EMBO/sd-character-level-ner huggingface dataset:
Figure 4A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates. A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of four independent experiments. >200 bacteria counted per coverslip. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. C) TBK1-Nap1-NDP52 complex. N- and C-termini, domains and binding partners are indicated. A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.
Figure 1(A) Study design for testing the effects of CMA in NAFLD patients. Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.
Inverse correlation between acetyl‐ and SUMO2‐FXR levels during the development of diet‐induced obesity A Experimental outline.Figure 4A Experimental outline. B-D Effects of feeding a HFD as a function of time on mRNA levels of the indicated genes (B), in mouse liver (pooled from 3 mice). Values in (B) are presented as mean ± SEM; two‐tailed Student's t‐test, *P 0.05, **P 0.005, n = 3. B-D Effects of feeding a HFD as a function of time on levels of SUMO2‐modified FXR (C) in mouse liver (pooled from 3 mice). B-D Effects of feeding a HFD as a function of time on levels of acetylated and SUMO2‐modified proteins (D) in mouse liver (pooled from 3 mice).
Figure 5A) Structural comparison of the CA-CCCT with CENPN-NT (left) and CA-CCpep with CENPN-NT (right). The cryo-EM density maps of the CENPC-CT fragment, CM peptide, and CENPN-NT are indicated as a surface representation. The cryo-EM density maps indicate that the structure of the CENP-A nucleosomes bound with two CENPC-CT fragments symmetrically (CA-CCCT complex) comprised approximately 94% of total analyzed particles. The RG loopCENPA is occupied by CENPC-CT in the CA-CCCT complex, and thereby the CENPN-NT could not access the same side of the nucleosome. The minor fraction (approximately 6% of total analyzed particles) comprised asymmetric CA-CCCT-CNNT complexes, in which CENP-C weakly associates with CENP-A nucleosomes through the CENPC motif. In the structures of the CA-CCpep-CNNT (right), CM peptide and CENPN-NT simultaneously bind to the CENP-A nucleosome. B) Side views of the CA-CCpep-CNNT and CA-CCCT complexes. The RG loopCENP-A binding site in each complex is indicated by a dotted circle. C) The left panel contains the structure of the RG loopCENP-A binding site in the CA-CCpep-CNNT complex. R81CENP-A of the RG loop is recognized by CENP-N. The right panel displays a superposition of the structure of CENPC-CT in the CA-CCCT complex on the CA-CCpep-CNNT complex shown in the left panel. The model of CM peptide in the CA-CCpep-CNNT complex has been removed for clarity. The CENPC motif region in CENPC-CT is shown in pink. Structural collision between the CM downstream region of CENPC-CT and CENPN-NT is shown at the RG loop of the CENP-A nucleosome.
H2AX staining is reduced both in stroma and epithelium of aggressive BC.Figure 4A Up, Representative views of H2AXimmunostaining from 117 BC patients of Luminal-A (LumA, N=33), HER2 (N=37) and Triple-Negative (TN, N=47) subtypes. Scale bars = 100 μm. Down, representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts (Left) or Epithelium (Right) cells. Scale bars = 25 μm.B Scatter plots of H2AX histological score (Hscore = staining intensity (0 to 4) x % of positive cells quantified from H2AXimmunostaining (as shown in A) in Fibroblasts (Left) or Epithelial (Right) cells from LumA, HER2 and TNbreasttumours.C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells (as shown in B), in HER2 (left) and TN (right) breasttumours. D Representative views of smooth muscle actin (SMA) immunostaining from BC patients (N=29) of Luminal-A (LumA, N=10), HER2 (N=10) and TN (N=9) subtypes. Scale bars = 100 μm. Zoom views are focused on the fibroblast compartment. Scale bars = 25 μm.E Scatter plots of SMA histological score (Hscore) evaluated from SMA immunostaining (as shown in D).F Correlation plot between SMA and H2AX Hscores in stromal compartment. G Scatter plot of the mitotic index, evaluated by pathologists, in LumA, HER2, and TN BC patients, as indicated. H Absence of correlation between mitotic index and epithelial H2AX Hscore, in LumA HER2 and TN BC.
Figure 4A. Mapping of enrichment across the positions targeted in dapF. The gray regions in the gene cartoon at the bottom highlights the windows containing targeted residues, with the enrichment map shown above for increasing AEC concentrations. Enrichments are color coded according the legend at the bottom. A histogram plot of enrichment is shown at the right B. Structure of the DapF dimer (PDB ID: 4IJZ) with the G210 and M260 sites highlighted in green. Diaminopimelate binding is shown in red. C. Zoom in the catalytic site highlighting the G210 and M260 sites relative to the catalytic cysteines. The G210D and M260Y substitutions are shown in the right panel D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samples E. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5 F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds
Melanoma cells show differential sensitivity to CDK7iA.Figure 1A. Protein lysates from the melanocytic-like melanoma cells 501mel, MM011, MM074 and MM117 or the mesenchymal-like melanoma cells MM029, MM047 and MM099 were immuno-blotted for proteins as indicated. Molecular mass of the proteins is indicated (kDa). B. Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. IC50 for each cell line is indicated. Melanocytic-type (MITF-High, proliferative) melanoma cells are shown in red, while mesenchymal-like (MITF-low, invasive) melanoma cells are shown in blue. Data information: data are presented as mean values + Standard Deviation (SD) for three replicates (n=3).
Figure 6Model for the potential roles of RNA in the assembly and maintenance of membrane-less organelles containing RNA and protein.
GEMC1 controls the MCC transcriptional program in vivo.Figure 5A. Gene ontology (GO) analysis of microarray data from the trachea and oviduct. GO scores (negative enrichment) of differentially expressed genes from the trachea and oviduct of Gemc1-/- mice (additional information in Dataset EV2). B. Geneset enrichment analysis of the microarray data using standard and custom genesets (additional information in Appendix Fig S4 and Appendix Table S1). Gene rank lists from the individual tissues were analyzed against genesets for gene ontology (GOSlim, top row), a custom geneset containing all known or suspected ciliopathy genes (second row), the Ciliome database (DB), the SYSCILIA consortium gold standard cilia gene list version 1 (SYSCILIA GS)(Roosing et al, 2015; van Dam et al, 2013), E24F target genes(Lee et al, 2011; Ma et al, 2014) and FoxJ1 target genes(Choksi et al, 2014a). Several additional genesets generated from analysis of ciliopathy patient data(Geremek et al, 2011; Li et al, 2015) or multiciliated cells(Hoh et al, 2012; Treutlein et al, 2014) were also queried (detailed information in Appendix Table S1). Negative enrichment scores are shown for genes downregulated in Gemc1-/- tissues. C. The most differentially expressed genes related to cilia in the trachea (Tr) and oviduct (Ov) of Gemc1-/- mice are plotted in a heatmap. Normalized, centered and scaled Affymetrix probeset intensities are plotted. D. Heatmap of differential expression of known human ciliopathy genes in the trachea and oviduct of Gemc1-/- mice compared to wild type littermates. E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).
TCR activation results in mitochondrial fragmentation and cristae remodelling in T cells.Figure 1A. AICD was induced in hPBT cells as described in Materials and methods. Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown. Data represent mean ± SE of six independent experiments. B. AICD was induced in Jurkat cells as described in Materials and methods. Apoptotic cells were detected as in (A). Data represent mean ± SE of five independent experiments. C. hPBT cells were transfected with mtYFP and after 24h AICD was induced. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 30' after AICD induction is shown. Scale bar, 5μm. D. Morphometric analysis of mitochondrial shape of cells treated as in (B). Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment). E. Jurkat cells were transfected with mtYFP, 24h after AICD induction cells were fixed. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.F. Morphometric analysis of mitochondrial shape in cells treated as in (E) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment). G. Representative electron micrographs of hPBT cells treated as indicated. 30min after AICD induction cells were fixed and processed for electron microscopy. Scale bar, 2μm.H. 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy. Representative electron micrographs of cells treated as indicated are shown. In EM images white arrowheads point to autophagic vesicles (late autophagosomes or most likely autophagolysosomes, Aφ); black arrows point to mitochondria (mito) with remodelled cristae, specifically touching a representative disorganized crista. Scale bar, 2μm. I. Representative confocal microscopy images of hPBT cells transfected with mtYFP, fixed 30' after AICD induction and immunostained with an anti-cytochrome c antibody (red).J. Cytochrome c localization index was calculated from 30 randomly selected cells treated as in (I). Data represent mean ± SE of three independent experiments.K. Representative confocal images of Jurkat cells transfected with mtYFP, fixed at 32h after AICD induction and immunostained with an anti-cytochrome c antibody.L. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of five independent experiments.
a–d) Levels of mitochondria-shaping proteins during starvation.figf2(a-d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times. (e) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments. (h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.
Figure 2A Whole mount immunofluorescence of a P8 Syne4-/- organ of Corti showing intact hair cells, as labelled with myosin VIIa. B Whole mount immunofluorescence of WT and Syne4-/- organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14. C Inner and outer hair cell counts of Syne4-/- organ of Corti at P8, P10, P12, and P14. D FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.
a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed.figf1(a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS-PAGE and visualized with autoradiography (top) or western blotting (WB; bottom). (b) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting. (c) HA-Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (-A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting. (d) HA-Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA-Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography , HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining. (e) HA-Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. In vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography, HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining. (f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions. (g) HA-Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (-Glu) or amino acids (-A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.
Figure 8(A) Scatter plots showing the effect of the epcr1/2 and arid2/3/4 mutations on CG, CHG, and CHH methylation at the co-upregulated TEs and genes in the epcr1/2 and arid2/3/4 mutants. TE methylation and gene DNA methylation refer, respectively, to methylation of the TE body and methylation of the 1-kb-gene-promoter region. (B) Box plots of CG, CHG, and CHH methylation in the wild type, arid2/3/4, and epcr1/2 mutants at the co-upregulated TEs and genes in the arid2/3/4 and epcr1/2 mutants. Asterisks indicate statistical significance as determined by paired student t test. CHG methylation of co-upregulated TEs is significantly increased in arid2/3/4 (P=1.2610-4) and epcr1/2 (P=6.310-9); CHH methylation of co-upregulated genes is significantly decreased in arid2/3/4 (P=3.510-6) and epcr1/2 (P=6.410-7). Horizontal lines represent the median, the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range. (C) Overexpression of DRD1 fails to restore the silencing of EPCR1/2 target loci in the epcr1/2 mutant background. (D) Model for the role of the PEAT complexes in chromatin regulation. In heterochromatin regions, the PEAT complexes either directly mediate heterochromatin condensation or interact with the histone deacetylases HDA6 and HDA9 and thereby mediate heterochromatin condensation by histone deacetylation. Thus, the PEAT complexes repress transcription mediated by both Pol II and Pol IV. The repression of Pol II transcription is required for transcriptional silencing in heterochromatin regions. Moreover, the repression of Pol II and Pol IV transcription prevents overproduction of siRNAs that are responsible for DNA methylation. In euchromatin regions, we predict that the PEAT complexes may regulate histone acetylation by interaction with the histone acetyltransferases HAM1 and HAM2. However, further studies are required to elucidate how the molecular mechanisms of the regulation.
Colloidal osmotic lysis and membrane permeabilization caused by the GBS pigment/lipid toxin.Figure 1The GBS pigment also known as granadaene is an ornithine rhamnopolyene (Rosa-Fraile et al, 2006). Human red blood cells (RBCs) were incubated with 400 nM pigment or control ΔcylE extract, and kinetics of K+ and Hb release was monitored. Data shown are the average and SEM of six independent experiments. The time to 50% K+ and 50% Hb release with pigment was 4.8 min and 8.4 min, respectively; n = 6, P < 0.0001, extra sum-of-squares F test. Role of osmoprotectants in pigment-treated RBC. HumanRBCs were pre-incubated with GBSpigment for 2 min at RT, centrifuged, and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0.40 nm (PEG200), 0.56 nm (PEG400), 0.89 nm (PEG1000), 1.1 nm (PEG1500), or 1.6 nm (PEG3000), respectively. Release of Hb was measured after 1 h of incubation at 37°C. Data shown are the average and SEM of three independent experiments. Characteristics of membrane permeabilization by the GBSpigment in artificial lipid bilayers. Lipid bilayers were generated using diphytanoylphosphatidylcholine (DPhPC) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract. In the pigment-treated sample, channel conductance indicating disruption of the membrane is seen within 45 s. Erratic and non-discrete fluctuations in current are observed, suggesting the formation of multiple, small membrane defects. The bilayer eventually breaks at 120 s. In lipid bilayers treated with the control ΔcylE extract, the mean ionic current trace remains constant at 0 pA, showing no membrane disruption. Data shown are representative of three independent experiments.
Figure 4Percentage (%) of γ-H2AX positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of γ-H2Ax positive cells were plotted as Mean % ± SD. Percentage (%) of hyper-RPA positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of hyper RPA positive cells were plotted as Mean % ± SD. Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618) Relative γ-H2AX intensity in cells transfected with either Empty Vector (EV) or HA-E2F1 treated with Chk1i for 20 hours. γ-H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA. HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.
Figure 5(A) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP, dCTP, dGTP, or dTTP. (B) Outline of the method used to isolate and sequence replicated leading strands from reconstituted replication reactions. (C) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in (B). Three individual experiments were performed, two in the presence and one in the absence of Pol δ (10 nM), giving 99, 97, and 95 successful sequencing reads respectively.