EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Total RNAs or proteins from tail-tip of wild-type and JFKTG mice were extracted and analyzed for Jfk expression by qPCR or western blotting with the indicated antibodies, respectively. Error bars represent mean ± SEM (n = 6, ***p < 0.001, paired two-tailed Student's t-test). (B) Measurement of the body weight of wild-type and JFKTG mice under ND in the indicated time of age. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (C) Representative images of wild-type and JFKTG mice fed on ND. Arrows indicate abdominal SAT under MRI analysis. EWAT and PRAT were dissected, and the sections from EWAT were stained with H&E. Scale bar, 100 μm. (D) 17-week-old wild-type or JFKTG mice fed on ND were fasted for 6-12 h prior to intraperitoneal injection of glucose or insulin. The blood was collected from the tail vein at different time points for plasma glucose measurement. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (E) The body weight of male wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (F) Representative images of wild-type or JFKTG mice fed on HFD. Arrows indicate abdominal SAT under MRI analysis. EWAT and PRAT were dissected and the sections from EWAT were stained with H&E. Scale bar, 100 μm. (G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.
Figure 7A. Double fluorescence in situ hybridization images indicating co-labeling of Slc4a4 (green) and GLAST (red). Scale bar: 100 μm. B. Schematics showing induction of temporally-controlled astrocyte-specific conditional null alleles. C. In situ hybridization confirms deletion of Slc4a4 in the cortex. GFAP immunostaining intensity was decreased significantly in Slc4a4 icKO at P12 whereas Sox9+ cell number remained unchanged. Scale bar: 100 μm. D. Co-immunoprecipitation revealing that Slc4a4 associates with Daam2 in vivo. E. Schematics showing induction of temporally-controlled astrocyte-specific conditional null alleles. F-J. Representative images of GFAP immunostaining (F) and quantification (G-J) in controls, Daam2 icKO, Slc4a4 icKO, and double icKO 6 weeks after tamoxifen injection. Scale bar: 20 μm. Data is presented as mean ± SEM. n=10-24 images, N=3-4 mice per genotype. Scale bar: 20 μm. Two-way ANOVA is used for statistics. [*] and [#] indicates comparison with control and double icKO, respectively. **p<0.01, p***<0.001, ##p<0.01.
Figure 2Numbers of DEGs from INK128 (200 nM) treatment or Rptor KO mESCs in 2iL. FC > 1.5, FPKM mean > 1, and P < 0.05. Hierarchical RNA-seq clustering of DEGs as in (A). RNA-seq heatmaps of the indicated pluripotency gene sets in control and INK128 (200 nM) treated mESCs in 2iL.   RNA-seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs in 2iL.   GO analysis of RNA-seq data of INK128-induced paused state mESCs as in (A). Cell cycle analysis of INK128 (200 nM) treated mESCs in 2iL (mean values ± SD). n = 3 biological replicates.
Figure 2(A). Overview of the experimental design. Rif1+/+ and Rif1F/F mESCs were grown for two days in medium supplemented with 4-Hydroxytamoxifen (OHT) to induce the translocation into the nucleus of the Cre-recombinase, leading to Rif1 deletion in the Rif1F/F cells (Rif1-/-). The embryoid body (EB) differentiation protocol was then started to trigger XCI. OHT was kept in the medium during the first 24 hours of EB differentiation. Cells were differentiated up to 4 (RNA analysis) or 7 days (H3K27me3 IF). (B). Representative western blot to monitor RIF1 levels after Cre-mediated Rif1 deletion and EB differentiation. SMC1: loading control. (C). Time course analysis of Xist RNA expression by RT-qPCR during EB differentiation of Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) cells at the indicated timepoints. Rif1+/+ (solid line) and Rif1-/- (dashed line), female (black) and male (grey). Data are presented as mean ± standard deviation from three (female lines) or two (male lines) independent experiments. Statistical significance was determined using 2-way ANOVA comparing female Rif1+/+ to female Rif1-/- cell lines (****p ≤ 0.0001). Xist RT-primers Xist ex7 F and R were used. Values are normalised to a geometric mean consisting of the expression of Gapdh, Ubiquitin and β-Actin. (D). Bar plot summarising the number of cells showing H3K27me3-marked Xi as a percentage of total cells counted, in Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) female mESCs at the indicated days of EB differentiation. Averages ±standard deviation from three (day 4 and 7) and two (day 2) independent experiments (n>200).
Some residues that differ between Siz2 and Siz1 contribute to SUMO conjugation of RPA.Figure 7Fluorescence polarization assays performed with 50 nM 6-FAM-ds20 and serially diluted Siz2∆CT or Siz2∆CT mutant. Histograms derived from the curves presented in (A) and presenting apparent dissociation constants (Kd) for each Siz2∆CT variant. Histograms derived from the gels presented in (D) and presenting Rfa1 SUMO conjugation rates for RPA complexes in the presence of different Siz2∆CT variants. SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.
Figure 4 A Heat map of DAVID analysis showing enriched annotation clusters (e.g., gene ontology and pathways) in the genes showing differential expression between the indicated mutant and wild type. Each annotation cluster is represented by a DAVID annotation cluster score (the higher the number, the stronger the enrichment). DAVID analysis of genes whose expression is lower in a mutant relative to wild type are highlighted with green text (four left hand columns), whereas DAVID analysis of genes whose expression is higher in a mutant relative to wild type are highlighted with red text (four right hand columns). Rows are labeled with each annotation cluster, with Z-scores (color scale at graph bottom) representing significant deviation in the annotation cluster score between genotypes across the row for each annotation cluster. Yellow cells indicate higher-than-average enrichment of that annotation cluster (row) for that genotypic comparison (column). Annotation clusters (rows) are further clustered along the Y axis according to the Pearson average linkage method  B Heat map of log fold change (logFC, color scale at graph bottom) in the expression of C. elegans CYP genes between the indicated mutant and wild type. Genes whose expression is lower in a mutant relative to wild type are colored blue, whereas genes whose expression is higher in a mutant relative to wild type are colored yellow. Rows are labeled for the different CYP families and subfamilies. Specific genes (rows) are clustered according to sequence alignment (CLUSTAL W) to maintain the relationship of their molecular evolution  C Schematic representation of the synthesis pathway for mono-unsaturated fatty acids (MUFAs), poly-unsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), and their derived eicosanoids in C. elegans. The gene encoding each biosynthesis enzyme in the pathway is indicated next to the arrow representing its catalyzed reaction  D Abundance of the indicated free ω6 PUFA-derived eicosanoid, normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. E Abundance of the indicated free ω3 PUFA-derived eicosanoid, as in (D), normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM
USP28 regulates ∆Np63 protein stability in SCC tumour cell lines Immunoblot of endogenous USP25, USP28 and ∆Np63 immunoprecipitated (IP) from A-431 cells and co-precipitated ∆Np63 or USP28.Figure 3Immunoblot of endogenous USP25, USP28 and ∆Np63 immunoprecipitated (IP) from A-431 cells and co-precipitated ∆Np63 or USP28. Beads were coupled to specific USP25, USP28 and ∆Np63 antibodies or non-specific rabbit IgG as control. The input corresponds to 10% of the total protein amount used for the IP (TUBULIN as loading control). Inducible depletion of USP28 in A-431 upon treatment with doxycycline (1µg/ml) for 96h, western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) was performed. Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization. Cycloheximide (CHX) chase assay (100µg/ml) of control or inducible sh-USP28 A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right). Doxycycline induced murine USP28 overexpression (EtOH or 1ug/ml dox for 96 hours) in A-431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping). TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon overexpression of mUSP28 for 96 hours (EtOH or 1µg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization. CHX chase assay (100µg/ml) of control or inducible mUSP28 overexpressing A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well quantification of relative protein abundance (VINCULIN as loading control). Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63 and USP28 protein abundance in LUDLU-1adh (VINCULIN as loading control), followed by qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN. Doxycycline induced mUSP28 overexpression (EtOH or 1µg/ml dox for 96 hours) in LUDLU-1adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).
Ubiquitination of insoluble protein aggregates in the atg7 and nbr1 mutants under heat stress.Figure 13Leaf tissues from wild type (WT), atg7 and nbr1 mutants were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.
a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose.figf4(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose. (b) Effect of methylpyruvate (MetPyr) on ATP levels in WT p53−/− HCT116 cells depleted of glucose. ATP levels were measured as in a, in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr. (c) Effect of autophagy on the ATP levels of WT and p53−/− HCT116 cells depleted of glucose. Cells were transfected with siRNAs specific for Beclin 1 and AMPKα, or they were treated with rapamycin for 6 h, followed by measurement of ATP levels as in a, before or after glucose withdrawal (means ± s.d. of triplicates, 3 independent experiments). (d) Metabolic stress-induced cell death is attenuated in the absence of p53. HCT116 cells were transfected with control, Atg5-, Atg10-, or AMPKα-specific siRNAs and were subjected 48 h later to metabolic stress (cultured for 48 h in nutrient-free, hypoxic conditions) and stained with DiOC6(3) and PI. The black and white portions of the columns refer to the DiOC6(3)low PI− (dying) and DiOC6(3)low PI+(dead) population, respectively. (e) Metabolic stress-induced decrease of clonogenic survival was attenuated in the absence of p53. HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival. (f) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion. MCF7 cells that carry a tetracycline-repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it, transfected with a control siRNA or a p53-specific siRNA, subjected to metabolic stress, and finally stained with DiOC6(3)/PI. (g) Effect of p53 and autophagy on the survival of metabolically stressed MEFs. WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays. Results in d-g are mean ± s.d. of 3 separate experiments (*P 0.05); Co, control.
Figure 3A. Quantification of in vitro matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001. B. Quantification of in vitro matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENA∆v6 expressing vectors, demonstrating that the overexpression of hMENA∆v6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.
VP1390 is a periplasm-targeting toxin that leads to cell lysis.Figure 4A Toxicity of periplasm-targeted proteins in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide (perisfGFP, periVP1388, and periVP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated. B VP1389 interacts with VP1390. Co-immunoprecipitation of FLAG-tagged VP1390 or BC3020 using Myc-tagged VP1389 when co-expressed in V. parahaemolyticus Δvp1389 (input). Precipitated proteins (output) were detected by immunobotting using α-Myc and α-FLAG antibodies. Source data are available online for this figure. C VP1390 induces cell lysis in E. coli. Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 (perisfGFP, periVP1388, and periVP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 µm.
Figure 4A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test. B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test. C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells.E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+.G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test.
β-catenin regulates the expression of HR and FA pathway genes β-catenin regulates the expression of HR and FA pathway genes in HPAF-II cells.Figure 4 β-catenin regulates the expression of HR and FA pathway genes in HPAF-II cells. HPAF-II cells were transfected with siRNA against β-catenin for 48 hours. The relative expression of genes as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates  Stabilized β-catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition. Mice bearing HPAF-II xenografts without or with stabilized β-catenin were treated with ETC-159 (37.5mg/kg bid) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT-PCR. ETC-159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β-catenin. The horizontal lines represent mean of replicates  Stabilized β-catenin reduces the sensitivity of HPAF-II cells to olaparib and ETC-159. HPAF-II cells without or with stabilized β-catenin were plated in soft agar in 48 well plates. The cells were treated with a combination of olaparib and ETC-159 as in Figure 1A and the total number of colonies were scored after 2 weeks. Data shown are average colony counts ± SD from three replicates  Wnt inhibition reduces the expression of HR and FA pathway genes in APC-mutant COLO320HSR cells. COLO320HSR cells were seeded in 6 well plates, treated with control siRNA, siβ-catenin, DMSO or G007-LK for 72 hours and then the expression of the indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates  E, F. G007-LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation. COLO320HSR cells were plated at a low density and treated with G007-LK, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. (E) Representative image from two independent experiments is shown. (F) The Combination Index (CI) values of olaparib and G007-LK calculated from two independent experiments using the Chou-Talalay CompuSyn software
Figure 1A, B. Comparisons of RNAseq and ChIPseq data. Wt strain (A) and ∆rnjA (B). The x-axis shows the relative RNAP occupancy at a given gene (normalized ChIPseq coverage). The y-axis shows the relative abundance of the transcript (normalized RNAseq coverage; each dot is one gene). The genes are color-coded, ranging from yellow (low RNA abundance) to red (high RNA abundance) in wt. The ∆rnjA strain has higher RNAP occupancy mainly among the genes with less abundant transcripts. The color coding in (B) reveals no dramatic overall changes in the vertical direction (RNA abundance). If anything, some of the low abundance transcripts decreased further in level in the mutant strain. The main difference (mostly among the low abundance transcripts) is their shift in the horizontal direction to the right (towards higher occupancy with RNAP). Data represent mean values of three independent experiments. C. Relative expression of all sigma factors in wt (LK1371) versus ΔrnjA (LK1381). Wt levels of each sigma factor were set as 1 (indicated with the horizontal line). D. Sigma-dependent genes and correlation with expression of sigma factors (significantly changed) in ΔrnjA (LK1381). The most down-regulated sigma factor was sigD and almost all sigD-dependent genes were downregulated. A similar trend is visible for the rest of sigma factors and their respective dependent genes. The x-axis shows expression of each sigma factor, the y-axis shows expression of genes for each sigma regulon. The violet (dark blue) line: regression line.
Figure 4Sashimi plots indicating combined exon usage of NUP58 mRNA in HAP1 WT and mutant clones across four biological replicates. Numbers of reads per exon-exon junction are indicated below introns. WT canonical isoform is colored in black; mutant isoforms in grey. NUP58 gene structure is reported over the sashimi plot; red asterisks and exons highlight the positions of the identified mutations. Western blot for NUP58 protein expression in mutant clones and control cells. Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Blots were loaded with wild-type HAP1 cell line (WT), empty vector (EV), non-targeting siRNA (siNT) and NUP58 targeting siRNA (siNUP58), followed by NUP58 mutant clones (C1 / C2 / C3 / C4 in left panels, C5 / C6 in right panels). Actin was used as loading control. Asterisks indicate possible NUP58 alternative isoforms.
Translation and topogenesis of LepB.Figure 2Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2. Fitting of time courses (filled circles) of the translation of LepB mRNA constructs of increasing length (Appendix Fig S3) by delay-exponential functions (red lines). Kinetic model used for fitting of both translation and FRET time courses. Ribosome pausing at intermediates P1 and P2 is modeled as off-pathway states. Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting Intrinsic fluorescence intensities (IFI) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting (red squares). Confidence contours (shaded areas) were calculated using the Fitspace algorithm