EMBO/sd-character-level-ner huggingface dataset:
Figure 3(a) Top: Schematic illustration of the experimental design for the translocation assay in CH12F3-2A cells. Bottom: PCR amplification scheme to detect IgH/c-Myc chromosomal translocations. Triangles represent the positions of nested PCR primers used to amplify the rearranged regions. The position of the Myc probe used in the Southern blot hybridization is shown as a horizontal black bar. (b) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. CH12F3-2A cells were transfected with the indicated siRNAs. (c) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test). Analysis of the IgH/c-Myc translocation frequency in CH12F3-2A cells expressing Tet-inducible miRNA against Top1 mRNA. Schematic illustration of the experimental design for the translocation assay (d). Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right). Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments (f). Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test) (g). (h-j) Analysis of the IgH/c-Myc translocation frequency in WT and Samhd1 KO CH12F3-2A cells. (h) Schematic illustration of the experimental design for the translocation assay. (i) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (j) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test). (k-m) Analysis of the IgH/c-Myc translocation frequency in spleen B cell derived from WT (Samhd1+/+) or Samhd1 KO (Samhd1-/-) mice. (k) Schematic illustration of the experimental design for the translocation assay. (l) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (m) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).
Figure 4Abundance changes of proteasome proteins and their transcripts during aging. Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots, orange dots represent significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range. Heat map showing transcript and protein fold changes for members of the 26 proteasome (19S and 12S complexes). Genes are annotated according to significance of their changes at the level of transcript (adj. p < 0.05: black, adj. p > 0.05: white) or protein (adj. p < 0.05: green, adj. p > 0.05: white). The between 20S and 26S+30S proteasome abundance assessed by immuno blot on native gels (Figure EV5A). ** p<0.001, Wilcoxon Rank Sum test. The bars indicate mean +/-SD. In-gel proteasome assay following native gel electrophoresis (top) and immunoblotting of proteasome complexes (30S, 26S and 20S) (bottom) in young (5wph), adult (12 wph) and old (39 wph) killifish brains. For additional samples and low exposure pictures see Figure EV5B and EV5C. Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%. Percentage (%) of chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing or caspase-like (PGPH) proteasome activities in brain extracts of killifish of different ages. n >=6 per sample group; error bars indicate standard error of the mean. ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of each activity in young samples (5 wph) was set to 100%. Age-related changes of proteins involved in the ubiquitin cycle. All the displayed proteins showed significant protein level changes in the 39 wph vs. 12 wph comparison (adj. p < 0.05).
Figure 9A HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in young (8-week-old) and old mouse (20-month-old) heart tissue, 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. * p = 0.0087, n = 5 in each group. B HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. * p = 0.0021, n = 5 in each group. C Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in young and old mice. * p = 0.0164, n = 6 mice per group. D Comparison of heart weight / body weight (HW/BW) in WT and Cuedc2-/- mice. ** p = 0.036, n = 15 in young WT and Cuedc2-/- mice, n = 10 in old WT mice and n = 12 in old Cuedc2-/- mice. E Comparison of the left ventricle function in WT and Cuedc2-/- mice by transthoracic echocardiography. *p = 0.0384, **p = 0.0389. n = 15 in young WT and Cuedc2-/- mice, n = 10 in old WT mice and n = 12 in old Cuedc2-/- mice. F Left ventriclecardiomyocytes apoptosis was tested by TUNEL staining. *p = 0.0017, n = 5 in each group. G Left ventricle myocardial fibrosis was tested by Masson staining in WT and Cuedc2-/- mice. The represent results were shown in (G) and the quantitative analysis of myocardial fibrosis was shown in (H). *p = 0.0177, n = 5 in each group.
The N‐terminal 61 residues of USP45 mediate binding to ERCC1 A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full‐length or indicated fragments of USP45.Figure 3A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full‐length or indicated fragments of USP45. Thirty‐six hours post‐transfection cells were lysed and GFP immunoprecipitated. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue or subjected to immunoblotting with the indicated antibodies. GFP empty vector was used as a negative control. B Sequence alignment of the first 62 residues of USP45 from the indicated species. Conserved residues that were mutated in part (C) are indicated in red. The residues whose mutation in (C) is shown to inhibit binding to ERCC1 are marked with an asterisk. Residue Thr37 (marked with a solid triangle) was found to destabilise expression when mutated to Ala. C, D HEK293 cells were transiently transfected with constructs expressing the indicated wild‐type or mutant GFP-USP45 [1-62] fragment (C) or GFP full‐length USP45 (D) or empty GFP (C and D) as a control and processed as described in (A). LICOR-Odyssey quantitation of results shown in (C) is shown in Supplementary Fig S4. E As in (C) except HEK293 cells were transfected with the indicated N‐terminal Znf‐UBP domain‐containing DUBs. Similar results were obtained in at least two separate experiments.
Crystal structure of Sir4 H‑BRCT A) Domain architecture of Sir4.Figure 1Domain architecture of Sir4. Crystal structures have been determined for the Sir4 Ku80-binding motif (KBM) in complex with Ku80, the Sir4-interacting domain (SID) in complex with Sir2, and the Sir4 self-dimerization coiled-coil module (CC) (PDB 5Y59, 4IAO, and 1NYH, respectively). Sir4 H‑BRCT (orange box, this work) locates to the first half of the partitioning and anchoring domain (PAD) which recruits silenced chromatin to the nuclear envelope. ClustalO (Sievers et al, 2011) multiple sequence alignment of various yeast Sir4 proteins focusing on the putative new structural domain and comparison with S. cerevisiae Dbf4 H‑BRCT. Sir4 sequences: ScSir4 (Saccharomyces cerevisiae, Uniprot P11978), NdSir4 (Naumovozyma dairenensis, G0W8P4), NcSir4 (Naumovozyma castellii, G0VBN1), KnSir4 (Kazachstania naganishii, J7S2D5), KaSir4 (Kazachstania africana, H2AVL7). Scheme of the Sir4 H‑BRCT crystal structure in two orientations rotated by ~120 ° around a vertical axis. Colors gradually vary from blue (N-terminus) to red (C-terminus) while the surface is shown in grey (transparent). Termini and secondary structural elements are labelled. Superposition of Sir4 H‑BRCT (orange) onto the H‑BRCT domain of Dbf4 (PDB 3QBZ, cyan) in two orientations as in (C). Secondary structural elements, termini and the elongated Sir4 loop connecting helix α3 and strand β4 are labelled.
Figure 1A. Structure and domain organization of the motor domain of cytoplasmic dynein (PDB 4RH7 Inset shows zoom of MTBD (white) and coiled-coil stalk (grey), which consist of two helices, CC1 and CC2. Well conserved residues, that were used as anchor points to define CC1 and CC2 in this study are depicted as red spheres. Numbering is based on yeast cytoplasmic dynein. B. Table showing location, number of inserted or deleted residues and motility phenotype of all 18 stalk mutants. Quantification and classification are based on three technical repetitions. C. Example kymographs for 'Directional - WT like', 'Diffusive like', 'No movement', and 'Transient binding'. Magnified area for 'Diffusive like' motion shows run of a single molecule. D. Table showing the type of movement found for wild-type, mutant 2, mutant 5, and mutant 13 in a modified single-molecule assay with and without ATP. Classification of type of movement is based on two repetitions of different dynein preparations.
Figure 7Patients with colon cancer were divided into four groups based on tumor stage. mRNA expressions of Dectin-3 in tumor tissues were detected by qPCR. The box represents the 25th to 75th percentile, and the whisker plots represent the minimum and maximum percentiles. Patients with colon cancer were divided into two groups based on fungal burden in the feces. mRNA expressions of Dectin-3 was compared between these two groups. Overall survival rate was shown by K-M survival curve. Tumor tissues were stained for IL-22 and p-STAT3. The percentages of IL-22-positive and p-STAT3-positive cells were quantified. mRNA expressions of IL22 and STAT3 were detected by qPCR. Scale bars, 25 µm.
A dynamic model of FtsZ abundance predicts division timing.Figure 7Pulsing experiment was repeated in the presence of protease inhibitor (PI) that reduced the lag time for a given TI feedrate (f = 0.28 mmol/g/h). The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line The sets of proteins that are actively degraded and division-related intersect at FtsZ and FtsN A schematic of how FtsZ abundance changes. FtsZ is repressed by the transcriptional factor, PdhR. PdhR is activated by Crp-cAMP. FtsZ is also degraded primarily by the ClpXP protease complex. An approximate FtsZ threshold model poses a basal synthesis rate (α0), a feedrate-dependent synthesis (α1f), and a degradation term (the Michaelis Menten term) to explain changes in FtsZ abundance with and without pulsing. Per the model, FtsZ would deplete via degradation during starvation, be synthesized with glucose pulsing, and engender division when its abundance reaches the threshold concentration Analytical solution of the model (Appendix Supplemental Information) plotted against data from Figure 2A (R2 = 0.86). Lag time axis is log scaled
CHD7 regulates PAQR3 expression in human cells.Figure 4A. qPCR analysis of PAQR3 expression in a CHD7-mutation positive patient compared to parental controls set (N=4; ****p<0.0001; Student's t-test). Fold change was calculated according to the 2(−ΔΔCt) method, using HPRT1 and RPS1 as housekeeping genes for normalization. All data were expressed as mean fold change ± SD across replicates, relative to control parents set to 1 (dotted line), N is the number of experimental repeats. B. Schematic view of PAQR3 exon 1 and proximal promoter on chromosome 4 (hg19 assembly), obtained with the UCSC genome browser (https://genome.ucsc.edu/) and showing the sequence amplified in ChIP-qPCR assays in LCLs (thick black line) along with a previously described CHD7 ChIP-seq peak (thick grey line) and signal (green) in H1-hESC (ENCODE3). C. ChIP-qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7-mutation positive patient (N=3) compared to parental controls (N=6); *p< 0.05; Student's t-test, All data were expressed as mean fold change ± SD, N is the number of experimental repeats.
Figure 4XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experiments Same as in (A) but cells were fed with 8 µg of A9M peptide bead and exposed to 12.64-Luc Reporter T cells. Experiments were combined by normalizing RLU values to the average of the negative control I-Ab. Error bars show SD of 3 independent experiments. * p < 0.05 for Rab39a or β2m siRNA vs negative control I-Ab using ANOVA
Domains of Grk2 required for Hh pathway activity.Figure 7A Schematic representation of the zebrafish Grk2 protein showing the location of residues assayed for the effects of their mutation on rescuing activity. B Prox1a (green) and Eng (red) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding: Grk2K220R-GFP; Grk2K220M-GFP; Grk2D335N-GFP; Grk2ΔN7aa-GFP and mGrk2L4AV7AL8A-GFP. Merged panels show nuclear staining with DAPI in blue. (n=20 for each sample). Scale bar, 50μm. C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).
Figure 1(A-B) qRT-PCR analysis of TGF ligands, TGF receptors, and TGF target expression in control and FRS2 knockdown HASMCs. (NS: not significant compared to control, *p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test). -actin was used for sample loading normalization. Histogram of qRT-PCR results are representative of three independent experiments. (C) Left: Immunoblot analysis of TGFRs, phosphorylated Smad2 (p-Smad2), and phosphorylated Smad3 (p-Smad3) in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of p-Smad2, p-Smad3, TGFR1, and TGFR2 were normalized to Smad2/3 or GAPDH and expressed as a fraction of a control value. Results are expressed as means ± SD (*p<0.05; ***p<0.001 compared to control; unpaired two-tailed Student's t test).
Figure 4A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation. B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection. C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.
Fetal organoids resemble adult organoids after one month in culture.Figure 3Relative expression detected by Real-time qPCR in fetal organoids ( ) and adult organoids ( ). Neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids. Mature markers E Sis, F Treh and G Arg2 increase in fetal organoids to adult levels (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four independent organoid cultures with similar results).Enzyme activity assay of fetal ( ) and adult ( ) organoids. H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28, while I sucrase, J maltase, K trehalase and L arginase increase to adult levels. Activity is given in µM glucose/µg protein·min-1 (n=3 independent organoid cultures).
Figure 5Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells. repo-GAL4 was used to drive the expression of UAS-dRasG12V in glia. Images shown are volumetric reconstructions of confocal stacks. The various anatomical structures of the larval brain are annotated. VNC refers to the 'ventral nerve chord', which is distinct from dorsal hemispheres of the brain. GFP staining represents the total glial membranes in the larval brains. Scale bar, 100μm. Bar graph showing relative glial cell volume in larvae of the indicated genotypes. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes. He-GAL4 was used to drive the expression of UAS-dRasG12V in macrophages. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Representative Western blots generated using fat-body extracts isolated from 3rd instar larvae of the indicated genotypes. cg-GAL4 was used to drive the expression of UAS-HRASG12V in larval fat-bodies. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.
Figure 7(A-C) Embryos co-expressing CIBN-PatJ::GFPpm, RhoGEF2-CRY2::mCherry and the membrane marker GAP43::mCherry were photo-activated at the cell base after cells had already apically constricted. (A) Prior to photo-activation, cells were apically constricted and elongated. (B) In the non-activated region, within 6 min cells expanded their base and shorten. (C) In the photo-activated region, cell shortening was inhibited. (D-F) Cellular features extracted from the reconstructed cell shapes displayed in boxplots (pre A: pre activation (npre A = 107), initial time point; NR: non-activated region (nNR = 10); AR: activated region (nAR = 37)). The central mark, bottom and top edges of each box indicate the median, 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point and the '+' symbol indicates an outlier. Notches indicate comparison intervals. (D) The mean cell length at the initial time point was 45 µm (blue). 6 min after, the non-activated cells (green) shrank to about half of the size (26 µm), whereas photo-activated cells (red) only shrank to 39 µm. ANOVA result: F(2,151)=110.2, p=3e-30; Cohen's D: d > 1.5 for all comparisons. (E) The basal area in the pre-activation control of 30 µm enlarged to 60 µm in the non-photo-activated cells, but remained at 30 µm in the activated region. ANOVA result: F(2,151)=52.02, p=6.5e-18; Cohen's D: dpreA:NR, dAR:NR > 2, dpreA:AR < 0.5. Please note the logarithmic y-axis. (F) The cell volume did not change between the different conditions. There were no statistically significant differences between group means as determined by one-way ANOVA (F(2,151)=2.227, p=0.11). In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.
Figure 1(a) Schematic representation of the p62 architecture. Mutations tested in this study are indicated. PB1: Phox and Bem1p domain, ZZ: Zinc finger, LIR: LC3-interacting region, UBA: ubiquitin-associated domain (b) Schematic representation of the constructs employed in the clustering assay (c) Representative micrographs of a clustering assay with the proteins indicated on the left. GFP and mCherry fluorescence are consistently displayed in cyan and magenta, respectively (d-g) Quantification of particle number, size and estimated volume determined from the data shown in (c) (h) Quantification of cluster formation by the indicated p62 mutants. For all the graphs, averages and SDs from at least three independent replicates are shown
Figure 1 (A) Experimental setup for scRNA-seq analysis. E11.5 CD133+ spinal cord progenitor cells were isolated using magnetic cell sorting (MACS) whereas E15.5 cells were randomly isolated from dissociated spinal cord tissue. Bioinformatic analyses, including t-SNE, revealed clustering of 350 E11.5 CD133+ progenitor cells and E15.5 cells. Colors in the graph indicate cell origin (pink for E15.5 cells and brown for E11.5 cells) (B) Walktrap community (Igraph) analysis of the cells revealed 7 distinct cell clusters within the t-SNE graph (colored according to the Walktrap clusters), highlighted in blue, yellow, green, red, light-blue, magenta and orange. Differential expression analysis revealed genes specifically expressed in each cell cluster. Examples of two to three specifically expressed genes from each cell cluster are presented (C) Figures show the presence of differentially expressed genes within the analyzed single cells. Black color indicates cells with RPKM values above 40 for Pax3, 300 for Stmn2, 200 for Slc1a3 and 100 for Mbp (D) GO-analysis of the differentially expressed genes in each Walktrap cell cluster (E) Overlap between differentially expressed genes in three Walktrap cells clusters (marked by blue, green and red) and genes expressed in adult murine astrocytes, oligodendrocytes and neurons. Adult neuronal and glial expression profiles were derived from two individual studies [14,17]. P-values (phyper, R) were calculated from the total number of genes for the mm10 assembly (23´389). Non-significant overlaps were excluded