EMBO/sd-character-level-ner huggingface dataset:
Figure 7Figure 7. The RPA1-binding ability of SLFN11 is required for its function in DNA damage response. (A-C) A SLFN11-deficient SF268 cell line to express Flag-tagged wild-type SLFN11, the K605M/D668A mutant, or the Δ5 mutant 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 fixed and processed for RPA2 or RAD51 immunofluorescence. Scale bar, 10 μm. Error bars are s.d.; n=3. Figure 7. The RPA1-binding ability of SLFN11 is required for its function in DNA damage response. (A-C) A SLFN11-deficient SF268 cell line to express Flag-tagged wild-type SLFN11, the K605M/D668A mutant, or the Δ5 mutant 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 fixed and processed for RPA2 or RAD51 immunofluorescence. Scale bar, 10 μm. Error bars are s.d.; n=3. (D) The ability of SLFN11-deficient SF268 cells to maintain DNA damage checkpoint was disrupted by the re-expression of wild-type SLFN11 and the K605M/D668A mutant, but not the Δ5 mutant. Cells were treated with CPT (1 μM) for 1 hr, then washed and shifted to fresh medium. 24 hr later, cells were harvested for immunoblotting with the indicated antibodies. (E) Re-introduction of wild-type SLFN11 and the K605M/D668A mutant, but not the Δ5 mutant, resensitized the SLFN11-deficient SF268 cells to DNA-damaging agents. 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. . (F) A proposed model for the role of SLFN11 in the regulation of DNA damage response. Please refer to the main text for details.
Figure 1A) Schematic representation of kinesin constructs used in the inducible trafficking assays. Kinesin constructs contain a motor domain and a coil (MDC). (B) Principle of inducible peroxisome-trafficking assay. COS-7 cells were transfected with truncated motor constructs of kinesins fused with FRB and GFP (summarized in (A)) and peroxisomes labeled with PEX-mRFP-FKBP. Upon addition of rapalog, kinesin motors are recruited to peroxisomes, and kinesin transporting features can be measured as peroxisome displacement. (C) Exemplary images of COS-7 cells expressing PEX-mRFP-FKBP before (upper panels) and 30 minutes after (middle panels) rapalog addition in the presence of a translocating motor - KIF1C-MDC-FRB and a non-translocating motor - KIF3B-MDC-FRB. Yellow lines indicate COS7 cell outline. Lower panel represents the overlay of sequential binarized images color coded by time (see also Appendix Figure S1). Scale bar = 20 µm (D) Plots representing intensity time traces of R90% for COS-7 cells transfected with PEX-mRFP-FKBP and KIF1C-MDC-FRB (n =13) or KIF3B-MDC-FRB (n =10). R90% is the radius for each time point that includes 90% of the total fluorescent intensity. Error bars indicate SEM. E) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS-7 cells transfected with kinesin constructs summarized in (A). The number of analyzed cells is summarized in Appendix Table S1. Kinesins were considered translocators, if they displaced peroxisomes ≥ 5 µm (marked with a blue dashed line) in 30 minutes after rapalog addition. Error bars indicate SEM. (F) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90% (summarized in Appendix figure S2, Appendix Table S1). Error bars indicate SEM.
Figure 8Double immunolabeling of SYCP3 (red) and TRF1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres. J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance. S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.
Oxidative stress response in IDH wildtype and IDH mutant glioma.Figure 5A) NADPH levels are reduced in IDHm compared to wildtype tumors, resulting in a reduced NADPH/NAD+ ratio (n=3/ sample). p-value from T test: *<0.05. NADPH, NADP: reduced, oxydized nicotinamide adenine dinucleotide phosphate, NADH, NAD: reduced, oxydized nicotinamide adenine dinucleotide. B) MALDI images for glutathione (GSH) and cystathionine on IDHwt (P3) and IDHm (E478) PDXs. C) Quantification of MSI signals for GSH and cystathionine from 6 PDXs (n=3 / group). D) LC-MS quantification of independent samples for GSH, GSSG (oxidized GSH), GSSG/GSH ratio and cystathionine from 6 PDXs (n=3/ sample / group). Statistical significance between IDH groups calculated by T test, pv: p-value. E) Schematic of GSH synthesis, cysteine supply and its degradation pathways. GSH: reduced glutathione; GSSG: oxydized glutathione; SAM: S-adenosylmethionine, SAH: S-adenosylhomocysteine, CSA: cysteine sulfinic acid. Key enzymes in red: CBS: cystathionine-beta-synthase, GCL: glutamate-cystine ligase, xCT: cysteine-glutamate antiporter.
a) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h, fixed and processed for electron microscopy.figf7(a) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h, fixed and processed for electron microscopy. Scale bar, 2 μm. (b) Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype. Where indicated, cells were starved for 5 h. Scale bars, 0.5 μm. (c) Morphometric analysis of cristae density in cells of the indicated genotype. Experiments were carried out as in a. The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle. Data represent mean ± s.e.m. of five independent experiments.
Figure 7Phenotypes of the kup9-1/ProWOX5:AMI1 (A) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm. Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control. Phenotypes of the kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm. Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.
FKK5 and FKK6 inhibit NF-κB activation in a PXR-dependent manner.Figure 2A, PXR (luciferase) reporter assay in HEK293T cells transiently transfected with PXR plasmids (wild-type and ligand binding domain mutant C285I/C301A). RLU, relative light units are shown normalized to beta-galactosidase (β-Gal) expression. The histogram represents one experimental data (of n > 2 independent experiments each performed in quadruplicate) mean (95 %CI); n.s. not significant; * p < 0.05, two-way ANOVA. B, Histogram represents fold change in mRNA expression, normalized to GAPDH, by RT-qPCR from Caco-2 cells exposed to compounds. DMSO, 0.1% DMSO vehicle; 9.3 is 9.3-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biologic replicates, n = 4 technical replicates). Points and error represent mean (95 %CI) values for control (TNFα 5 ng/ml) normalized NF-κB (luciferase) reporter activity in LS180 cells after C, Passage 13 (P13) and D, Passage 8 (P8) and C, D, Line graph depicts one representative experiment of a series of experiments (n > 4) performed in four consecutive passages of cells. Data was expressed as mean with 95% CI of four technical replicates. E, LS174T PXR-KO (knockout) cells transiently transfected with pNL3.2.NF-κB-RE vector. mean (+ SD). *, ** p < 0.05, two way ANOVA with Tukey's multiple comparisons test.
Figure 1(A) Schematic representation of engineered perimCherry/cytoGFP E. coli cells that express mCherry in the periplasmic space (between the outer and inner membrane) and GFP in the cytosol. (B) Structured illumination microscopy image of perimCherry/cytoGFP E. coli confirming localization of mCherry (red) in the periplasm and GFP (green) in the cytosol. Scale bar = 3 µm. (C) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of perimCherry/cytoGFP E. coli bacteria exposed to (different concentrations of) human serum. Inner membrane damage correlates with killing (samples where bacteria are killed are indicated with gray shadings and a cross, see CFU data in Fig EV1b). Data information: (C-E) Data represent mean ± SD of 3 independent experiments. (D) Serum-induced inner membrane damage (% Sytox positive) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001. , E Serum-induced killing (CFU/ml) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.
Figure 8Figure 8a. Experimental outline for measurement of transcription elongation rates. AS CDK12 HCT116 cells were treated with DRB for 3.5 h to synchronize RNAPII at gene promoters. The cells were either pretreated (+) or not (-) with 3-MB-PP1 0.5 h prior DRB wash off. After DRB wash off (0 h) fresh medium either supplemented (+) or not (-) with 3-MB-PP1 was added and samples were taken at indicated time points for analyses of pre-mRNA expression by RT-qPCR. Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs.
Figure 4A. An immuno-precipitation was performed using whole cell lysates from cells expressing Flag-tagged wild-type STAT2, and samples were analyzed by the Western method. The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type, calculated in comparison with 293T cells. B. HME cells expressing wild-type STAT2 were treated with IFN-β (100 IU/ml). Whole cell lysates harvested at the times indicated were analyzed by the Western method; C. HME cells expressing wild-type STAT2 were seeded in complete medium. On the second day, the cells were re-fed with medium lacking supplements. On the third day, the cells were treated with each supplement for 4 h, and total lysates were analyzed by the Western method. 1. EGF, 2. Insulin, 3. Hydrocortisone, 4. GA-1000, 5. BPE. D. HME cells were pretreated with hydrocortisone (0.04mg/ml and 0.4mg/ml) for 3 h and then treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments. E. Consensus sequences for CDK-catalyzed phosphorylation, compared to residues 383-391 of STAT2. F. HCC827 and LnCAP cells expressing wild-type STAT2 were treated with CDK inhibitors (1 µM) for 4 h and whole-cell lysates were analyzed by the Western method. 1. Flavopiridol, 2. AT7519, 3. Dinaciclib.
Figure 3A. Immunofluorescence of collagen matrix sections co-stained for cleaved collagen neo-epitope Col1 3/4C and DAPI (left panels). Second harmonic generation (SHG) images of collagen fibers within 3-dimensional collagen matrices at 15 µm and 45 µm below the cell surface (middle panels). Scale bar = 20 µm. Graphs indicate the mean ± SEM fibrillary collagen areas at 2.5 µm increments below the cell/collagen interface (n=5). SHG fibrillar collagen (red); cells (green).B, C. Grey level correlation matrix (GLCM) texture analysis of SHG correlation (n=6) and contrast (n=5). Mean ± SEM at each distance. D. Transmission electron microscopy of a collagen matrix section. Scale bar = 1 µm. Positions of magnified areas indicated by corresponding colored squares. E. Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid. Yellow arrows indicate long collagen bundles, Red arrows indicate short collagen bundles. Scale bar = 0.5 µm.
Figure 7The schematic depicts a proposed model for the miR-499/Fnip1/AMPK circuit that orchestrates mitochondrial function to match muscle contractile machinery during fiber type transition.
Figure 5(A,B) Cumulative risk for ICU admission (A) and cumulative survival (B) in COV patients (n=111), grouped for cut-off value of S1P serum level of 0.60 μM. Statistical analysis was performed by Cox regression. Exact p values are reported.
Figure 1(A) Immunoprecipitation of A3A. U2OS-HiLo cells with doxycycline-inducible, haemaglutanin (HA)-tagged A3A (U2OS-A3A) and parental cells (U2OS-HiLo without transgene) were treated with dox for 72 hours. Immunoblot detection of A3A-HA after immunoprecipitation (IP) of cell lysates with anti-HA antibody is shown. (B) Comparison of mass spectrometry results from three (n=3) replicates. IP followed by mass spectrometry was performed in triplicate for parental U2OS-HiLo and U2OS-A3A cell lines. Venn diagram shows the overlap of proteins identified in three biological replicates of HA-immunoprecipitation from U2OS-A3A cells. Number of proteins identified in each replicate is displayed along with the percentage of protein overlap identified between replicates. (C) All eight subunits of the CCT chaperonin complex were enriched in U2OS-A3A samples. Proteins detected by MS are displayed by fold enrichment (log2) from U2OS-A3A cells compared to parental cells (x-axis), and by protein abundance within the U2OS-A3A samples (y-axis). Significantly enriched proteins (p<0.05 by one-tailed t-test, n=3) are denoted in dark blue. Components of the CCT complex are highlighted and labeled. (D) Network of specific interactors of APOBEC3A. Only proteins significantly enriched in A3A samples over HiLo were considered for network analysis (log2 fold change>0; one-tailed t-test p<0.0.5), with the exception of CCT6B and CCT4 (t-test p>0.05). Node colors denote enrichment (indicated by heatmap legend) of interacting protein in A3A samples compared to HiLo. Nodes are grouped into boxes according to gene ontology enriched biological processes for interacting proteins (STRING database, FDR<0.01). Gray edge lines indicate observed interactions between proteins. CCT complex members are denoted by a star.
Figure 2A Simplified lignin biosynthetic pathway. B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation. C Measurements of Pst DC3000 (AvrRpm1) growth. Data are shown as means ± SD (n = 3). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation. D Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). E Quantification of leaves (n ≥ 30) with spreading cell death as in (D). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation. F Quantification of total and free SA in Col-0 and palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 5; 6 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid dpi, days post-inoculation.
Figure 6A HEK293T cells were transiently co-transfected with ARL13B-FLAG and GFP or GFP-RAB35. Cells were cultured either in full growth medium (+serum) or serum-starved for the final 8 h before harvesting. Immunoprecipitations (IP) were performed using anti-GFP beads. Cell lysates (2.5% of input) and immunoprecipitates were loaded on the same gel, subjected to immunoblot (I) analysis with indicated antibodies and detected with the same exposure. B Schematic representation of the human ARL13B structure depicting the guanine nucleotide-binding (G), coiled coil (CC) and proline-rich (PR) domains as well as the point mutations affecting N-terminal palmitoylation or C-terminal ciliary targeting RVEP motif. C HEK293T cells were transiently co-transfected with mCherry-RAB35 together with GFP or indicated ARL13B-GFP wild type, mutant or truncation constructs. Cells were serum-starved for the final 8 h before harvesting. IPs were performed using anti-GFP beads and interacting proteins detected by immunoblotting (IB). D HEK293T cells were transiently transfected with ARL13B-FLAG and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with GFP-RAB35 bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0333). E HEK293T cells were transiently transfected with mCherry-RAB35 and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with ARL13B-GFP bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0466)
Figure 4HEK-293 cells expressing V2R, B2R, B2RΔG/L370Tor B2RL370T along with βarr1-mCherry and YFP-tagged intrabody30 (Ib30-YFP) were stimulated with their respective agonists (AVP, 100nM for V2R) and (bradykinin, 1μM for B2R) and the localization of βarr1 and Ib30 were visualized. Although βarr1 gets recruited for all four constructs, Ib30-YFP does not colocalize with βarr1 either at the surface or upon internalization in case of B2RWT. Representative data from 3-5 independent experiments are shown and the scale bar is 10μm. Visual scoring from 3-5 independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 82% of the cells for V2R (691 cells), 10% for B2RWT (254 cells), 63% for B2RL370T (273 cells) and 83% for B2RΔG368+L370T (158 cells). Pearson's correlation coefficients (PCC) were measured to assess the colocalization of βarr1 and Ib30 using JACoP plugin in ImageJ, and the mean±sem for the basal, surface and internalized panels, respectively, are presented here. B2RWT - 0.32±0.02 from 16 cells, 0.18±0.01 from 32 cells, 0.25±0.02 from 31 cells; B2RΔG/L370T - 0.32±0.03 from 13 cells, 0.92±0.01 from 18 cells, 0.90±0.01 from 16 cells; B2RL370T - 0.26±0.01 from 10 cells, 0.85±0.01 from 11 cells, 0.87±0.01 from 25 cells.