autoproteolysis. LRR = Leucin Rich Repeat Domain; ZU5 (N and C) domains = Domain present in ZO-1, Unc5-like netrin receptors and in ankyrins; UPA domain = conserved in UNC5, PIDD, and Ankyrins; DD = death domain. (B) A549 cells of the indicated genotypes were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated PIDD1-V5 derivatives and subjected to immunoblotting. N = 2 independent experiments. (C) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced (mock), or transduced with PIDD1-V5 lentiviral vectors and co- stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm. (D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.
Steady-state abundance of lncRNAs is not directly affected by miRNA loss (A) Percentage of mRNAs (n=6701, red) and predominantly cytosolic (n=57, blue) and nuclear lncRNAs (n=175, grey) with experimental evidence of binding by ribosomes (Translation Efficiency>0) in mESCs.Figure 1(A) Percentage of mRNAs (n=6701, red) and predominantly cytosolic (n=57, blue) and nuclear lncRNAs (n=175, grey) with experimental evidence of binding by ribosomes (Translation Efficiency>0) in mESCs. (B) Density of HEAP-AGO2 peaks across cytosolic lncRNAs (n=62, blue) and the 3'UTR regions of mRNAs (n=8798, red) with experimental evidence for AGO2 binding in mESCs (>0 AGO2 peaks). Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution. (C Small RNA and Poly(A)-selected RNA sequencing based estimates of the fold difference (y-axis) in (C) miRNA relative to day 0, during a 12 days' time-course (x-axis) following treatment of DTCM23/49XY mESCs with 4-OHT and consequent loss of DICER function. Points represent the average miRNA or mRNA expression and error bars the standard deviation based on three independent biological replicates. D) Small RNA and Poly(A)-selected RNA sequencing based estimates of the fold difference (y-axis) in (D) mRNA expression, respectively, relative to day 0, during a 12 days' time-course (x-axis) following treatment of DTCM23/49XY mESCs with 4-OHT and consequent loss of DICER function. Points represent the average miRNA or mRNA expression and error bars the standard deviation based on three independent biological replicates. (E) Cumulative distribution plot of the fold-difference in expression after 8 days of 4-OHT treatment for mRNAs, relative to day 0 of treatment (tpm≥1) with (n=6034) and without (n=7887) evidence of AGO2-binding by HEAP (Li et al., 2020). (D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.
The effect of Atg14L localization on its function.Figure 5.The effect of Atg14L localization on its function. (a) Atg14L knockout mouse ES cells were transfected with GFP, GFP-Atg14L, GFP-Atg14L4C4A, or GFP-ER-Atg14L4C4A of the pCAG vector and subjected to starvation. The cells were fixed and immunostained with anti-Atg16L or anti-LC3 antibodies. (b and c) The number of Atg16L-positive puncta (b) and LC3-positive puncta (c) per cell was counted in at least 10 GFP-positive cells, and mean ± SD values are presented. Bars, 10 µm.
Anti-mutant calreticulin antibody binds to cell surface and inhibits TPO-independent proliferation A.Figure 1A. Schematic showing wild type C-terminal calreticulin protein sequence and neoepitope sequences for 52 bp deletion or 5 bp insertion and peptide sequence used for immunization. B. Diluting concentrations of 4D7 to bound full-length 30 amino acid peptide compared to scrambled, C-terminal and N-terminal 11 amino acid peptides as shown in A (n=3 biological replicates). C. Scatchard analysis showing dissociation constant of 4D7 using I125-labelled and unlabelled 4D7 bound to full-length 30 amino acid peptide (n=3 biological replicates). D. Histogram showing fluorescence intensity of 4D7 conjugated to phycoerythrin at a 4 µg/mL TF-1 TpoR CALRdel61 vs TF-1 TpoR CALRWT compared to unstained. E. TF-1 TpoR cells with endogenous wild type CALR cultured in the presence of TPO and 2, 10 or 20 µg/mL 4D7 anti-mutant CALR antibody or 20 µg/mL control IgG antibody (n=3 biological replicates). Data information: bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate, with a one-way ANOVA with Bonferroni correction used to determine statistical significance. F. Proliferation curves of factor-independent TF-1 TpoR CALRdel61 cells cultured with 2, 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). G Proliferation curves of factor-independent TF-1 TpoR CALRdel52 cells cultured with 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). Data information: bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate, with a one-way ANOVA with Bonferroni correction used to determine statistical significance.
Figure 6A. mRNAs enriched in optimal (red) codons have longer poly(A)-tails, higher translation efficiency (TE) and higher stability compared to genes enriched in non-optimal codons (blue). B. Heatmap showing the codon optimality code underlying the genetic code in zebrafish and (C) Xenopus. Based on the consistent identification by two methods (rCSI) and (CSC) we defined: Optimal codons in red and non-optimal codons in blue. The intensity of the color represents the strength (light, weak; dark, strong) based on different CSC values, which corresponds to different cumulative effects in mRNA half life (Fig 1F).
Figure 5(A) Normalized GE values of plant SR proteins in a 48-hour time course. In the first day, plants were kept at 20°C (red) and shifted to 4°C (blue) for the second day. Plants were under a 12-hour dark/light regimen (gray/yellow bars). Warm- and cold-expressed genes are indicated. (B) A. thaliana SR proteins divided in their subfamilies and classified as warm- or cold-expressed. (C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots.(E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80
Figure 4A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex. B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site. C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 . D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101. E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).
Arrangement of dynactin's shoulder subunits A.Figure 3The structure of the lower subdomain from the shoulder showing the organization of p150 (purple), p24 (yellow) and the two copies of p50 (p50-A in red, p50-B in pink). The N and C termini of each chain are shown. A Gaussian surface rendering of the lower subdomain is colored by feature (blues and magenta, inset). The dimerization domain of the shoulder. Subunits from the lower subdomain are colored, with p50-A in red, p50-B in pink and p150 in purple. * marks equivalent features in D. Helical bundle break in the lower subdomain arm, showing how the three helices in the arm break and reform after a twist. p24 (yellow) forms a short loop at the break, whereas p50 forms a longer loop, which is ordered in p50-A (red), and disordered in p50-B (pink), modelled by a dotted line. # marks equivalent features in D. D. Secondary structure diagrams for the segments of p150, p50 and p24 within dynactin's shoulder. Helices H1-8 and S1 are labelled for p50-A and p50-B.
Confocal analysis of the subcellular localization of HA–PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h.f2Confocal analysis of the subcellular localization of HA-PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h. Organelle markers used were as follows (from top to bottom panels): Lamp1 (late endosomes/lysosomes), giantin (Golgi-TGN), Vps35 (retromer-positive endosomes), GFP-Rab5 (early endosomes), GFP-UVRAG (early and late endosomes). Insets show high magnification of the selected areas. The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.
Structural comparison between 2019-nCoV S and SARS-CoV S.Figure 2.A single RBD "down" monomer of 2019-nCoV S is shown in ribbons, colored according to Figure 1. A monomer of SARS-CoV S is also shown in ribbons, colored white (PDB ID: 6CRZ). The following structural domains from 2019-nCoV S have been aligned to their counterparts from SARS-CoV S; NTD (top left), RBD (top right), SD1 and SD2, (bottom left) and S2 (bottom right).
Figure 2Plots showing the RT-qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands. Results are expressed as the mean ± s.d (n=10 for PBS, n=5 for WT and n=4 for CV2) of the 2-ΔΔCt relative expression values of the indicated interleukins; the value from each individual animal was calculated from three technical replicates. The values obtained in the PBS group were used as control for normalization of gene expression (=1). Statistical analysis was performed using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test: (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005).
Figure 6(A) Schematic diagram of the truncations of p38IP, in which the numbers indicate the residues of the truncations and the red box indicates T1B. (B) HEK293T cells transfected with N-terminal (left panels) or C-terminal (right panels) deletion mutants of p38IP as indicated in (A) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1. (C) Ribbon representation of a model structure of the p38IP-T1B (pink) and TAK1 (green). (D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1. (E) HEK293T cells transfected with YFP-p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1. (F) Jurkat TAg cells transfected with YFP vector or YFP-T1B were stimulated with 10μg/ml anti-CD3 for indicated times, then lysed and immunoprecipitated with anti-TAK1, followed by immunoblotting with the indicated antibodies. (G) Primary human PBMCs transfected with YFP vector or YFP-T1Bwere stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 for the indicated times, then lysed and analyzed by immunoblotting. (H) Intracellular cytokine staining analysis of TNF-α (upper) and IL-2 (lower) in primary human PBMCs transfected and stimulated as in (F) for 3 hours (TNF-α) and 8 hours (IL-2).
Figure 3(A) Examples of local field potential (LFP) traces in response to light flashes at 595 nm (light stimulus is shown as orange bar; 1.25 × 1017 photons cm-2 s-1). (B) Spike raster plots in response to 200 repetitions of the same light stimulus. (C) Averaged spike responses shown as peristimulus time histograms (PSTH, bin size: 5 ms) based on the spike raster plots. Top-row: WT-mice, middle row: rd1 control mice, bottom-row: ReaChR-treated rd1 mice. (D) ReaChR-induced cortical responses (spikes and LFP) as a function of light intensity at (595 nm) demonstrating that ReaChR-treated rd1 mice (n = 4) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina. Data presented as mean ± SEM.