EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 3(A) Fluidigm analysis of expression of genes involved in sphingolipid metabolism in human dermal fibroblasts (HDFs) upon indicated stimulations for 4 h. Heatmap shows means of 4 independent experiments. The color scale represents the relative gene expression compared to control from lower (blue) to higher levels (red). (B) Lipidomic profiling of sphingolipid metabolites in HDFs upon indicated stimulations for 2 h. Fold changes of each metabolite were calculated against corresponding controls. Heatmap shows means of 3 independent experiments. The color scale represents the relative levels of various lipids from lower (blue) to higher abundance (red). (C) ELISA analysis of cytosolic S1P production in HDFs upon indicated stimulation for 4 h. (D) qRT-PCR analysis of IL6 expression in HDFs upon indicated stimulations for 4 h in absence or presence of the inhibitor of sphingosine kinases-SKI II (SPHKi, 50 µM). ELISA analysis of IL6 in supernatants of HDFs (E) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases. , HeLa NOD1 cells (F) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases. HeLa NOD2 cells (G) upon indicated stimulations for 20 h in absence or presence of the inhibitor of sphingosine kinases. (H) qRT-PCR analysis of Cxcl2 expression in WT, Sphk1 KO or Sphk2 KO BMDMs upon indicated stimulations for 4 h. (I) Multiplex analysis of CXCL2 production in supernatants of WT, Sphk1 and Sphk2 KO BMDMs upon indicated stimulations for 20 h. (J) ELISA analysis of IL8 production upon Shigella infection. HeLa scrambled control cells and SPHK1/2 double KD cells were infected with Shigella flexneri M90T for 3 h and 6 h, or stimulated with TNFα for 6 h.
Figure 6A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times. B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag. C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis. D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed. , qRT-PCR (above) and Western blot (below) analyses for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. F, ChIP analysis of H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. I, A proposed working model for the p53-USP7-H2Bub1 axis in the regulation of SLC7A11 expression.
Figure 7(A) Prediction of ESM1 and β-catenin interaction using HADDOCK. (B) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM. (C) Cell extracts of 22Rv1-M transfected with either Scramble-shRNA or ESM1-shRNA were immunoprecipitated with an β-catenin antibody and blotted with anti-TCF4 and anti-BCL9 antibody. (D) Prediction of the ESM1, β-catenin, and TCF4 interaction using HADDOCK. The surface representation of ARM is colored red and blue for negative and positive charges, respectively, and grey color represents neutral residues. (E) Upper, Schematic diagram of ESM1-targeting short peptides. Lower, HEK293T cells were transfected with ESM1-HA constructs. Cell extracts were treated with the short peptides, pulled down with purified GST-ARM and blotted with anti-HA and anti-TCF4 antibody. (F) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM and blotted with an anti-TCF4 antibody. (G) ChIP-qPCR validation of β-catenin binding to the CCND1 and c-Myc promoter in 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Bars are the mean ± SD of three independent experiments. (H) Upper, Schematic diagram of ChIP-qPCR primers targeting regions upstream of the CCND1 and c-Myc transcription start sites (TSSs). Bar graphs show β-catenin and ESM1 binding at the CCND1 and c-Myc promoter regions. Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments. (I) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. (J) The effects of ESM1 point mutants on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.
Enhanced microglial activity in Grn-/- mice and in a patient with Grn haploinsufficiency.Figure 4A. TSPO-µPET in rodents: Axial slices (A1, A2, A3) of averaged %-TSPO-PET differences between Grn-/-or Trem2-/- mice and wt indicate increased microglial activity in the cerebrum of Grn-/- mice (hot color scale) and decreased microglial activity in the cerebrum of Trem2-/- mice (cold color scale). Scatter plot depicts single TSPO-µPET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=8 at 11 months; wt n=10 at 8-11 months). Statistics derive from one-way ANOVA with Tukey post-hoc test. B. PGRN serum levels of the patients subjected to TSPO-PET confirmed the GRN mutation and non-GRN mutation carrier. PGRN was measured by ELISA and normalized to serum levels of a healthy control. C. TSPO-PET in human FTLD patients: Visual interpretation of axial TSPO-PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices (white arrows) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation. PET results are illustrated upon an MRI template; 60-80 min p.i. emission recording; global mean scaling. Both patients were low-affinity binder.
Figure 1(A, B and C) Polyphenols TA , PGG and EGCG activate rhpR-lux in wild type but not rhpS deletion Psph strains. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates). (D, E and F) Polyphenols repress T3SS induction in P. syringae in MM. Transcripts of the indicated genes in the presence of 100-μM TA (D), 100-µM PGG (E) or 2-mM EGCG (F) were determined by RT-qPCR 6 hours after culturing in MM. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).
Figure 6A) Four-week-old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored. Data represents mean values ± SEM (n=20;***, p<0.001; paired t-test). Three biological experiments were done showing similar results. B) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p<0.01; paired t-test). Experiments were done at least twice with similar results. C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p<0.05; **, p<0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.
Figure 3Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.
Figure 2A-C Protein AD/non-AD fold changes plotted vs statistical significance for Sweden (A), Magdeburg/Kiel (B), and Berlin (C) cohort. Proteins associated with the GO annotation neuron projection labelled in orange. Proteins above the dashed green line are statistically significant (p < 0.05) and those above the black curves have a q-value below 0.05
Heat map representation of potential positive and negative immune modulators identified from the RNAi screens.Figure 3Heat map representation of potential positive and negative immune modulators identified from the RNAi screensDifferential scores were used to identify positive immune modulators (yellow) the knockdown of which enhance CTL-mediated cell killing and negative immune modulators (blue) the knockdown of which reduce CTL-mediated cell killing. Differential scores prior to filtering are shown for all genes tested in the 3 different screens (see Materials and Methods). Selected representative clusters of high-confidence hits are displayed herein.
Figure 4(A) A schematic diagram shows the design of the Aff4flox allele and the strategy for knocking out AFF4. The deletion of exons 10 and 11 ablates AFF4's entire Lilli-like segment. Representative skin phenotypes of wild-type (WT) mice shown macroscopically The phenotypes compared are as follows: hair coats, 4-month-old adults Representative skin phenotypes of Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.
A heterologous amphipathic helix can functionally substitute for the N‐terminus A Helical wheel projection showing the amphipathic helix from hepatitis C virus protein NS4B.Figure 3A Helical wheel projection showing the amphipathic helix from hepatitis C virus protein NS4B. The hydrophobic face is indicated by an arrow. Colour scheme as in Fig B. B Growth of strains DWA350 (Δnoc ΔminCD) and DWA307 (Δnoc ΔminCD, Pxyl‐HCVAH‐NocNΔ10) on nutrient agar plates at 30 and 48°C in the absence and presence of 0.5% w/v xylose, as indicated. C-F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5-95. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm. G, H Cellular localisation of HCVAH‐NocNΔ10‐YFP (G) in strain DWA193 (Δnoc, Pxyl‐HCVAH‐nocNΔ10‐yfp) and overlay showing DAPI‐stained DNA (H). The strain was grown at 30°C in CH medium. Inset shows the corresponding phase contrast light microscopy image. Scale bar, 5 μm.
Figure 3(A) Cartoon depicting the experimental design. A PIP5Kγ construct fused to FKBP12 is expressed together with an FRB module fused to Rab7 and the GFP-tagged PLCδ1PH reporter for PI(4,5) P2. Addition of rapamycin causes recruitment of PIP5Kγ to the endosomes where it makes more PI(4,5) P2 and attracts the PI(4,5) P2 reporter. (B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5) P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm. (C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5) P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates). (D) PI(4,5) P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates). (E) The response of PI4K2A K/O cells was significantly reduced Means ± S.E.M. are shown from three experiments performed in triplicates. (F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).
Figure 5(A) Structure of the yki2-only allele compared to wild type yki locus. Introns surrounding exon 3 are removed. This allele does not contain any exogenous sequence. (B) Western blot of adult males and females showing disappearance of Yki1 isoform in the two yki2-only fly lines. (C) Comparison of posterior/total area ratio of females wings upon depletion of B52 in a yki+ (wild type) or yki2-only background. Each point corresponds to a single wing, bars represent mean with SD (unpaired two-tailed t-tests: **** p-value <0.0001). (D) Analysis of embryonic lethality according to maternal and paternal genotypes. Bars represent mean and error bars represent SD. Numbers of counted embryos are indicated in each column. Unpaired two-tailed t-tests: **** p-value <0.0001. (E and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.
Generation of the Apopt1 knockout mouse model A Strategy employed for the targeted disruption of mouse chromosome 12 Apopt1 coding exon 2 by CRISPR/Cas9.Figure 1 A Strategy employed for the targeted disruption of mouse chromosome 12 Apopt1 coding exon 2 by CRISPR/Cas9. The mutation selected for the characterization of the mouse model produced an indel (c.188delAinsTG) resulting in a frameshift and truncated protein (p.Asp55Valfs*20)  B Chromatograms generated by Sanger sequencing of Apopt1-/- (KO) and Apopt1+/+ (WT) genomic DNA highlighting the mutated position in comparison with the wild-type sequence  C Relative mRNA expression of Apopt1 normalized to the expression of GAPDH in skeletal muscle and liver of wild-type, heterozygous and knock-out mice of three months of age. Data are presented as mean ± SEM (n = 6 mice per genotype). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ***P = 0.0001 (WT vs KO), *P = 0.0310 (WT vs het), *P = 0.0395 (het vs KO), Liver - **P = 0.0022 (WT vs KO), *P = 0.0101 (WT vs het)
IRF2BP proteins negatively regulate expression of the EGF receptor(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1.Figure 4(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls. (B) Model. IRF2BP1 is a SUMO-dependent transcriptional repressor of immediate early genes (IEGs). Transcription of IEGs is repressed by SUMOylated IRF2BP1, which binds to its proximal promoter. EGF receptor signaling yields at least two signals to induce IEG expression, one of which is the transient deSUMOylation of IRF2BP1. (C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates. (D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates. (E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates. (F) GSEA of the highly significant enriched EGFR signaling pathway. IRF2BP1 knockdown correlates with an increase of genes involved in EGFR signalling. (G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies. (H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM.
Figure 4Figure 4. SLFN11 inhibits maintenance but not initiation of DNA damage checkpoint. (A) Analysis of the kinetics of CHK1 and CHK2 phosphorylation in cell lines expressing SLFN11 at high (SF268 and DU145) and very low or undetectable levels (HeLa and U2OS), respectively. The indicated cells were treated with CPT (1 µM) for 1 hr. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies. (B-C) HeLa or U2OS cells overexpressing exogenous SLFN11 display a specific defect in checkpoint maintenance. A HeLa or U2OS cell line to express Flag-tagged SLFN11 under the control of a tetracycline-inducible promoter was generated. The resulting cells were treated with CPT (1 µM) for 1 hr. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies. (D) Overexpressing of exogenous SLFN11 rendered both HeLa and U2OS cells more sensitive to CPT treatment. A HeLa or U2OS cell line stably expressing Flag-tagged SLFN11 was generated. The resulting cells were treated with various doses of CPT for 24 hr, then shifted to fresh medium and permitted to grow for 14 days before staining. Error bars are s.d.; n=3. (E) Downregulation of SLFN11 delays the decline of phosphorylated CHK1 and CHK2 in SF268 cells. SF268 cells were transfected twice with control siRNA or siRNAs specific for SLFN11. 48 hr after transfection, cells were treated with CPT (1 µM) for 1 hr. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies. (F) SLFN11 knockout SF268 cells were treated with CPT (1 µM) for 1 hr. Cells were then washed, shifted to fresh medium (time 0), and harvested at the indicated time points for immunoblotting with the indicated antibodies. ( (G) A SLFN11-deficient SF268 cell line stably expressing Flag-tagged SLFN11 was generated. The resulting cell line was treated with CPT (1 µM) for 1 hr. Cells were then washed and shifted to fresh medium. 24 hr later, cells were harvested for immunoblotting with the indicated antibodies.
Figure 7(A) The E406-R479 (GltPh Q318, R397) salt bridge locks HP2 in the open state in EAAT1. Image shows the region around residues 406 and 479 (shown as spheres) with HP2 open (gray cartoon). Both residues sterically prevent HP2 closure by forming a salt bridge. (B) Contour plot of the HP distance vs. the R479-E406 (GltPh Q318, R397) distance distribution. Gray lines indicate thresholds for the HP2-closed and salt bridge-formed states. (C) Probability density distribution for HP2 opening in WT and R479A EAAT1 (GltPh R397). (D) Probability of HP2 closure calculated by integrating the densities (Mean ± SD from 1000 bootstrap samples). Statistical significance was tested by comparing bootstrapped confidence intervals (*, 95% confidence intervals do not overlap). (E) Representative transport current recordings from a cell expressing R478A EAAT2 (GltPh R397) in the absence of internal K+. Transport currents were elicited by voltage jumps to -140mV before and after superfusion with 5 mM l-serine. Transient capacitive currents during the first 5 ms after a voltage jump were blanked. (F) Time-dependent changes in steady-state R478A EAAT2 (GltPh R397) uptake currents during two consecutive serine applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus time. Serine application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace. (G) Current-voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments. (H) Mean net transport currents for WT and R478A EAAT2 (GltPh R397) induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.
Figure 2(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse colon. (B) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse brain. (C) AEP activity assay with colon lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; ***P = 0.0002 (*** P ＜ 0.001), **** P ＜ 0.0001 compared with control, one-way ANOVA. (D) AEP activity assay with brain lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; *P =0.0103 (*P< 0.05), ****P < 0.0001 compared with control, one-way ANOVA. (E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; **P =0.0071 (**P < 0.01)，***P =0.0009 (3xTg vs 3xTg AEP KO) , ***P =0.0004 (3xTg vs WT) (***P < 0.001) compared with control, one-way ANOVA. (F) Human Aβ1-42 concentration in brain lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; **P=0.0097(**P< 0.01), ***P =0.0006 (***P < 0.001) compared with control, one-way ANOVA.