EMBO/sd-character-level-ner huggingface dataset:
Figure 6A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3). B) A model of the dynamic change of binding partners of the CENP-A nucleosome during the cell cycle progression in chicken (upper) or human (bottom). During the interphase, chicken CENP-C without phosphorylation at T651 is dissociated from the CENP-A nucleosome. Then, CENP-N can bind to the RG loopCENP-A of the CENP-A nucleosome in interphase cells. In mitosis, the CENP-C C-terminal region is phosphorylated by CDK1, and this phosphorylation facilitates CENP-A-CENP-C interaction. The stable CENP-A-CENP-C interaction excludes CENP-N from the RG loop. This phospho-regulation by the CENP-C C-terminal region is conserved in chicken and human. In humans, the second CENP-A binding region (central domain, CD) might associate with the CENP-A nucleosome throughout the cell cycle. However, it is still unclear how CD is involved in formation of centromeric chromatin structure (bottom).
Figure 6Expression of PID-2::eGFP(xfSi145) (A) in perinuclear granules in the germline. PGL-1::mTagRFP-T (A) mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm. Expression of , PID-4::mTagRFP-T (B) in perinuclear granules in the germline. DEPS-1::GFP (B mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm. Expression of PID-5::mTagRFP-T (C) in perinuclear granules in the germline. DEPS-1::GFP mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm. Expression of PID-4::mTagRFP-T (D) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm. Expression of PID-5::mTagRFP-T (E) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm. F) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. Expression of PID-2::eGFP (xfSi145) together with PID-4::mTagRFP-T (G) The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µ Expression of PID-2::eGFP (xfSi145) together with PID-5::mTagRFP-T (H). The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µm.
S100A9 expression inversely correlates with MoLC maturation and susceptibility to HIV-1 infection(A) MoLC from two different donors (donor #26 and #30) were left untreated (NT) or incubated with 1µg/ml of LPS for indicated time points before infection with HIV-GFP vectors for 48h.Figure 2MoLC from two different donors (donor #26 and #30) were left untreated (NT) or incubated with 1µg/ml of LPS for indicated time points before infection with HIV-GFP vectors for 48h. The percentage of GFP+ cells was analysed by flow cytometry (left dot-plots) and a graph representing the fold change of GFP+ cells, normalized to untreated infected MoLC is shown on the right. Non-treated (NT) and LPS-treated MoLC, as before, were analysed by flow cytometry for the expression of CD83 and HLA-DR. RT-qPCR and western blot analyses of S100A9 expression in MoLC from the two donors treated as above. RT-qPCR data were represented as means ± SD. MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).
a) THPN (yellow) can only be fitted in site C in structure I.figf3(a) THPN (yellow) can only be fitted in site C in structure I. Structure II has a different conformation, and site C in structure II is too narrow to accommodate THPN. (b) Site C of LBD is critical for Nix binding, as determined by the in vitro pull-down assay. The purified bead-bound GST or GST-Nix proteins were incubated with different mutants as indicated and THPN (20 μM) for 3 h; the proteins were then analyzed by western blotting. IB, immunoblotting. (c) Footprint on the surface of LBD upon which Nix binds. Left, binding of THPN to Site C forms a fitting surface for the interaction with Nix. Right, THPN binding also results in a conformational change in residues Arg563 and Ser553 as compared to the apo structure. (d) The S553A mutation creates a dent on the surface of LBD, as compared to the native structure in c. (e) DPDO cannot facilitate Nix binding to TR3. Top, the structure of DPDO. Middle, DPDO binding could not form the interacting surface for Nix, unlike THPN. The structures of Arg563 and Ser553 are the same as those in the native structure. Bottom, DPDO does not enhance the interaction between TR3 and Nix. Different plasmids were transfected into A375 cells as indicated, and cells were treated with DPDO (20 μM, 3 h). The TR3 interaction with Nix was determined by co-immunoprecipitation. Images of full blots are shown in Supplementary Figure 7. IP, immunoprecipitation; HA, hemagglutinin.
Figure 1(A) Purification of the Halo-tagged cohesin tetramer. Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo-tag (cohesinHalo488). (B) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test). (C) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit. (E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm. (F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).
Plasminogen activation induces VN cleavage and reduces cell adhesion in several human cancer cell lines.Figure 6A The SMB domain is released by a wide variety of cancer cell lines. The cancer cell lines of epithelial origin belonging to the NCI-60 panel (n = 38) were seeded on VN in the absence or presence of 30 nM Plg and 100 nM a2AP. The supernatants were harvested after 16 h and the level of SMB-containing VN-fragments was measured by immunoassay. Dots represent the concentration measured for the individual cell lines or the value obtained analysing control wells (no cells) from 10 independent experiments. Means ± SD are shown. The statistical significance was probed using paired Student's t-test (two-tailed). B PLAU mRNA expression levels correlate with the SMB release. The levels of SMB released in the presence of Plg and a2AP were used as a query pattern and correlated with the expression levels of about 26000 genes across the NCI-60 panel, using the CellMiner web tool [32]. The table shows the 10 most strongly correlated genes. The rank, gene name and Pearson correlation coefficient (r) are reported. C Plasminogen activation induces a reduction in cell adhesion of human cancer cell lines. The epithelial cancer cell lines of the NCI-panel were seeded on a VN-coated E-plates and allowed to adhere for 4 h before the treatment with 30 nM Plg and 100 nM a2AP or vehicle. The graph shows the average normalized cell index of the 38 cell lines. The time of the addition (4 h) and the time at which the reduction of cell adhesion has been quantified (20 h) are depicted by dashed vertical lines. D Correlation between gene expression and basal cell adhesion to VN and reduction in cell adhesion upon Plg treatment. Adhesion 4 h after seeding the cells on VN (expressed as cell index) was measured in the experiment described in panel C. The extent of cell adhesion reduction was calculated at the indicated time point in panel C as percentage of the cell index value measured in control wells treated with vehicle. Both parameters were correlated with the expression levels of the indicated genes. Gene expression data were downloaded from the CellMiner web tool. The table shows gene name, Pearson correlation coefficient (r) and p-value. E The negative regulation of cell adhesion correlates with the amount of released SMB. Scatter plot showing the correlation between the reduction in cell adhesion (panel C and Appendix Fig S7C) and SMB release (panel A and Appendix Fig S7B). The extent of cell adhesion reduction was calculated at the indicated time point in panel C as percentage of the cell index value measured in control wells treated with vehicle. Samples size (n), Pearson correlation coefficient (r) and p-value are reported.
Figure 5A Salivary glands dissected from 14 hours after puparium formation (top left) control (fkh-GAL4/+; pmCherry-Atg8a/+), and those with salivary gland-specific knockdown of (middle left) sec5 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec5IR/+), (bottom left) sec15 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec15IR/+), (top right) sec3 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec3IR), (middle right) sec8 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec8IR), (bottom right) exo84 (fkh-GAL4/w; pmCherry-Atg8a/UAS-exo84IR) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test (** p< 0.001, *** p< 0.0001).
Figure 4A Heatmap of RNA Pol II levels around TSSs of MYC promoter-target genes in BT168FO cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in tags/50bp. B Tracks of Pol II binding signals (ChIP-seq) at NCL, miR-17-92, OLIG2, HDAC1, and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h. The y-axis shows Pol II binding signals as tags/500bp/million reads. The x-axis displays genomic positions; introns as lines, exons as boxes. Arrowheads denote direction of transcription. Blue and green bars denote the presence of MYC or Omomyc peaks. Grey boxes are TSS regions. C Transcript levels (FPKM from RNA-seq data) in BT168FO cells of the genes shown in B. Data are means of 3 independent biological replicates. D Ratio between fold enrichments (specific antibody / IgG signal) derived from qChIPs of Pol II-Ser2p and Pol II-Ser5p (n = 3; mean ± SD). E RNA Polymerase II traveling ratio distribution from the Pol II ChIP-seqs, in the presence or absence of Dox.
Figure 6Autoinhibited state whereby the Ubl domain masks the E2 binding site on the RING1 (R1) domain as described by Chaugule et al (2011) and supported through crystallographic studies. Optimization step controlled by PINK1 phosphorylation of ubiquitin (Ub) and subsequent binding of pUb to the RING1/IBR interface resulting reorganization of the RING0/RING1 interface and movement of the IBR domain. Steps (A) and (B) have been previously described. PINK1 phosphorylation of the Ubl domain causes its dissociation from the RING1 binding site allowing it to sample a large conformational space in solution including weak binding to the RING0 domain favouring an uncovering of the catalytic cysteine C431. E2~Ub binding to the RING1/IBR domains making use of a cryptic ubiquitin binding site uncovered through dissociation of the pUbl domain. Re-modelling of the RING0/RING1/RING2(Rcat) interface with the tether region occurs based on NMR chemical shift data and HDX experiments. Fully activated state of parkin utilizes synergistic binding of pUbl domain to the RING0 domain observed in crystal structures (Gladkova et al, 2018; Sauvé et al, 2018) and E2~Ub binding that re-models the RING0/RING1/RING2(Rcat) interface to maximize accessibility to the catalytic cysteine.
Figure 3(A) PLX4032 IC50 (µM) of sensitive A375 and SKMEL5 and resistant (A375-R, SKMEL5-R) cell lines, as well as of GOKA and ESPcells, two BRAFi-resistant patient-derived short-term cultures, as determined by ATP assay (n=3, mean ± SD). For ESP, IC50 was >8µM. (B) Western blot analyses of ZEB1, TWIST1 and FRA1 in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. GAPDH was used as a loading control. (C) Quantitative PCR analyses of ZEB1and MITF in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. mRNA expression levels are represented as arbitrary units (a.u). Statistical difference relative to sensitive A375 cells is shown (n=3, mean ± SD, Student's t-test). (D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.
USP17 is necessary for EGF-mediated peripheral lysosome traffickingA HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2.Figure 2A HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.
Figure 6mRNA expression analysis of lipoprotein receptors in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments. Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control). glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control). Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control). Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control). Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control). Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm.
Figure 8A The percentages of WT or Arhgap45−/− CD45.2+ cells (Key: left side) among CD45.2 + WT CD45.1 cells found in the BM of control and Arhgap45−/− competitive BM chimeras are shown. B The percentages of WT or Arhgap45−/− CD45.2+ cells among CD45.2 + WT CD45.1 cells found in the blood of control and Arhgap45−/− competitive BM are shown. C The percentages of WT or Arhgap45∆T/∆T CD45.2+ cells among CD45.2 + WT CD45.1 cells found in the BM of control and Arhgap45∆T/∆T competitive BM are shown. D The percentages of WT or Arhgap45∆T/∆T CD45.2+ cells among CD45.2 + WT CD45.1 cells found in the blood of control and Arhgap45∆T/∆T competitive BM are shown. E The percentages of WT or Arhgap45−/− CD45.2+ cells among CD45.2 + WT CD45.1 cells found in the thymus of control and Arhgap45−/− competitive BM chimeras are shown. F The percentages of WT or Arhgap45−/− CD45.2+ cells among the CD45.2 + WT CD45.1 cells found in mesenteric LNs (mLN) and spleen of control and Arhgap45−/− competitive BM chimeras are shown. G The percentages of WT or Arhgap45∆T/∆T CD45.2+ cells among CD45.2 + WT CD45.1 cells found in thymus of control and Arhgap45∆T/∆T competitive BM are shown. H The percentages of WT or Arhgap45∆T/∆T CD45.2+ cells among CD45.2 + WT CD45.1 cells found in mesenteric LNs (mLN) and spleen of control and Arhgap45∆T/∆T competitive BM are shown. Data information: Each dot represents an individual mouse and results in (A) to (H) are representative of two experiments with a total of 5-10 mice per group. Consistent with previous reports (Jafri et al, 2017), competitively transplanted mice exhibit a bias in favor of CD45.2 reconstitution. *P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001; unpaired Student's t test. Mean and SD are also shown.
Non-apical mitosis is a feature associated with the primitive streak.Figure 6(a) Representation of the position of the AVE-opposed area (green) and the AVE-adjacent area (pink) in WT (left) and RhoAVE-deleted (right) embryos. (b, c) 3D reconstruction (b) and confocal Z-slice (c) of E6.25 WT (left) and RhoAVE-deleted (right) embryos stained for an AVE marker (Cerberus 1, yellow), F-actin (Phalloidin, grey) and mitosis (Phh3, magenta). Scale bar: 25 μm. Blue dotted line delimitates the pro-amniotic cavity (apical side of the epiblast). Red arrowheads point to non-apical mitosis. White lines delimitates the boundary between extraembryonic (up) and embryonic (down) regions of the embryo. (d) Percentage of AVE migration in WT and RhoAVE-deleted mutants. Data information: Values are shown as Mean ± SEM. WT: n=31 embryos; RhoAVE-deleted: n=17 embryos. (e) Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalised by the volume in μm3) in percentage, in AVE-adjacent and AVE-opposed regions from WT and RhoAVE-deleted embryos.
Figure 2(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting. (B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting C) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay (D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test). E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).