EMBO/sd-character-level-ner huggingface dataset:
Figure 4A IL-6 secretion in HEI-OC1 cells treated with 100 pg/mL LPS or 20μM cisplatin (n=3 or 4 independent biological replicates). B IL-6 secretion in TLR4-/- cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20μM cisplatin (n=6 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test). C Il6 and Tlr4 transcript levels in HEI-OC1 cells following treatment with 20μM cisplatin for the indicated times (n=3 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).
Increased chromosomal instability and DNA damage in ICL in vitro lines and tumors (A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD).Figure 8(A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD). (B) Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10μm, p<0.0001 for Ch9, Ch10 and Ch12, p<0.05 for Ch14; n=25 for each dataset and error bars denote SD). (See also Figures S5A and S5B) (C) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20μm, p<0.0001 for Ch10 and p<0.05 for Ch14).
Expanded polyglutamine repeats cause mitochondrial dysfunction.Figure 2Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 (top panel, left) and Q74 (bottom panel, left) and in brain tissues from wild-type (WT) (top panel, right) and HD transgenic mice (bottom panel, right). An indicated portion of each image (in yellow) is expanded to illustrate the mitochondrial morphology. Scale bars: 1 μm. Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045. Mitochondrial membrane potential measured by using JC-1 dye in models of HD and control cells (MMP, ΔΨm). n = 3. *P = 0.008; **P = 0.044; ***P = 0.025; #P = 0.005. Mitochondrial respiratory function in PC12 cells, represented by respiratory control ratio (RCR; [state3]/[state4]). n = 2. *P = 0.028. ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004. Western blots showing cytochrome c release from mitochondria. n = 2. *P = 0.003; **P = 0.003; ***P = 0.01; #P = 0.008. Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models. n = 3. *P, **P, ***P, #P < 0.005. Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson's correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.
VHHASC blocks the extracellular pro-inflammatory function of ASC specks.Figure 1(A) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed BMDMs (200 ng ml−1, for 2h), that were stimulated with in vitro-generated human ASC specks (200 µg ml−1, O/N). ASC specks were pre-incubated with 200 µg ml−1 of VHHASC, its mutated variant, mutVHHASC or 50 µg ml−1 of polyclonal ASC antibody (a-ASC pAb, AL177) for 15 min at RT. Data is pooled from at least two independent experiments, each represented by a different symbol. Data is displayed as floating bars with the max/min values and mean (thicker band). (B) Immunoblot analysis of pro- (p31) and cleaved (p17) IL-1β, and pro- (p45) and cleaved (p20) caspase-1 in cytosolic fractions of ASC deficient (Pycard-/-) immortalized murine macrophages that were primed with LPS (200 ng ml−1, for 3h). Fractions were incubated with recombinant ASC specks in the presence of 2, 20 or 200 µg ml−1 of VHHASC or mutVHHASC. One representative of three independent experiments is shown. (C) Quantitative analysis of band densitometry of three independent experiments as shown in B. Data represents the fold change from all conditions (lanes 1- 9) .vs Pycard−/− lysates incubated with ASC specks alone (lane 2). ns, p > 0.05; *, p<0.05; **, p<0.005; ***, p<0.0002, One-way ANOVA, multiple comparison (Dunnet test). Data is displayed as floating bars with the max/min values and mean (thicker band).
Figure 1Genetic screen for retrograde IFT defects uncovers a temperature-sensitive (ts) mutation in C. elegans CHE-3 (Dynein-2 heavy chain). Paraquat-resistant strains were first screened for dye-filling defects (Dyf phenotype) typically indicative of cilia structure anomalies (examples of normal and defective DiI dye-filling are shown). Three different IFT reporters were then introduced into the dyf mutants (CHE-11::GFP shown as an example) to reveal candidate retrograde IFT mutants showing strong IFT protein accumulation at cilia tips. Whole-genome sequencing revealed the likely causative mutation in one of the strains, namely in the retrograde IFT dynein motor CHE-3 (DYNC2H1 human orthologue). A variable Dyf phenotype at 20ºC suggested the possibility of a ts mutation. Scale bar, 20 µm The Dyf phenotype of the ts-che-3 mutant was confirmed, with animals grown at the permissive temperature (15ºC) showing the ability to uptake dye, while those raised at the restrictive temperature (25ºC) are Dyf. Scale bar, 20 µm Using the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µm The ts-che-3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm-9 mutant is included as a positive control. n = 50 animals; error bars are standard error
Figure 5Interactions between WT or interface mutant FAK with a PI(4,5) P2 containing membrane was probed using a pulldown assay with membrane coated silica beads. Plotted are bound protein relative to the binding for WT at the highest PI(4,5) P2 concentration. Mutant naming is as in Table 2. For WT the non-specific binding to a PC membrane is shown for comparison. Plotted are mean values from at least three independent experiments. Error bars: s.d.
Fig.Figure 8RAW 246.7 macrophages expressing the dynein-binding protein (mTagBFP2-BicD2-FKBP12) for rapalog-induced lysosome repositioning to the MTOC, also co-expressed HsLAMP1-mCherry with or without a C-terminal FRB* (LAMP-FRB* or LAMP, in red). Cells were treated with 0.1% ethanol or 250 nM rapalog for 2 h. Only the tripartite complex of FKBP12-rapalog-FRB induced clustering of lysosomes at the MTOC (A, B). This clustering was selective for lysosomes because ER and mitochondria (KDEL-GFP and MitoTracker Green) were not affected (C). (A, B) Phagocytosis of IgG-3-μm beads was reduced by immobilizing lysosomes at the MTOC, n = 80-564 cells; ***P<0.001. Activation of dynamin-2 with Ryngo 1-23 or expression of constitutively active dynamin-2 (S764A mutant, expression confirmed by the grayscale image) restored phagocytosis of IgG-3-μm beads (n = 34-51 cells), but had no effect on the phagocytosis of IgG-6-μm beads, n = 36-72 cells (E); ***P<0.001 EtOH Ctrl vs rapalog Ctrl, ###P<0.001 rapalog Ctrl vs rapalog Ryngo/Dyn2S764A.
Figure 2A. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. B. Percent testis to body weight ratios from control (n = 8), Plk1 cHet (n = 4), and Plk1 cKO (n = 7) adult mice. C. TUNEL staining of paraffin embedded testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. D. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Plk1 cHet, and Plk1 cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate. E. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Aurka cHet, and Aurka cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. F. Percent testis to body weight ratios from control (n = 5), Aurka cHet (n = 3), and Aurka cKO (n = 5) adult mice. G. TUNEL staining of paraffin embedded testis sections of adult control, Aurka cHet, and Aurka cKO mice. H. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Aurka cHet, and Aurka cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate. I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.
Figure 1Schematic representation of HSA-Ucp1-transgenic (TG) mouse line as model of compromised skeletal muscle-specific mitochondrial OXPHOS capacity via respiratory uncoupling. Uncoupled (LEAK) mitochondrial respiration of soleus (SOL) and extensor digitorum longus (EDL) muscle of wild type (WT) vs. Ucp1-TG (TG) mice (WT n=9, TG n=5). Relative mRNA expression in quadriceps (Quad) of ISR components (n=8 per genotype). Representative immunoblots of ISR component eIF2α and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression in quadriceps skeletal muscle (WT n=3, TG n=4). Multi-tissue transcriptomic profiling of Gdf15 gene expression. Heatmap is shown as raw ct expression values (n=4 per genotype). Quantification of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT littermates (WT n=5, TG n=4). Skeletal muscle (Quad and Gastroc) GDF15 protein content normalized to total protein content (WT n=9, TG n=18). Ex vivo secretion of GDF15 from SOL and EDL muscle of WT vs. TG mice (n=6 per genotype) after 2h incubation normalized to muscle wet weight (mg). Mouse GDF15 plasma levels in post-absorptive state of male and female WT vs. TG mice at 20wks of age (male WT n=8, TG n=9; female WT n=5, TG n=6). Mouse GDF15 plasma levels in post-absorptive state of male WT vs. TG mice fed low-fat diet (LFD) or high-fat diet (HFD) at 24wks of age (LFD WT n=6, TG n=5; HFD WT n=5, TG n=5). Mouse GDF15 plasma levels in post-absorptive state of young (10wks) vs. old (95wks) male WT and TG mice (young WT n=10, TG n=13; old WT n=13, TG n=13). Representative scheme of in vitro study design in C2C12 myocytes. Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control (Ctrl) or chemical mitochondrial uncoupler (FCCP, 1µM vs. 5µM) for 5hrs (n=3 biological replicates)
Figure 4(A) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5:GFP and ProQC25:GUS. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bars, 10 μm. (B) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h. The statistical method is described in Materials and Methods. Data are means ± SE (n = 150, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.
Figure 3Schematic of the OptoCAR‑T cell experiment to follow the dynamics of ERK phosphorylation. Dimerizer was added to cell conjugates at 0 min, with samples (denoted by triangles) taken at defined points. Where sample illumination was used, this is shown by blue region. Samples were rapidly fixed and stained for doubly phosphorylated ERK (ppERK) before measuring the distribution of ppERK levels in cell conjugates. OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3). A subset of ppERK distributions from individual datasets used to create plots in (B-D), respectively. Blue boxes denote samples that were illuminated.
ISG20 degrades dsDNA and ssDNA and is enriched on deoxyuridine(dU)-containing ssDNA.Figure 6A 3D-PCR amplicon was digested in vitro with recombinant ISG20 and increasing concentration of manganese chloride. A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated. Recombinant ISG20 and mA3A (mutant E72A, C72A) were mixed with indicated oligomers and analyzed in a single point binding mobility shift assay. dHepG2H1.3 cells were treated for one day with 1000 U/ml IFNγ and harvested proteins were subjected to an affinity purification-mass spectrometry assay (n=4 technical replicates). Oligomers used for pulldown were single-stranded or double-stranded DNA with and without dU as indicated.
The T557I point mutation in Sir3 abolishes Sae2-Sir3 interaction A.Figure 4A. Schematic representation of the assay used to screen for SIR3 mutants deficient for Sae2 interaction while maintaining interaction with Sir4. The SIR3SaID fragment (464-728) was mutagenized by PCR, cloned in the pACT2 two hybrid plasmid and transformed into the reporter strain along with plasmids expressing LexA-BD-SAE2C and GAL4-BD-SIR4C fusion proteins. The reporter strain (yKD1991) bears a Gal4 binding sequence (Gal4BD) upstream of a HIS3 reporter gene, and a LexA binding sequence (LexABD) precedes a LacZ reporter gene. Transformants in which the gal4-BD-Sir4C and Sir3SaID-gal4-AD fragments interact were selected for HIS3 expression on -HIS + 3AT medium and subsequently screened for LacZ expression upon X-gal coloration. Cells showing no LacZ expression were collected and the mutated sir3SaID-GAL4-AD was retrieved and sequenced. B. Representative images of two hybrid assays in the yKD1991 strain testing the interaction of the WT or the mutant SIR3SaID fragment isolated from the screen with SAE2C or SIR4C. C. Representative images of two hybrid assays testing the interaction of the WT or the mutant SIR3SaIDT557I fragment with SAE2C or SIR4C.
Figure 3(A) Gene ontology enrichment of stress- and Cdc55-dependent phosphorylation sites using DAVID v6.8. (Huang et al., 2009). Sets were defined according to phosphorylation site fold-changes in setup SR igo1∆igo2∆ (see Figure 2F). GO levels (of the category "GOTERM_BP_DIRECT") were required to be between level 3 and level 6, and GO terms should have at least two counts in the given set. The heat-map illustrates the respective fold enrichments for the resulting GO-terms in each set. The heat-map was sorted according to the enrichment values in Set1, and secondly according to the sum of the enrichment values in Set2 and all quantified phosphorylation sites in SR igo1∆igo2∆. (B) Overrepresented phosphorylation-sequence motifs calculated with MotifX. Phosphorylation sites with a fold change ≥ 2 in setup SR and ≤ 0.5 in setup SR igo1∆igo2∆ were compared to the whole set of phosphorylation sites quantified in setup SR and setup SR igo1∆igo2∆ as background. (C) Bar plots illustrating the phospho-threonine enrichment of stress- and Igo1/Igo2 dependent S/T-P motifs. Phosphorylation sites quantified in both setup SR and setup SR igo1∆igo2∆ were analyzed. Grey bars show the percentage of phospho-threonine, white bars the percentage of phospho-serine. The enrichment was assessed using Fisher's Exact test. (D) Results of the M-Track proximity assay. Cdc55 was fused to the protA-H3-HA prey-tag and putative substrate proteins were tagged with the HKMT bait-tag. Background proximity signal was defined using a strain expressing only Cdc55-protA-H3-HA and no tagged bait protein (noHKMT) and is shown as a dashed horizontal line. Western blot signals were quantified as described in the methods section and proximity signals are displayed as log2 fold over background. Dots/triangles show signals of individual replicates (n ≥ 3; if more than 3 replicates available, the sample size is indicated in the figure), horizontal bars indicate average proximity signals. Proximity signals that differ significantly from background are marked as black (q < 0.01) or white dots (q < 0.05), others as grey triangles (q > 0.05). AU, arbitrary units. Note that the differences in the variation and signal intensity between Series 1 and Series 2 are due to batch effects of the methylation-specific antibody used (see Methods).
Divergent AR binding triggered by agonist and antagonist in LNCaP cellsHeat map showing two distinct types of AR binding locations.Figure 1Heat map showing two distinct types of AR binding locations. Agonist (DHT) induced AR binding in 34,442 agonist‐responsive regions. Antagonist (bicalutamide and enzalutamide)‐enhanced AR recruitment in 3,292 antagonist‐responsive loci. The normalized average signal density of reads 2 is also shown for each condition.
Figure 2(A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases. (B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs (C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).
a mutants with a loss of CSR function show defects in protein-protein interactions.Figure 7(A) Amino acid sequence of mouse Phf5a protein. The 12 Cys residues indicated in red are part of the PHD finger-like domain. Each CxxC motif is underlined and color coded according to their pairing with another CxxC motif to form a zinc finger (ZF). A pair of CxxC motifs hold the zinc atom in each ZF. Arrangement of 4 Cys residues in ZF1-3 are schematically shown below the Phf5a sequence. (B) CH12F3-2A cells were transfected with siRNAs along with empty vectors or vectors expressing various zinc finger mutants of Phf5a (Phf5aR-MF). Twenty-four hours after transfection, the cells were stimulated by CIT for another 24 h, and then examined for CSR. The efficiency of IgA switching was calculated relative to the value obtained for siControl and empty vector-transfected cells. The data are compiled from three independent experiments. The values are presented as mean ± sd (n=3). Statistical significance was performed in reference to siControl by two-tailed unpaired Student's t-test ( *** p ≤0.001). See Supplementary Fig.S8 for a representative data set. (C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot. (D) CH12F3-2A cells transfected with WT or a mutant Phf5a (Phf5aR-MF) were subjected to anti-p400 immunoprecipitation. Associations with relevant interacting proteins were examined by immunoblot analysis, and are indicated next to each blot. (E) ChIP assay derived from CH12F3-2A cells transfected with the indicated siRNA and Phf5a (Phf5aR-MF) expression constructs. Antibodies used for ChIP are indicated on the top of the respective plot. The data are summarized from 2 independent experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.
Figure 2A) HeLa nuclear extracts were subjected to immunoprecipitation with anti-FUS antibodies or BSA antibodies (negative control) and the co-precipitated snRNAs as well as 7SL RNA were quantitated by RT-qPCR. The respective RNA amounts detected in the IPs are expressed as percent of the input. The precipitated RNA levels measured in the negative control were subtracted from those in the FUS-RNA-IP. The data before background subtraction is shown in Appendix Figure S1. Error bars indicate standard deviations (SD) of three biological replicates, each measured in duplicates. B) Enrichment of FUS after U11 snRNP affinity purification with a biotinylated antisense oligonucleotide (AS-U11) complementary to U11 snRNA from HeLa nuclear extracts. As control, incubation of the magnetic strepatvidin beads with AS-U11 was omitted. After biotinylated antisense oligonucleotide pulldown, the purified complexes were eluted from the beads and subjected to 4-12% NuPAGE gels. The blots were incubated with mouse anti-FUS-IRDye800CW, goat anti-hnRNPH, rabbit anti-U11-59K, and rabbit anti-SmD3, followed by detection with species specific IRDye680LT or IRDye800CW labelled secondary antibodies to confirm the presence of FUS, hnRNPH, U11-59K, and SmD3 in the AS-U11 enriched fraction. Input corresponds to 9% of the used material for pulldown.
Figure 2RNA-seq revealed 111 DEGs in HO-1-/- ECs. The identified DEGs separated HO-1-/- and HO-1+/+ ECs by (B) hierarchical clustering and (C) PCA. Selected GOBP terms enriched in GSEA analysis in HO-1-/- ECs. Heatmap of selected (E) hematopoietic factors and (F) adhesion molecules expression in HO-1-/- and HO-1+/+ ECs. RNA-seq revealed 100 DEGs in HO-1-/- CARs. The identified DEGs separated HO-1-/- and HO-1+/+ CARs by (H) hierarchical clustering and (I) PCA. Selected GOBP terms enriched in GSEA analysis in HO-1-/- CARs. Heatmap of selected (K) skeletal biology and (L) hematopoietic factors expression in HO-1-/- and HO-1+/+ CARs. Correlation similarity metric and average linkage clustering were used in the presented heatmaps.