EMBO/sd-character-level-ner huggingface dataset:
Figure 6Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 (10μM) or Bortezomib (100nM) Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Alternatively, ubiquitination was blocked for 4 hrs using TAK243 (10μM) (E,F) Representative immunofluorescence images (E) and quantification (F) of PML NBs (green) and SUMO 2/3 (red) in J-Lat 9.2 cells left latent or reactivated for 24 hours with 10μM TPA. Cells were imaged by STED microscopy, scale bar 2 μm. (G,H) Representative immunofluorescence images (G) and quantification (H) of PML NBs (green, maximal projection) and SUMO 2/3 (red, maximal projection) during mock and productive HIV-1 infection of primary CD4+ T-cells at 7dpi. Scale bar 10μm. I) Schematic representation of the interplay between metabolic imbalances induced by HIV-1 replication and PML stability. Upon HIV-1 infection of activated CD4+ T-cells, ROS content is increased by HIV-1 replication and potentially by iron through the Fenton reaction, leading to oxidative stress. Oxidative stress is accompanied by PML NBs SUMOylation and PML protein degradation via the ubiquitin-proteasome axis. Upregulation of Nrf2-mediated antioxidant responses in infected cells facilitates cell survival and reversal to a resting state where ROS content is decreased and PML reformed, while latency reactivation reignites oxidative stress and PML degradation.
Mithramycin does not lead to general transcription inhibition and instead favors pro-survival pathways.Figure 5A-C: Volcano plot showing gene expression trends at 8h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q-value threshold. D-F: Volcano plot showing gene expression trends at 18h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q-value threshold. G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h. The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. J: fgsea analysis of gene ontology terms upregulated in primary AT/RT tumors as described in (Wang et al., 2017) following 8h of mithramycin exposure. Mithramycin downregulates the expression of these pathways. P-values derived from the fgsea analysis package. fgsea analysis of hallmark pathways downregulated after 8-hours of MMA exposure P-values derived from the fgsea analysis package. L: fgsea analysis of hallmark pathways upregulated after 18-hours MMA exposure P-values derived from the fgsea analysis package.
Figure 7A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin. B Confocal images of PHcrac (left) and PHcrac merged with LimE (right, PHcrac as red, LimE as green) in a giant cell after RacGEF1ΔN recruitment. C Kymograph of LimE (top, green) and PHcrac (bottom, red) across the dotted line in B. D Confocal image of RalGDS (left) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph (right). E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex. F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment. Red dashed lines in F indicate rapamycin addition at time 0. G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0. H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments). I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each). J Confocal images of GflB (top) and RapGAP1 (bottom) in waves at the basal surface of a giant cell. K Intensity plots of GflB (blue) and RapGAP1 (orange) across the green dotted arrow in J. L Scatter plots of first peak distances, of GflB relative to LimE, RapGAP1 relative to LimE, and RapGAP1 relative to GflB in the same giant cells (red lines indicate median, n=19 cells each). M Confocal images of GflB (top) and RapGAP1 (bottom) at the basal surface of the same giant cell after RacGEF1ΔN recruitment. N kymographs of GflB (top) and RapGAP1 (bottom) across the green dotted lines in m. All scale bars represent 20 μm. (o) Cartoon highlighting positive and negative feedback loops from CEN to STEN.
Figure 2A Structure of autoinhibited ScV1. The catalytic A (pink) and non catalytic B (green) subunits are arranged as a heterohexamer around the central rotor subunits D (red) and F (cyan). The three peripheral stalks (EG heterodimers 1-3, blue and orange, respectively) are bound at the periphery of the B subunits. The inhibitory H subunit (yellow) is unique to the eukaryotic V-ATPase. Unless otherwise stated, the coloring scheme used here for the V1 subunits will be used from here on. B Interaction of the peripheral stalks with the catalytic hexamer. Each peripheral stalk crosses a non-catalytic A/B interface on its way from the top of the V1 to the base of the catalytic core. Representative view of the interaction is shown for EG3. C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer. The peripheral stalks are bound to the top of the B subunits via their C-termini (boxed). The electron density (contoured at 1.2 σ) for the C-terminal domains of EG1 and EG2 is shown in the panels to the left and right.D The structure of autoinhibited ScV1rotated 90° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3. Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the "bulge" in subunit G (indicated with an arrow). The box shows electron density (contoured at 1.2 σ) for the EG3 N-termini contacting the C-terminal domain of the A subunit of a closed catalytic site (see asterisk). The horizontal lines on the structure indicate the sections shown in panels E (upper two lines) and F (lower two lines). E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks (EG1-3). View is rotated 90° from the view in A, looking up the central rotor. The A/B pairs forming the three catalytic sites are designated (AB) 1-3 according to which of the three peripheral stalks is bound to each B subunit. For example, (AB) 1 has peripheral stalk EG1 bound to its B subunit. The black arrowheads indicate the non-catalytic A/B interfaces. F Section through the ScV1 electron density map at the level of the N-termini of the peripheral stalks. Note the change in position of the N-termini of the peripheral stalks, which have each crossed a non-catalytic interface (black arrowheads in E) and are now in proximity to the C-termini of the catalytic A subunits (ACT) of the adjacent A/B pairs. While EG1 and EG2 are now near ACT from (AB) 3 and (AB) 1, respectively (see double headed arrows), only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from (AB) 2 (see asterisk).
| ChIP-Seq and RNA-Seq analysis reveals a complex VxrB regulon.Figure 2(A) Overview of VxrB-binding profiles across the V. cholerae genome after β-lactam exposure (PenG 100μg/ml [10x MIC] for 3 hours). Red peaks indicate VxrB binding sites above the significance cutoff (q-value<10-10; MACS). (B) RNA-Seq MA plot is presented as fold change between VxrBD78E overexpressed sample and control sample. Red dots indicate differentially expressed genes after VxrBD78E overexpression (P-value < 0.001). Representative VxrB target genes are marked on both chromosomes and on the MA plot. (C) Venn diagram depicting overlap between RNA-seq and ChIP-Seq datasets. (D) Regulatory modes of representative VxrB-controlled genes in response to PenG. Upper plot shows VxrB binding patterns (assessed via ChIP-Seq), lower panel shows transcriptional response upon overexpression of VxrB D78E (assessed via RNA-Seq). RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG (100 µg/ml for 3 hours). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.
Figure 5A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm. D. Quantification of wildtype (WT), double and misplaced PPBs. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. E. Quantification of PPBs with prominent perinuclear microtubules (MTs). Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett's multiple comparisons test (** P < 0.01 and **** P < 0.0001). F. Quantification of spindles with lagging chromosomes. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (** P < 0.01 and ns = non-significant).
A3A deaminates HBV cccDNA dependent on transcription but is not sufficient to induce its degradation.Figure 1dHepaRG cells were infected with HBV at a multiplicity of infection (MOI) of 100 virions/cell and transfected with siRNA against A3A (siA3A) or control siRNA (-) on day 8 post infection (p.i.). Cells were treated with 300 U/ml IFNα from day 9 to 15 p.i. and HBV cccDNA was determined thereafter by qPCR relative to the prion protein (Prnp) gene (n=4 biological replicates). dHepaRG-TR-X cells were infected with an HBx-deficient HBV (HBV∆x) at an MOI of 200 virions/cell. Cells were treated with tetracycline to induce expression of HBx (+HBx) and/or 1000 U/ml IFNα from day 10 to 17 p.i. HBeAg (B) was measured by ELISA (OD: optical density) and HBV cccDNA (C) by qPCR relative to Prnp (n=3 biological replicates). dHepaRG-TR-X cells were infected with an HBx-deficient HBV (HBV∆x) at an MOI of 200 virions/cell. cccDNA amplicons were analyzed by differential DNA denaturation PCR (3D-PCR) (D) to visualize deamination. dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Marked amplicons were sequenced and analyzed for nucleotide composition (F) in comparison to the wildtype HBV sequence (n=3-5 biological replicates). HBV infection level was determined by measuring cccDNA, intracellular HBV DNA by qPCR relative to Prnp or HBeAg by ELISA (G) (n=6 biological replicates from two independent experiments).
Figure 2A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas. B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2. C, Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) mass-spec analysis (spectral counts/total peptides identified) and (right) verification of associated protein by western blot. D, Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown. E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes. F, HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively. G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates. H, Complementation analysis of H3K14ac. Total cell extracts of siRNA treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by western blotting. One representative experiment out of four biological replicas is shown.
Fig.Figure 5  Fig. 5: Deletion of VE-cadherin results in fragmentation and distension of the intestinal lacteals. A-E Deletion of VE-cadherin was induced in mice at various ages (neonatal (C), 16 weeks (D) or 1 year (E)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages).Control mice of the same age were subjected to an identical regimen (A, B). Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins, scale bars = 100 μm. The data represent wholemount stainings from 3 (D), 5 (B,E) and 6 (A, C) animals analyzed for each genotype.
Figure 2A The recovery kinetics of heterochromatin in MEFs infected with Flag or Gadd45a on day 3. The ratio of MF at 120 s post-bleaching is shown at the right panel. More than 20 cells were analyzed for each group. (***p≤0.001) B The recovery kinetics of heterochromatin in MEFs infected with Flag alone or SKO plus Flag or Gadd45a on day 3. The ratio of MF of heterochromatin is shown at the right panel. More than 19 cells were analyzed for each group. (*p≤0.05) C ChIP-PCR analysis of H3K9Ac, H3K9me2, H3K9me3, H3K27Ac, H3K27me2, or H3K27me3 modification levels in Oct4 binding sites of MEFs infected with SKO plus Flag or Gadd45a on day 3. (**p≤0.01; n=3)
Fig.Figure 4Fig. 4 Higher-order assemblies identified by proteome-wide cross-linking mass spectrometry, biomolecular modeling and negative-stain electron microscopy. (A) Distance distributions of identified cross-links on top of the modeled protein complexes and identification of novel interactions. Satisfied distances are shown in blue and over-length cross-links are shown in red. (B) Negatively stained electron micrographs of fractions 3-7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions. Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted. (C) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative-staining electron micrographs; shown for fatty acid synthase, 20S proteasome, 60S and 40S ribosome (the number of particles per image per fraction is indicated below the class averages). (D) Simplification of lysate (collecting only the flow-through from anion exchange chromatography) prior to SEC separation allows class-averaging of structural signatures from complex fractions that were previously too low abundant. Proteins identified in this simplified fraction are found in Table S4.
Figure 7A Time-course analysis of [Ca2+]cyt dynamics between 10-day-old wild type (Col-0) and ost1-3 mutant after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment. C MST assays of the calcium-binding affinity of GST-AtANN1, GST-AtANN1S289A, and GST-AtANN1S289E. GST was used as a control. D A proposed working model. Under normal conditions, OST1 protein kinase interacts with PP2C, which inhibits the kinase activity of OST1. AtANN1 is localized in the cytosol and at the plasma membrane. Under cold stress, OST1 is activated and phosphorylates AtANN1, which, on the one hand, enhances the Ca2+ transport activity of AtANN1, and, on the other, promotes the Ca2+-binding activity of AtANN1. This dual role of phosphorylation results in increased cold stress-induced [Ca2+]cyt. Consequently, AtANN1 indirectly facilitates the expression of CBFs and CORs to positively regulate plant freezing tolerance.
The number of Venus-positive neurons in the lateral amygdala is larger in fear-conditioned KO mice A.Figure 6A. A schematic image of the basolateral complex of the amygdala. B. Numbers of Venus-positive neurons in the lateral nucleus (LA) and the lateral side of the basal nucleus (BAl) of Naive, Unpaired, and Paired groups. Open and filled bars represent AV-WT and AV-KO mice, respectively. Positive neurons of AV-WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA (upper panel). AV-KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV-KO mice were increased compared to the same groups of the AV-WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV-WT and AV-KO mice The error bars represent S.E.M. (n =5/group). C. Numbers of Venus-positive neurons classified by fluorescence intensities in the LA of the AV-WT and AV-KO mice. Weak positive neurons were increased in the Paired group compared to the Naïve group in AV-WT Both Weak and Middle positive neurons were increased in the Paired group compared to the Naïve group in AV-KO The increment of the Middle Venus-positive neurons was larger in the AV-KO compared to AV-WT
Figure 2A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2). B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively. C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen. D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen. E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control. F. Sre2 cleavage assay for indicated yeast strains was performed as in (D). G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.
Figure 3A, MCF10A Wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours, and cells were harvested for immunoblotting. B, MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 µg/ml) for 6 hours with lipofectamine 3000. Immunoblotting was performed with indicated antibodies. MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates. MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates. MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means p value < 0.001). Data are presented as mean + standard error of the mean of three biological replicates. MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.