EMBO/sd-character-level-ner huggingface dataset:
FXR1 inhibits aberrant assembly of cytoplasmic Nups.Figure 2A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. B HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3). HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C) HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. 1800 cells were analysed for each graph (mean ±SD, *P < 0.05; **P < 0.01; N = 3). G, H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. 1600 cells were analysed in (G) and 2100 cells were analysed in (H) (mean ±SD, *P < 0.05; *** P<0.001; N = 3).
Figure 2(A) Total RNAs or proteins from tail-tip of wild-type or JfkKO 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) The body weight of wild-type or JfkKO mice under ND were measured at 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 or JfkKO mice fed on ND. Arrows indicate abdominal SAT under MRI. EWAT and PRAT were dissected, and the sections from EWAT were stained with H&E. Scale bar, 100 μm. (D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (E) Representative images of wild-type or JfkKO mice fed on HFD. Arrows indicate abdominal SAT under MRI. EWAT and PRAT were dissected, and the sections from EWAT were stained with H&E. Scale bar, 100 μm. (F) GTT or ITT in 17-week-old wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test). (G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm. (H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm. (I) Wild-type, JfkKO, or JfkKO mice with liver-specific reconstitution of JFK were housed with free access to water but no food during indirect calorimetry. The white or gray horizontal bars depict the light or dark period in a 12: 12 h light-dark cycle, respectively. EE or RER was calculated and plotted at 1 h intervals and averaged. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test).
Figure 6(A) Drug administration protocol for intravital microscopy (IVM) experiments. Male, 10 week old C57BL/6 mice (n=3/group) were pretreated with vehicle or mixed inhibitor (Daminiozed+GSKJ4, 50 mg/kg each) at 16 hrs and 1 hr prior to TNF-α treatment. Inflammation was induced by an intraperitoneal injection of TNF-α (5 μg/mouse) and IVM was performed 4 hrs later. (B) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries. Margins of vessels are indicated with dashed lines. White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G. (C) Quantitative analyses of leukocyte adhesive interactions in the femoral arteries. Data are shown as means ± SE (n=3/group). *P < 0.05 compared to the vehicle + TNF-α group. Statistical differences were analyzed by the Student's t test. See also Movies EV1 and 2.
Figure 2(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD. (C) Pml+/+ and Pml-/- BMDMs were treated with AT-406 at the concentration indicated for 24 h, and cell viability was then determined by ATP assay. Data show mean ± SD of three biological replicates. (D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.
Figure 2A. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction. B. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. 100 parasites found mid-invasion were counted in triplicate and compared to extracellular, freshly egressed parasites. One-way ANOVA was used for statistical analysis. **** p value <0.0001. Data information: Error bars represent standard deviation. and Scale bars represent 5 µm. C. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. Indicated parasites were analysed. For adf cKD induced and non-induced conditions were compared, as indicated. 30 parasites were counted for each condition in triplicate. One-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. *** p value 0.0009. Data information: Error bars represent standard deviation. ; and Scale bars represent 5 µm. D. Time-lapse microscopy analysis of indicated parasites invading HFFs cells. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds E. Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations. Individual stages were named in regard to the invasion step (Penetration initiation, Speed decrease and finalisation). Invasion was analysed with Icy Image Processing Software (Pasteur Institut) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurements F. Time-lapse analysis demonstrating abortive invasion of indicated parasites. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds
Cay1 and Rap1 genetically interact to maintain telomere homeostasisTelomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen).Figure 4Telomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen). Telomere length analysis of ApaI‐digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. qRT-PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1+ mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. **P 0.01 relative to wt. ChIP analysis of H3K9ac and total H3 for the indicated strains. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to wt.
Pre-activation of the DDC allows Rad9-dependent repression of late origins.Figure 3Wild type and rad9∆ cells were synchronized in G1 and released into S phase with the addition of pronase in medium containing IdU and 200 mM HU to activate the DRC. Then, DSBs were induced by the addition of 100 μg/ml Zeocin to activate the DDC. HU was washed away 45 minutes later to inactivate the DRC and cells were resuspended in fresh medium containing CldU and 100 μg/ml Zeocin, in order to keep the DDC active. Samples were collected at the indicated times. Analysis of Rad53 phosphorylation by Western blot. Analysis of DNA content by flow cytometry. Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb. Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb. 2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing. Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.
Salt tolerance of slhak20 from the salt-sensitive tomato and oshak4/17 from rice.Figure 5Generation of SlHAK20Hap2 mutations using CRISPR/Cas9 system. The sequences of SlHAK20Hap2 in wild type (TS-670), slhak20-3 and slhak20-4 mutant tomato are shown. Salt-tolerance assay of TS-670 and slhak20 mutant plants. TS-670, slhak20-3, and slhak20-4 grown under normal growth conditions for 23 days, and then treated with 150 mM NaCl for 5 days. Control plants grown under normal growth conditions for 28 days. Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stressPlant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. resh weight f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype). Generation of oshak4/17 mutants using CRISPR/Cas9 method. Sequences of OsHAK4 and OsHAK17 in wild type (NIP), oshak4/17-1, oshak4/17-2 and oshak4/17-3 mutants are shown. Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative plants ere analyzed among these genotypes. ontrol plants were grown under normal growth conditions for 28 days Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative fresh weight of shoots were analyzed among these genotypes. Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured. Values are means ± SD (n = 12 plants of each genotype for fresh weight Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative survival rate were analyzed among these genotypes.
TFEB promotes redistribution and docking of lysosomes to the plasma membrane in autophagy-deficient PD fibres.Figure 6Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).Muscle was transfected with mCherry-LAMP1 only.Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).
XBP‐1‐mediated ER stress suppresses the rpl‐43 phenotype A, B Phsp‐4::GFP is very weakly expressed in wild‐type animals.Figure 3A, B hsp‐4::GFP is very weakly expressed in wild‐type animals. C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype. D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals. F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation. H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals. J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals. L Functional interaction of ER stress‐induced genes. M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.
SUMM2 is required for the constitutive defense phenotype of pat1 mutantsElevated PR gene expression in pat1 mutants is suppressed by summ2‐8.Figure 6Elevated PR gene expression in pat1 mutants is suppressed by summ2‐8. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2‐8. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). 14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test. Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col‐0 and mkk1/1‐summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.
Figure 9A) Motif counts for abundant TF binding sites at the specific DHSs in TM, TB and TB+. B) Total motif counts for 5 inducible motifs (AP-1, NFAT, EGR, NF-B and CREB/ATF) and 5 constitutive motifs (ETS, RUNX, KLF, GATA, E-box) in the specific DHSs in TM, TB and TB+. The motifs used here are defined in dataset EV5. C) Overlaps between ETS-1 and RUNX1 ChIP peaks and the 2882 pDHSs in TN compared to TB. D) Mechanisms of pDHS and iDHS regulation in T cells.
Figure 5A. Representative, detrended Cry1-luc traces of emitted bioluminescence (photon count values) from 4 SCN slices, pre-transfected with AAV pCry1-DIO-Luciferase after addition of (left) pProk2.Cre.T2A.mCherry (red, cohort n=10) or (right) pProkR2.Cre.T2A.Venus (green, cohort n=9) transfection. B. Raster plots (yellow=trough, blue=peak) of Cry1-luc bioluminescence oscillations from a representative slice of each cohort. The black arrow marks the time-point of pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus AAV transfection, thus resulting in the evolution of rhythmic Cry1-luc bioluminescence. C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively).E. Representative raster plots of single-cell bioluminescence (yellow=minimum, blue=maximum) from Prok2Cry1-luc (left) or ProkR2Cry1-luc (right) cells in single slices. F. Within-slice cellular synchrony depicted in representative Rayleigh plots of the phases of peak Cry1-luc bioluminescence for Prok2Cry1-luc (red) and ProkR2Cry1-luc (green) cells. The Rayleigh plots can be read as the face of a clock with CT0 marking the top and CT12 the bottom of the plot circle. The black arrow depicts the mean peak phase across the individual oscillators. G. The mean Rayleigh vector lengths for Prok2Cry1-Luc and ProkR2Cry1-luc (p>0.99) do not differ between the cohorts (n=5 SCN slices per group). H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN. I. After registering the respective peaks of [Ca2+]i fluorescence to the peaks of Cry1-luc bioluminescence in each respective cohort, phase-alignment between the peaks of Prok2Cry1-luc and ProkR2Cry1-luc bioluminescence shows that the intracellular clocks of Prok2+ cells are phase advanced by ~0.6h to those of ProkR2+ cells (**p<0.01) (n=5 SCN slices per group).
Fig.Figure 4(A) qPCR analysis of mitochondrial DNA (mtDNA) on nuclear DNA (nDNA) in C2C12 myotubes treated for 48h with C26CM and ferric citrate. (n=6). Data information: For all data, n represents the number of biological replicates. Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group. (B) Western blot of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with C26CM and ferric citrate. (n=3) Profile of oxygen consumption rate OCR in C2C12 myotubes after 48h treatment with C26 CM and ferric citrate supplementation. Data normalized to protein content (n=9-12). Data information: For all data, n represents the number of biological replicates. Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group. Profile of maximal OCR (E) and OCR used for mitochondrial ATP production (F) in C2C12 myotubes after 48h treatment with C26 CM and ferric citrate supplementation. Data normalized to protein content (n=9-12). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test or one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group. (G-H) Representative microscopic pictures and diameter of C2C12 myotubes stained for myosin heavy chain (G) or human myoblast-derived myotubes (H) after 48h treatment with C26 CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group. (I) Diameter of C2C12 myotubes treated with rotenone and ferric citrate (n=3). Data information: For all data, n represents the number of biological replicates. Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.