EMBO/sd-character-level-ner huggingface dataset:
Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C.Fig 1A) Workflow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (D) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16). (G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Figure 3(A) A schematic representation of the TaDa analysis performed in Drosophila NSCs. (B The binding profile of Pr-set7 on cdk1 gene region (B) in NSCs. The green peaks represent positive binding and red means no binding, which are based on the log2 ratio between Dam-Pr-set7 and Dam control (Y-axis). C) The binding profile of Pr-set7 on ebd1 gene region (C) in NSCs. The green peaks represent positive binding and red means no binding, which are based on the log2 ratio between Dam-Pr-set7 and Dam control (Y-axis). (D) qPCR analysis of pr-set7, cdk1, ebd1 and arm in larval brains of control (ctrl; yw) and pr-set7716/Df(3R) ED5644 (716/Df) at 24h ALH. n=7 as biological replicates. Statistical analyses were done comparing between control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and pr-set7716/Df, using Mann-Whitney test. The mRNA levels of control were normalized to 1. Data are presented as mean ± SD calculated from all replicates. Data information: Data are presented in (D, as mean ± SD. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (D *P < 0.05 (E) Western blot analysis of larval brain extracts of control (ctrl; yw) and pr-set7716/Df at 24h ALH. Blots were probed with anti-Cdk1 antibody and anti-GAPDH1 antibody (loading control). (F) Quantification of normalized Cdk1 intensity against GAPDH in (E). n=7 as biological replicates. Data information: Data are presented in as mean ± SD. Statistical analyses were done comparing two different genotypes using Mann-Whitney test F, G) Larval NSCs of control (ctrl; yw) and pr-set7716/Df at 24h ALH were stained for Ebd1, Dpn and Mira. Yellow dotted boxes, zoomed-in area. White arrows point to NSCs. Data information: Scale bars, 15 μm (G). (H, I) Quantifications of nuclear Ebd1 (H) and the corrected total cell fluorescence (CCF) (I) In (H), n=756, 9 BL for control; n=765, 9 BL for pr-set7716/Df. In (I), CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 1102.2±493.2 (n=99) for control (ctrl; yw) and 144.0±88.0 (n=98) for 716/Df. Data information: Data are presented in , I, s mean ± SD. n numbers in (H) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test , H, I, ***P < 0.001. (J) Western blot analysis of larval brain extracts of control (ctrl; yw) and pr-set7716/Df at 24h ALH. Blots were probed with anti-Arm antibody or anti-GAPDH1 antibody (loading control). (J) Western blot analysis of larval brain extracts of control (ctrl; yw) and pr-set7716/Df at 24h ALH. Blots were probed with anti-Arm antibody or anti-GAPDH1 antibody (loading control). (K) Quantification of normalized Arm levels against GAPDH in (J). n=5 as biological replicates. Data information: Data are presented in K) as mean ± SD. Statistical analyses were done comparing two different genotypes using Mann-Whitney test K). ***P < 0.001.
Fig.Figure 1(A) GO functional analysis of the differential genes in the fasted and refed condition. (B, C) Body weight of the WT and L-HRD KO mice at the age of 12 weeks. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test. (D) Body heights and tibia lengths of the WT and L-HRD KO mice. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test. (E) Phosphorylation of STAT5 in WT and HRD1 LKO liver. (E) Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
Tyramine (Tyr) treatment reduces autophagy induced by proteasome inhibition.Fig 5(A-C) Time course of effect of temperature shift on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals starved 6 hrs prior to analysis. (A) The number of ubiquitin aggregates peaks at 18-24 hrs PS, and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (B) The number of GFP-Atg8a punctae peaks at 12-24 hrs PS and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (C) Larval locomotion, measured as the time to crawl 3cm, gradually and significantly worsens from 18 to 48 hrs PS (n = 6 and p-values shown relative to 0 hrs PS. (D-E) tyramine (Tyr) treatment reduces GFP-Atg8a punctae in larval muscle. UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals were placed on 100mg/ml Tyr (D) or control food (E), shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. (F-H) Effect of Tyr treatment on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and Dmef2-Gal4, UAS-GFP-Atg8a animals, shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. Aggregate (F) and GFP-Atg8a punctae (G) numbers in muscles are significantly reduced with 100 mg/ml Tyr treatment in DTS but not control animals 18 hrs post-shift (PS). (H) 100 mg/ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS. n = 12 and p-values shown relative to non-Tyr treated animals. For all measurements, SEM is shown with *p<.05; **p<.01; ***p<.001. (I) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-G4 control with or without 100 mg/ml Tyr. After 6hrs starvation, Atg8a-II levels are reduced in DTS + 100 mg/ml Tyr relative to untreated DTS.
Pacs1 is required for normal numbers of circulating lymphocytes (A) Super-pedigree mapping of two mutations in Pacs1 that were linked to peripheral B cell deficiency.Figure 1A) Super-pedigree mapping of two mutations in Pacs1 that were linked to peripheral B cell deficiency. Insert shows peripheral B cell deficiency in the endive (N=22 REF, 28 HET, 10 VAR) and chicory (N=24 REF, 15 HET, 3 VAR) pedigrees. Horizontal bars indicate mean values. Ordinary one-way ANOVA with Tukey's multiple comparisons test. Ns = not significant, **P < 0.01, and ***P < 0.001. Protein domain model shows the encoded location of the ENU alleles. B) A 1 bp insertion in Pacs1 using CRISPR/Cas9 leads to loss of Pacs1 protein (red arrowhead). C) Peripheral blood immune cell counts from Pacs1+/+ and Pacs1−/− mice. Horizontal bars indicate mean values. Two-tailed unpaired t test, *P < 0.05, **P < 0.01, and ***P < 0.001. (D) Absolute numbers of lymphocytes subpopulations in the bone marrow, thymus, and spleen. B cell development in the bone marrow was assessed by FACS analysis for surface expression of: B220+CD43+CD19−IgM−IgD− (pre-pro B); B220+CD43+CD19+IgM−IgD− (pro B); B220+CD43−CD19+IgM−IgD− (pre B); CD19+IgM+IgD− (immature); CD19+IgM+IgD+ (mature). T cell development in the thymus was assessed by FACS analysis for surface expression of: CD4−CD8− (double negative, DN); CD4+CD8+ (double positive, DP); CD4+CD8− (CD4 single positive, SP); CD4−CD8+ (CD8 SP). Splenic B cell populations were assessed by FACS analysis for surface expression of: B220+CD21+CD23+ (follicular B cells, FOB); B220+CD21+CD23low (marginal zone B cells, MZB). Each symbol represents an individual mouse. Horizontal bars indicate mean values. Two-tailed Mann-Whitney test, ns = not significant, *P < 0.05, and **P < 0.01. E) Proportion of cell populations derived from Pacs1+/+;CD45.1 and Pacs1−/−;CD45.2 donors during competitive bone marrow reconstitution. Populations were determined based on the same markers as in (C) with the added congenic markers CD45.1 and CD45.2. Each symbol represents an individual recipient. Results are representative of two independent transplant experiments. Two-tailed unpaired t test, ns = not significant, *P < 0.05, **P < 0.01, and ***P < 0.001. F) Measurement of cell death with Annexin V staining in FOB and MZB cells from the spleens of Pacs1+/+ and Pacs1−/− mice. Results are representative of two independent experiments. Horizontal bars indicate mean values. Two-tailed unpaired t test, ns = not significant, **P < 0.01.
Figure 2(A-D'') Ventrolateral (A, C, D) and lateral (B) images of the cranial vessels in lyve1b:Kaede transgenic embryos with the dorsal (A) and ventral (C) portions of the VA-L photoconverted at 54 hpf and followed through to 72 hpf (B) and 78 hpf (D). Unconverted vessels are shown in green (A-D), while photoconverted vessels are shown in red (A'-D'). The extent of lymphatic and arterial contribution from the VA-L to the LFL (B'') or HA (D'') is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS (A'', C'') and LFL (B'', D'') demarcated (dotted line) from the adjacent vessels. Schematic included for anatomical reference. Note, for all images, the overlap of red and green fluorescence in the eye (labelled E) is due to a combination of natural pigmentation and auto-fluorescence; and is not indicative of photoconverted cells (C'', white arrowheads).
Figure 3(A) A cartoon representation of a crystal structure of the COP1 WD40 domain (grey) in complex with the TRIB1 tail (cyan) (PDB: 5IGQ). Point mutations introduced in the WD40 domain are shown in red. (B) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP-COP1 constructs and untagged TRIB1. (C) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (***p < 0.0001; ****p < 0.00001). (E) Cartoons summarizing the effects of TRIB1 binding and WD40 domain mutation/deletion on COP1 localization. (F) Proposed model for regulation of COP1 subcellular localization by an intramolecular regulatory site that competes with TRIB1 for binding to the WD40 domain.
Fig.Figure 1Chemical structures of IBL-301 (A) Chemical structures of IBL-302 (B). C. IC50 data of IBL-301, IBL-302 and IBL-202. IC50 values were calculated representing the percentage of inhibition against compound concentration and adjusting the experimental data to sigmoidal curve using the software Activity base from IDBS.
Figure 4Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal. Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal. Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal. Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal. Quantification of autophagosome-free lysosomes in (E). Experiments were performed three times, and one representative experiment is presented here. Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h.
TDP-25 inclusions are neither liquid-like nor solid-like.Figure 2(A-D) Primary rat hippocampal neurons were transduced with GFP-TDP-25 variants, GFP-NPM1 or Htt97Q-GFP and analyzed by fluorescence recovery after photobleaching (FRAP) at DIV5+8, except Htt97Q-GFP which was analyzed at DIV5+6 to avoid excessive toxicity. Example images with indicated bleach regions (dashed circles) are shown (A, C). Scale bar = 1.5 µm. Resulting normalized FRAP curves (relative fluorescence intensity over time) with values representing means ± SD are shown in (B, D). (B) Comparison of the averaged recovery fraction of the last six timepoints obtained from three independent experiments. GFP-NPM1 (0.982 ± 0.019, mean ± CI, n=37 cells) vs GFP-TDP-25 wt (0.097 ± 0.013, n=35 cells) vs GFP-TDP-25 mut (0.085 ± 0.006, n=38 cells): H(1)=74.134, df=2, ***p<2.2*10-16, η²[H]=0.674, Kruskal-Wallis Test. GFP-NPM1 vs GFP-TDP-25 wt: ***p<2*10-16, GFP-NPM1 vs GFP-TDP-25 mut: p<2*10-16, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.11, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. In (D), the recovery fraction averaged at >20 min timepoints was compared from four independent experiments. Htt97Q-GFP (0.094 ± 0.017, n=25 cells) vs GFP-TDP-25 wt (0.312 ± 0.041, n=23 cells) vs GFP-TDP-25 mut (0.271 ± 0.029, n=24 cells): H(1)=46.376, df=2, ***p=8.501*10-11, η²[H]=0.643, Kruskal-Wallis Test. Htt97Q-GFP vs GFP-TDP-25 wt ***p=1.9*10-11, Htt97Q-GFP vs GFP-TDP-25 mut ***p=4.3*10-12, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.12, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.
Figure 1A. Individual hESCs have the potential to generate another stem cell through self-renewal or to differentiate into a more lineage-specific cell type. B. Before differentiation, hESCs show uniformly high expression of OCT4. Treatment with BMP4 produces a mixture of OCT4+/CDX2- self-renewing hESCs and OCT4-/CDX2+ mesodermal cells. C. Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs. Cells were assigned to one of two distinct populations based on a 2-component mixed Gaussian distribution D. A fluorescent mCherry coding sequence was introduced into the endogenous OCT4 locus of H9 hESCs using CRISPR-mediated homologous recombination. E. Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration. Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity. F. Distribution of OCT4 levels in individual hESCs. A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell. G. Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration. H. Distribution of cell cycle durations for 120 hESCs.
H3.Figure 1Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. H3.3-GFP levels are increased at the E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom). Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ but there is little change in H3-GFP between Notch-OFF and Notch-ON nuclei ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom). Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). with H3.3core-GFP ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).
Figure 5A-B. Nucleosome occupancy at condensin binding sites during mitosis. Fission yeast cells expressing Cnd2-GFP were arrested in mitosis, mitotic indexes determined (A), and cells processed for MNase-seq. (B) NDRs are shown in purple and condensin binding sites in blue with a star indicating the peak maximum. Nucleosome patterns shown are representative examples of 3 biological replicates. C. Permutation test. 100,000 sets of 48 DNA segments of the same size of condensin binding sites were randomly drawn from the genome and the number of bp associated with a NDR by chance compared with the experimental value (purple bar). *** P<10-5. D. Metagene analysis of nucleosome occupancy within the NDR and positioning of the flanking nucleosomes. E. Coverage depth over NDR sequences plus upstream and downstream 200 bp. Boxes indicate the 25th, median and 75th percentile, whiskers indicate the upper and lower 1.5 IQR and circles outliers. *** P=10-14 by Kruskal-Wallis non parametric test. F. Cells arrested in mitosis were processed for ChIP against histone H3. Mitotic indexes are indicated in parentheses. %IP are averages and s.d. calculated from 6 ChIPs on 3 biological replicates. *** P<0.001, ** P<0.01, ° P>0.05. High-occup. and Low-occup. refer to condensin high-occupancy and low-occupancy binding sites, respectively.
Figure 5A) Fluorescent micrographs of a pachytene stage germ- cell nuclei from WT or zsp-1(gg682) animals expressing the indicated fluorescent proteins. Scale bar, 2μm. Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig. EV4B and Materials and Methods to determine; the size of P granules (panel B) Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed. Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine; the size of P or Z granules (panel C); or the number of P or Z granules surrounding a typical nucleus in wild-type and zsp-1(gg682) animals (panel D). Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed. ) Time-lapse imaging of the first embryonic cell division in zygotes of the indicated genotypes and expressing the indicated fluorescent proteins. Pronuclear meeting (pnm) time is defined as 0min. Times are relative to pnm. Scale bar, 5μm. F) Fluorescent micrographs of four cell embryos of WT or zsp-1(gg682) expressing the indicated fluorescent proteins. Scale bar, 5μm. G,H) GFP::ZNFX-1 (panel G) fluorescence or PGL-1::TagRFP fluorescence (panel H) in the anterior (A) or posterior (P) section of a one cell embryo. Anterior and posterior regions were determined based on corresponding DIC images. N=4 embryos for G, N=3 embryos for H. Error bars are +/- s.d.