EMBO/sd-character-level-ner huggingface dataset:
Figure 3A, B) Time course analysis and severity of the symptoms in wt and Sfrp1-/- mice after EAE induction. In A means are depicted with black (wt, n=19) and cyan (Sfrp1-/-, n=19) lines. In B data are expressed as % of the total number of analysed animals (n=19 per genotype).In A, Error bars represent Standard Error. C) Wt and Sfrp1-/- mice immunized with MOG and sacrificed 16 days post immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar 100μm. D) Quantification of CD4+ infiltrated lymphocytes, Iba1 normalised immunoreactivity, MBP normalised immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1-/- mice 16 days after immunization (n=16 acquisitions, white dots; from N=4 animals, black dots, per genotype). Error bars represent Standard Error. Statistic refers to number of acquisitions.
Figure 7A. Cartoon of reporter construct (Tang et al., 2013) in which induction of the mCherry-tagged FokI endonuclease (with 4-OHT) results in double-strand break (DSBs) in a region upstream of a doxycycline-inducible reporter gene (YFP-MS2). Ongoing transcription of the reporter gene can be visualized by the presence of a YFP-MS2 fusion protein that binds stem-loop structures in the nascent transcript. B. CDKL5 is recruited to FokI-induced DSBs. GFP alone (top panel) or GFP NLS-CDKL5 (middle and bottom panels) were stably expressed in U-2-OS 265 DSB reporter cells. Cells were mock treated or treated with 1 µg/ml doxycycline for 3 h to induce transcription of the reporter gene. An hour before DSB induction cells were treated with 0.3 µM PARG and 10 µM ATM inhibitor. Site specific DSBs were induced by treating the cells with 4-OHT and Sheild1 ligand. Cells were live imaged at 37 °C, between 15-25 minutes following DSB induction. Representative image showing the recruitment of GFP NLS-CDKL5 to FokI induced DSBs upstream of transcriptionally active (middle) but not the inactive (bottom) MS2 gene. GFP alone is used a control (top). White arrowheads mark the location of the mCherry-FokI upstream of the MS2 reporter cassette. Images are representative of multiple technical replicates of three independent experiments. Scale bar is 10 μm C. Representative image for U-2-OS 263 IFII cells harbouring the reporter construct and transfected with the siRNAs indicated. After addition of doxycycline, transcription was monitored in cells ± induction of FokI by quantification of YFP(-MS2) foci. Arrows indicate sites of FokI mediated DSB (mCherry) and YFP-MS2 transcript. "TRXN": doxycycline added; "TRXN/DSB": doxycycline added with 4-OHT. Scale bar is 10 μm D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.
FRH mutant interactions with the core clock components examined by race tube and immunoprecipitation assays.Figure 7(A) Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ= period in hours, σ = standard deviation, n = number of race tubes, ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes. (B) Pull-down assays evaluating interactions among FRH, FRQ, WC-1 and WC-2. Ratio of FRQ, WC-1 and WC-2 binding to the V5 tagged FRH mutants as compared to the inter-strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two-tailed t-test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.
Figure 1(a) A schematic of the workflow for Trim28-KO in mouse NPC cultures. Scale bars: embryo 1 mm, NPCs 20 μm. (b) Estimation of gene editing at the Trim28 loci using NGS-sequencing of amplicons. Black bars indicate % of frameshift indels. Columns show an average of two biological replicates per guide RNA and error bars show mean ± SD. (c) Western blot confirmed the loss of Trim28 expression upon Trim28-KO in mouse NPCs. (d) RNA-seq analysis of the expression of TE-families using TEtranscripts(f) CUT&RUN analysis of H3K9me3 in mouse NPCs of all full length MMERVK10Cs (right panel) RNA-seq analysis of the Trim28-KO and control samples, visualising full length MMERVK10C elements. The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram. (g) Example of transcriptional readthrough outside the full length MMERVK10Cs into a nearby gene.
Figure 1Relative expression of Rab39a in siRNA-treated cells after 48 hours of knockdown. Rab39a expression in I-Ab or Rab39a siRNA-treated cells was normalized to expression in cells treated with control β2m siRNA. Error bars show SD between three independent experiments. * p < 0.05 using two-way Student's t-test. XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments. XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-latex The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments. XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments. DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.
Figure 4A Protein O-glucosyltransferase activity of wild-type (WT) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat (hFIX-EGF). Wild-type shows higher O-glucosyltransferase activity than D233E, and this activity is dependent on the concentration of the acceptor substrate hFIX-EGF repeat (left) and on the concentration of donor substrate UDP-glucose (UDP-Glc) (right). Values indicate mean ± SEM from three independent assays. B O-glucosyltransferase activity towards five different single EGF repeats from mouse Notch1. Wild-type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E. The Y-axis shows the normalized activity relative to wild-type POGLUT1. EGF repeats were at 10 μM. Values indicate mean ± SEM from three independent assays. C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC. hFIX-EGF repeat was incubated with wild-type or D233E mutant POGLUT1 and donor substrate, UDP-Glc or UDP-xylose (UDP-Xyl), at 37ºC overnight. Values on top of the peaks indicate the measured masses. Addition of Glc (162 Da) or xylose (132 Da) to hFIX-EGF (5696.2 Da) by wild-type POGLUT1 caused a shift to an earlier retention time (left). Similarly, the products exhibited a similar shift after incubation with POGLUT1D233E (right). These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX-EGF repeats and thus has residual enzymatic activity.
Figure 2A, B. Either empty vector plasmids (EV) or those producing EGFP-PCM1 (A) or myc-Plk4 (B) were transfected into U2OS cells. Immunoprecipitation (IP) was performed with anti-GFP (A) or anti-myc antibodies (B), followed by immunoblotting with the indicated antibodies. DIC, the dynein intermediate chain. C. Bacterially expressed and purified His-PCM1 (a.a. 1-1128) was incubated with MBP alone or MBP-Plk4 (WT or KD) in vitro, and pulled down by magnetic beads coupled with an anti-MBP antibody, followed by staining with Coomassie Blue (left) or immunoblotting with an anti-His antibody (right). Asterisks show non-specific bands. The mobility difference between Plk4-WT and Plk4-KD was reported previously [43, 48]. We reproducibly observed the upward mobility shift of His-PCM1 upon binding to MBP-Plk4-WT, but not Plk4-KD. D. Overall structure of PCM1 and the Plk4-mediated phosphorylation site. The conserved serine residue that is phosphorylated in a Plk4-dependent manner is indicated in red. E. Total cell extracts were prepared from U2OS cells treated with PCM1 siRNA and transfected with RNAi-resistant EGFP-PCM1-WT*. Cells were cultured asynchronously (As) or arrested in G1, G2 or M phase, followed by immunoprecipitation with an anti-GFP antibodies. Pulled down samples were immunoblotted with the indicated antibodies.
Figure 4A) Segmentation of a prepupal eye imaginal disc from Drosophila (anterior to the left). The analysed developing photoreceptors are labelled in green. The cells adjacent to the photoreceptors are labelled in blue (these are "cone cells" in the most posterior clusters). B) Segmentation of a prepupa wing imaginal disc epithelium where myosin II levels have been reduced . C) Adult Biceps biopsy from a patient with neurogenic atrophy pathology. Fibres outlined with collagen VI antibody (green). D) Segmentation of the image showed in (C) highlighting two atrophic elongated fibres that present seven neighbours. E) Comparison of polygon distributions of different tissues with altered contractibility, tension or pressure balance within the tissue. Scale bars, 10 m in (A) and (B); 75 mm in (C).
Figure 6Morphological analyses of Spatc1l-KO epididymal sperm by SEM. Sperm were obtained from the vas deferens of 8-week-old WT (Spatc1l+/+) and KO (Spatc1l-/-) mice. Sperm neck regions are magnified in lower panels. AC, acrosomal cap; MP, mid piece; BP, basal plates. Scale bar = 2 μm (upper) and 0.5 μm (lower). TEM analyses of flagella in WT and KO testicular sperm. TEM images represent ultrastructures of cross-sections of the midpiece (upper) and end piece (lower) of WT and KO testicular sperm. Ax, axonemal microtubules; Mt, mitochondrial sheath; ODF, outer dense fibers. Scale bars = 200 nm (upper) and 100 nm (lower). TEM analyses of the development of the connecting piece in WT (left) and KO (right) sperm. TEM images represent ultrastructures of longitudinal sections of elongating spermatids (ES) and testicular sperm (TS) in WT and KO mice. An, annulus; BP, basal plates; Cp, capitulum; Mc, manchette; Mt, mitochondria; Nu, nucleus; PCV, proximal centriolar vault; PM, plasma membrane; SC, segmented column. Scale bar = 500 nm. Ultrastructures of epididymal sperm in KO mice. TEM images represent ultrastructures of separated sperm head (upper) and tail (lower) in the epididymis of KO mice. Images in the boxes are enlarged at right. BP, basal plates; Cp, capitulum; SC, segmented column. Scale bar = 500 nm. TEM analysis of morphologically abnormal KO sperm. Detached heads abnormally located to the midpiece of sperm flagella were observed by TEM (left) and confocal differential interference contrast (DIC) microscopy (right). PM, plasma membrane. Scale bars = 500 nm (left) and 5 μm (right).
Figure 2(A) The 35S:GFP-EVDint/ter-GUS fusion was made by introducing the EVD intron and proximal shGAG terminator (including the premature cleavage and polyadenylation site; PCAP) between the GFP and GUS coding sequence. Like EVD, it spawns full length unspliced and short spliced mRNAs. Red squares: stop codons. (B) Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. (*) = p-value < 0.05 (two-sided t-test against corresponding controls). (C) sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends. (D) Low molecular weight RNA analysis of the GFP- and GUS-spanning regions. tasiRNA255 is a control for the rdr6 mutation and miR159 provides a loading control.
Figure 4A, B. Tumors from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were harvested, and tumor single-cell suspensions were analyzed for tumor-associated myeloid cells by flow cytometry analysis. Representative graphs and quantification of F4/80+MHC-II+ immunosuppressive myeloid cells (IMCs)-M1 macrophages (A) and F4/80+CD206+ IMCs-M2 macrophages (B) gated on CD45+CD11b+ myeloid cells (n = 5). C. Representative flow cytometry analysis and quantification of Ly6C+Ly6G- IMCs-monocytic myeloid-derived suppressor cell (mMDSC) and Ly6CmidLy6G+ IMCs-PMN-MDSC frequencies gated on CD45+CD11b+ myeloid cells (n = 5). Relative expression of selected M1, M2 markers on flow-sorted M1 macrophages (D), M2 macrophages (E) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5). Relative expression of selected MDSC markers on flow-sorted mMDSCs (F), and PMN-MDSCs (G) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5). H-K. Representative flow analysis and quantification of CD8+ T cell activation markers: Granzyme B (H), IFN-γ (I), TNF-α (J), and PD-1 (K) (n = 5).
Figure 7A. Schematic diagram of the establishment of azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colorectal cancer model and drug therapy regimen. AOM/DSS-induced colorectal tumor mice were treated with vehicle, TP-16 (75 mg/kg, po, daily), PD-1 antibody (50 μg, ip, twice weekly), or TP-16 and anti-PD-1 combined. The endpoint was day 37 from treatment initiation. Representative resected colons (B) after 5 weeks of indicated treatment (n=7). total number of colon tumors per mouse (C, left) and tumor size distribution (C, right), and colon length (D) after 5 weeks of indicated treatment (n=7). E. Representative hematoxylin and eosin (H&E) staining and immunostaining for CD8, p-STAT3, p-AKT, and ARG-1 of tumors treated with vehicle, TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined. Scale bars, 500 μm (left images), 50 μm (right images). F. Quantitative polymerase chain reaction (q-PCR) analysis of the mRNA level of genes associated with immunosuppression (n = 6).
Figure 6(A) Representative hematoxylin and eosin (H&E) images of inflamed joint footpad at 6 dpc. Ed, region of edema; red arrow, synovitis; black arrow, normal synovial membrane; blue arrow, mild synovial hyperplasia; D, degeneration of muscle; N, necrosis of muscle; R, regeneration of muscle. (B) Histopathological scoring of edema, inflammation in different regions of the joint footpad and muscle pathology of WT-CHIKV challenged animals (n=5) at 6 dpc, performed blind. Three sections from each joint footpad were scored. The data are expressed as the means ± SD. Data were analyzed by one-way ANOVA with Tukey post-test; Edema (***P =0.000002 WT-CHIKV versus PBS, ***P =0.000004 RH-CHIKV versus PBS), Muscle necrosis (**P =0.005521 WT-CHIKV versus PBS, *P =0.02628 RH-CHIKV versus PBS), Muscle regeneration (***P <0.000001 WT-CHIKV versus PBS, ***P <0.000001 WT-CHIKV versus RH-CHIKV), Muscle inflammation (*P =0.040258 WT-CHIKV versus PBS, *P =0.040258 RH-CHIKV versus PBS), Synovial membrane inflammation (*P =0.014457 WT-CHIKV versus PBS, *P =0.014457 RH-CHIKV versus PBS), Subcutaneous area inflammation (***P =0.00004 WT-CHIKV versus PBS, ***P =0.000074 RH-CHIKV versus PBS).
Sir4 H‑BRCT structural comparison with Dbf4 H‑BRCT and Rad53 FHA1 binding A) Structure-based sequence alignment between H‑BRCT domains of Sir4 (top) and Dbf4 (PDB 3QBZ, bottom).Figure 2Structure-based sequence alignment between H‑BRCT domains of Sir4 (top) and Dbf4 (PDB 3QBZ, bottom). Secondary structural elements present in the Sir4 H‑BRCT crystal structure are displayed above the Sir4 sequence (α, α-helix; β, β-strand; η, 3.10 helix). Dbf4 residues strongly contributing to Rad53 FHA1 binding are highlighted with black (interface I) and blue (interface II) asterisks. The elongated loop connecting helix α3 and strand β4 in Sir4 H‑BRCT is labelled with an orange bar and the Tyr198 (Dbf4) to Lys1064 (Sir4) exchange is highlighted with colored arrows. Superposition of Sir4 H‑BRCT (orange) onto the Dbf4-Rad53-CDC7 complex (cyan, grey, blue) (PDB 5T2S) in two orientations rotated by ~180° around a vertical axis. Helices are displayed as barrels and protein-protein interfaces between Dbf4 and FHA1 of Rad53 are highlighted with dashed circles in black (interface I) and blue (interface II).
Figure 8The amount of CAPZB in mature sperm from WT (+/+) and KO (-/-) mice. Graph shows densitometric analysis of CAPZB shown in immunoblot analyses (Figure 7F), expressed relative to WT as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of α-Tubulin. The amount of CAPZB in mock and Spatc1lΔC-transfected cells. Anti-GAPDH antibody was used as control. Overexpression of SPATC1L in TM4 cells was confirmed by immunoblotting with anti-SPATC1L antibody. Graph shows densitometric analysis of CAPZB in pcDNA3.1 vector-transfected control cells (mock; white bar) and SPATC1LΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of GAPDH. Phosphorylation of CPAZB3 by cAMP-dependent protein kinase (PKA). CAPZB3 was incubated with PKA and 32P, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower). Phosphorylation of CPAZB3 in the presence or absence of SPATC1L. Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in the presence or absence of SPATC1L, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower). Graph shows densitometric analysis of phopho-CAPZB3 (p-CAPZB3) in reactions without (control; white bar) or with SPATC1L (black bar), expressed relative to control as a percentage. Observation of filamentous actin (F-actin) in the presence (lower) or absence (upper) of SPATC1L. F-actin in pEGFP vector-transfected control cells (mock; upper, green) and cells overexpressing SPATC1LΔC (lower, green) was visualized by staining using Phalloidin (red). White bar shows the length of transfected cell. Scale bar = 50 μm. The length of cell overexpressing SPATC1LΔC compared with control. Graph shows the average of measured cell length in pEGFP vector-transfected control cells (mock; white bar) and Spatc1lΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage. Proposed model for the function of SPATC1L in maintaining the stability of the sperm head-tail junction. SPATC1L is localized to the centrosome in a round spermatid, but separated from the centrosome in an elongating seprmatid. Finally, SPATC1L is localized to the neck region close to the centriole in testicular sperm. SPATC1L acts through interactions with RIα to increase PKA activity, and thereby increases the phosphorylation of CAPZB and stability of F-actin responsible for the integrity of the head-tail junction.
Figure 1A. Schematic representation of mapping chemoproteomic-detected amino acids (CpDAAs) to pathogenicity scores. B. Timeline of gene annotation database release dates and project-specific datasets, including Ensembl releases tested for compatibility to CpDAA coordinates based on canonical UniProtKB protein sequences and the database reference corresponding to the genomic pathogenicity scores C. Average database release cycle length for releases between August 2013 - July 2019. All values are mean ± SD. Total of 25 Ensembl, 13 GENCODE, 6 CCDS (homo sapien only), and 5 NCBI releases were counted. UniProtKB value was calculated by taking the average of release cycle lengths reported on the UniProt website.
FA transferred from adipocytes to melanoma cells by EV are released from lipid droplets by lipophagy A) Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV).Figure 4Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm). 1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization. 1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4). 1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group). 1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5). 1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).
Figure 2a,b An moi of 0.001 was used to infect VeroE6 with rescued or clinical SARS-CoV-2 (a,b). c,d An moi of 0.01 was used to infect FeA cells with rescued or clinical FeCoV (c,d).
Figure 3Diagram showed the full-length LOC113230 and the four ΔATG mutants (Δ1, Δ2, Δ3 and Δ1+2+3). Bottom: agarose gel image showed the in vitro-transcribes of ΔATG mutants (Δ1, Δ2, Δ3 and Δ1+2+3) were in correct sizes. ΔATG mutants (Δ1, Δ2, Δ3 and Δ1+2+3) showed no apparent effect on the cell migration. Scale bar represents 100 μM. Diagram showed the full-length LOC113230 and its truncations. Bottom: agarose gel showed that the in vitro transcribed full-length LOC113230 and the truncations were in correct sizes. Stable expression of LOC113230 300-800 resulted in decreased cell migration. Cell migration was detected by transwell assays. Scale bar represents 100 μM. Diagrams showed LOC113230 300-800 and its antisense strand. Bottom: agarose gel image showed the in vitro-transcribed LOC113230 and the antisense strand were in correct sizes. I)Stable expression of LOC113230 300-800 inhibited cell migration, while LOC113230 antisense showed no apparent effect on cell migration. Scale bar represents 100 μM.