EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Vertical section of macaqueretinal explant transfected with AAV2 hSyn:ReaChR-mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL. (B) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F-CAG:ReaChR-GFP after 3 days of incubation. GFP expression was primarily observed in the cell bodies but also in processes of RGCs; green: endogenous GFP fluorescence; blue: DAPI; outer nuclear layer (ONL). scale bars = 10 µm. (C) Averaged spike responses obtained from multi-electrode array recordings shown as PSTH of 4, 8 and 2 cells respectively of three different macaques (580 nm; 1.24 × 1017 photons cm-2 s-1). PSTHs are presented as mean ± SEM (dashed lines, calculated over responding cells). Raster plots of a representative cell from each individual macaque are shown below (10 stimulus-repetitions). (D) Action spectrum of ReaChR in primate RGCs from multi-electrode array recordings. Data is shown as mean ± SEM of the normalized firing rate, averaged from three individual macaques (28 cells, error bars calculated over cells), see also Fig EV2B for action spectrums of three individual macaques obtained from multi-electrode array recordings. (E) Responses (Hz) of a ReaChR-expressing RGC (patch clamp recording, in cell-attached mode) to flicker stimulations at increasing frequencies (2−22 Hz; 1.3 × 1016 photons cm-2 s-1).
Fig.Figure 6WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb for 24 h, Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test. WT and LRRK2 KO RAW264.7 macrophages were infected with Ca for 60 min Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test. WT and LRRK2 KO RAW264.7 macrophages were infected with Lm for 60 min. Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test. WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test. WT and LRRK2 KO RAW264.7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test. WT and LRRK2 KO RAW264.7 macrophages were infected with Lm WT or Lm Δhly. Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.
Minor intron splicing defects in CENATAC mutant cells fully correlate with mitotic defects.Figure 4RT-PCR (middle panel) and quantification (upper panel) of IPO5 minor intron 21 (U12-type) splicing on RNA extracted from HeLaEGFP-AID-CENATAC cells treated as in Figure 2C (three biological replicates, normalized to siCENATAC + IAA). The bottom panel shows RT-PCR analysis of IPO5 major intron 19 (U2-type). RT-PCRs of IPO5, SUDS3 and ZCCHC8 minor and adjacent major introns on RNA extracted from patient lymphoblasts or HeLaEGFP-AID-CENATAC cells depleted of GAPDH or CENATAC as indicated. u, unspliced; s, spliced. For each condition in (A), minor intron splicing (Figure 4A) was plotted against the percentage of cells showing a mitotic phenotype (Figure 2C). R2 and p-values are provided for the linear regression trendline (dotted line). RT-PCRs of ZRSR2 and GAPDH (to visualize ZRSR2 knockdown efficiency, upper) and RT-PCRs of SUDS3 minor intron 7 (middle) and major intron 5 (lower) on RNA extracted from HeLaEGFP-AID-CENATAC or DLD-1 cells depleted of GAPDH or ZRSR2 as indicated. Quantification of mitotic defects as in Appendix Figure S6 of H2B-mNeon-expressing HeLaEGFP-AID-CENATAC or DLD-1 cells treated as in (D) (three biological replicates, >103 cells in total per condition).
Figure 4(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium. (B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation). (D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers. (F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting (G) PiggyBac transposon was used to ectopically express HA-tagged IKKα in KDF1 deficient cells. WCL were collected and analyzed by immunoblots with different antibodies as indicated. (H) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification. Skin sections from grafted tissue were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bars = 50 µm.
Fig.Figure 2A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF. (C) Same as (A) in Cnx-KO MEF. (D) Same as (A) in WT MEF exposed to kifunensine (KIF). (E) Same as (A) in WT MEF exposed to Castanospermine (CST). (F) Same as (A) in sCNX MEF. (G) Same as (A) in Uggt1-KO MEF. (B) Radiolabeled ATZNNN enters in 2C1-positive polymers that are immunoisolated at the end of the chase times. BafA1 inhibits polymers degradation. Quantification for n=4. Unpaired two-tailed t-test, **** P<0.0001. (H, I) (H) Quantification of HA-positive or (I) of 2C1-positive endolysosomes (mean, n= 18, 14, 17, 13, 12, 12 cells for panels A, C-G , respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001. (J) Halo-ATZNNN in Uggt1-KO MEF mock-transfected (empty pcDNA3 plasmid). (K, L) (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1. (M) Quantification of Halo-ATZNNN-positive endolysosomes (mean, n=22, 10, 15 cells for panels J-L, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001).
Genetic inhibition of autophagy in the germline prevents DNA fragmentation.Figure 2.Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C-E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.
| Rotavirus NSP5 and NSP2 undergo phase-separation.Figure 4(a) Recombinantly expressed, Atto647-dye labelled NSP5, Atto488-dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their phase-separation properties. All labelled protein samples were mixed with the unlabeled NSP5 (1:10 molar ratio) to minimise the effect of labelling on LLPS. Top panel, left to right: NSP5-647/NSP2-488 droplets formed upon mixing of both proteins (10 μM each). Both channels are shown, along with an image of both channels overlaid. 4% (v/v) 1,6-hexanediol (1,6HD) dissolves these condensates. Scale bar, 10 µm. Middle panel, left to right: NSP5-647 (50 μM) + poly-arginine (polyArg, 5 μΜ); NSP5-polyArg condensate + 1,6-HD; NSP5-polyArg condensates + 0.5 M NaCl; NSP5 + R9 peptide (Arg-9, 5 μΜ). Bottom row, left to right: NSP5-647 sample (35 μM) alone; after addition of 10% v/v PEG-20K; NSP2-488 (25 μM) alone; after addition of 10% v/v PEG-20K. (b) Phase diagrams generated through droplet microfluidics for the coacervation of NSP2 and NSP5, in the presence of 0% v/v (left), 1.5% v/v (middle), and 4% v/v (right) 1,6HD. Phase diagrams were generated from N = 2206, 2035 and 1470 data points for each 1,6-hexanediol concentrations, respectively, and the data were used to construct the LLPS probability plots. (c) The number of viroplasms (black y axis on the left) and estimated cytoplasmic NSP5 concentration ([NSP5] µm, blue y axis on the right) at 2, 4, and 6 hours post infection. Viroplasms were counted in N = 595 ± 85 cells for each time point, and intracellular NSP5 concentration was determined by quantitative Western blotting
Comparative bulk RNA-seq analysis of microglia from brain and retina, cornea macrophages and bone marrow-derived monocytes.Figure 2Heat map of differentially expressed genes between rMG, bMG, cMΦ or bone marrow-derived monocytes (BM-Mo). The mean centered and s.d. scaled expression values for genes that were significantly and at least twofold more or less abundantly expressed are shown. See figure S6 for gating strategy. Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Principal component analysis of myeloid cell transcripts analyzed by RNA-seq. Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Comparison of functional gene clusters between BM-Mo, cMΦ and rMG in comparison to bMG (reference population). Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Left, spider plots showing commonly expressed genes across macrophages in comparison to bMG (reference population, center). Bold genes were plotted as bar graph on the right. Four samples were analyzed per cell type. Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Left, spider plots showing genes highly enriched in microglia in comparison to bMG (reference population, center). Bold genes are plotted as bar graphs on the right. Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Left, spider plots showing genes commonly expressed by monocytes and/or cMΦ in comparison to bMG (reference population, center). Bold genes are plotted as bar graphs (right). Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Left, bar graphs showing expression of the genes Mrc1, Itgax and Hexb in rMG, bMG, cMΦ or bone marrow-derived monocytes (BM-Mo). Right, validation of Mrc1 (CD206) in Cx3r1-GFP mice, Itgax in Itgax-GFP mice and Hexb in Hexb-Tomato mice in the cornea and the retina. Arrows point to CX3CR1+CD206+ cMΦ (top row) or Iba1+CD11c+ cMΦ or rMG (middle row) or Iba1+Hexb+ cMΦ or rMG (bottom row). Asterisks point CX3CR1+CD206- cMΦ (top row) or Iba1+CD11c- cMΦ or rMG (middle row) or Iba1+Hexb- cMΦ or rMG (bottom row). Data are derived from four independent experiments with 5-10 pooled mice per sample and shown as mean ± s.e.m. Scales bar represents 50 µm.
Figure 3Schematic representation of full-length PlexinD1 (PD1) and its deletion mutants. Co-IP from 293T cells 48 h after transfection. RhoJ-PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30-min stimulation with Sema3E. Arrows indicate PD1 Full (upper, 250 kDa) and PD1△RBD (lower, 1.7 kDa smaller than PD1 Full). PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation. Scale bar, 10 µm. Super-resolution images of subcellular distributions of ectopic WT-RhoJ (red) and endogenous PlexinD1 (green) in HUVECs 24 h after transfection. RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation. Scale bar, 1 µm. Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm. Quantification of areas of individual HUVECs. n > 100 cells per group. Data represent mean ± SEM. ***p < 0.001; NS, not significant, by Tukey-Kramer test. A model of Sema3E-induced cell contraction.
Figure 1A. A scheme for in vitro screening method of small molecules competing Dvl-CXXC5 binding. Briefly, purified Dvl PDZ domain was attached to the polystyrene surface of each well of 96-well plates. Then, PolyR-DBM (ployarginine conjugated Dvl binding motif tagged with FITC) (Kim et al, 2015) was added to each well and allowed to bind to the Dvl PDZ domain; 10 μM small molecule compound was added to each well, and the compounds competing with Dvl PZD-PolyR-DBM binding were measured by a microplate reader to screen the compounds reducing fluorescence signal. B . Screening results of compounds competing Dvl-CXXC5 interaction. Each 2 μM of the compounds was used for competing as described in Fig 1A. [n=3] C, D. Primary calvaria cells were treated with 2 μM of each compound for 2 days. The cells were subjected to immunofluorescence analyses to visualize β-catenin (C; green). The relative numbers of β-catenin-positive nuclei were counted (D). [n=3] E, F . The calvariae from 4-day-old mice were cultured ex vivo for 7 days with 2 µM of each compound. Representative calvaria sections were visualized by H&amp;amp;E staining (E). The calvaria thicknesses were measured from the images using Image Pro software (F). [n=3]
Microglial phagocytosis of photoreceptors in alternative mouse models of retinitis pigmentosa (RP) and in human histopathological specimens.Figure 4A-C Evidence of microglial phagocytosis of photoreceptors in other mouse models of RP. Histological analysis of a rd1mouseretina (A) demonstrates microglial phagocytosis of photoreceptornuclei in vibratome sections (left) and in flat-mounted retina (right). Phagocytosed nuclei were predominantly negative for TUNEL staining (arrows). Similar findings were found in the retinas of the rd16mouse (loss-of-function mutation in the photoreceptor-expressed CEP290 gene, 1 month old) (B) and the RPGRIP-deficient mouse (6 months old) (C). Scale bar, 20 μm. D-G Evidence of microglial phagocytosis of photoreceptors in other human histopathological specimens of RP. (D) Retinal section from a 30-year-old male donor with autosomal recessive RP (AR RP) showing extensive microglial infiltration of the ONL; expanded inset (right) shows multiple photoreceptor nuclei in phagosomes that were predominantly negative for TUNEL staining. (E, F) Retinal sections from two separate donors with autosomal dominant RP (AD RP1 = 68-year-old man, T17M rhodopsin mutation, AD RP1 = 50-year-old woman, Q-64-ter rhodopsin mutation) showing similar evidence of microglial phagocytosis. (G) Retinal section from a 46-year-old male donor with X-linked RP (XL RP). Arrowheads indicate phagocytosed photoreceptor nuclei. Scale bars, 20 μm.
| Application of MOFA to a study of chronic lymphocytic leukaemia.Figure 2 (a) Study overview and data types. Data modalities are shown in different rows (D = number of features) and samples in columns, with missing samples shown using grey bars (b) Proportion of total variance explained (R2) by individual factors for each assa (c) cumulative proportion of total variance explained (d) Absolute loadings of the top features of Factors 1 and 2 in the Mutations data (e) Visualisation of samples using Factors 1 and 2. The colors denote the IGHV status of the tumors; symbol shape and color tone indicate chromosome 12 trisomy status (f) Number of enriched Reactome gene sets per factor based on the gene expression data (FDR< 1%). The colors denote categories of related pathways defined as in Appendix Table S2
Spine density is selectively affected along the radial axis of CA1.Figure 3A, B: Spine density of deep CA1 pyramidal cells is significantly reduced in basal dendritic segments (A; unpaired Student's t-test, **p = 0.0067), while it is unchanged in midapical dendritic segments (B; unpaired Student's t-test, nsp = 0.4949). C: Representative images of basal and midapical dendritic segments from both radial layers. Brightness and contrast were adjusted to ensure a uniform appearance. Scale bar: 5 µm. D D, E: Spine density of superficial CA1 pyramidal cells is normal in basal dendritic segments (D; unpaired Student's t-test, nsp = 0.3004), while it is significantly reduced in midapical dendritic segments (E; unpaired Student's t-test, ***p = 0.0005). F: Schematic representation of affected regions dependent on position of the pyramidal cell along the radial axis of CA1.
Figure 5A Comparisons of NSP1 proteins from seven human coronaviruses. B Principal Component Analysis of the seven HCIP lists with bait NSP1 from different human coronaviruses. C Heat map of the NSP1 HCIPs obtained using SFB- TAP. NSP1 HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Six areas of the heat map are manually selected, enlarged and labeled as (1), (2), (3), (4), (5), and (6). These groups of proteins are enriched by NSP1 protein specific from one or limited human coronaviruses. Functional characterization of each protein is shown with the red circles below the enlarged images. D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1/PYCR2. Cells transfected with vector or construct encoding GFP were included as controls in these experiments.
Figure 2A A positive control region shows suppression signal. Average suppression score (y-axis) for TS alleles of query genes located on the left arm of chromosome XVI (TS allele index, sorted by chromosome coordinates, x-axis) across strains with a translocation that generates a duplication of the shaded area (blue markers), and strains without the translocation (grey markers). Dashed line: y = 0.75 (suppression cut-off in screen). B B Genotype and phenotype trees are concordant. Top: Hierarchical clustering (UPGMA) of the ten wild strains used in this study based on sharing segregating sites. Colours: global genetic cluster membership. Bottom: as top, but based on correlation distance between genetic suppression profiles. Strain abbreviations as in Figure 1A, with in addition: NC = NCYC110, UW = UWOPS87-2421, and 27 = 273614N. C C About a third of suppressed alleles can be explained by a deficiency in the reference strain that is not present in the majority of wild strains. The frequency (y-axis) of the number of wild strains that suppress a TS allele (x-axis) for the 187 alleles that could be suppressed by at least one strain, using a more lenient criterion (suppression > 0.5; no z-score cut-off) to avoid edge effects and winner's curse. D D Genetic suppression is consistent across different TS alleles of the same gene. Suppression score (colour scale) in crosses to different wild strains (x-axis) for TS alleles (y-axis) of GAB1 and NSE4 genes. E Genetic suppression is consistent across genes encoding members of the same complex. Strongest suppression score across TS alleles for a gene (colour scale, as in (D)) in crosses to different wild strains (x-axis) for genes (y-axis) that encode members of the GPI-anchor trans-amidase complex (bottom) or the nuclear condensin complex (top). The G GPI16 gene suppression was not estimated in the NCYC110 strain due to chromosome II copy number variation ("X").
The 53BP1 repair compartment is sensitive to hyperosmotic stress.Figure 1Asynchronously growing human U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC. Representative images from the QIBC analysis in (A). Scale bars, 10 μm. Cells were treated with 0.5 Gy of IR in absence or presence of 0.4 M sorbitol, fixed 1h later, and 53BP1 assembly at DSBs was analyzed by QIBC. Treatments as in (C); RIF1 accumulation at DSBs was analyzed by QIBC.
Wnt4‐independent canonical Wnt signaling during mammary gland developmentX‐gal‐stained whole‐mounts of Axin2::LacZ embryos at E14.Figure 5A X‐gal‐stained whole‐mounts of Axin2::LacZ embryos at E14.5 either Wnt4+/+or Wnt4−/− (n = 5 or 4, respectively). Arrows mark mammary buds. Scale bar: 5 mm. B Whole‐mount analysis of X‐gal‐ and carmine alum‐stained mammary glands harvested from 6‐week‐old littermates either Wnt4fl/wt;MMTV::Cre+;Axin2+/lacZ (n = 5) or Wnt4fl/flMMTV::Cre+;Axin2+/lacZ (n = 4). Left insets: higher magnification; right insets: histology section of the same gland. Note expression of reporter detected around the neck of TEBs in Wnt4‐deficient and littermate control is comparable. LN, lymph node. Scale bars: 1 mm, and inset scale bar: 100 μm.
a) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell.figf1(a) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell. U2OS cells were fixed and stained with anti-p62 (red) and anti-MKLP1 (green) antibodies. Scale bars are 10 μm. Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division. The arrowheads point to MRs, which are shown enlarged in the insets. The quantification is presented in Supplementary Information Fig. S1a. (b) siRNA-mediated depletion of p62 from U2OS cells results in moderate MR accumulation, compared with a control depletion (lamin A/C). For quantification, MRs were counted in control cells and in p62-depleted cells that were negative for p62 staining. Data are means ± s.d. of three independent experiments (n, total number of cells counted; P, probability of a two-tailed paired t-test).
a, Wild-type and two independent parkin-deficient D.f5a, Wild-type and two independent parkin-deficient D. melanogaster lines (parkc00062, parkf01950) were infected with L. monocytogenes by anterior abdomen injection. Atg8 processing was monitored in whole-fly protein lysates by western blotting. b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3-5, **P < 0.001 by Student's t-test). c, Survival of D. melanogaster was determined (n = 33, **P < 0.001 by log-rank test). d, e, Flies were infected with S. enterica serovar Typhimurium and bacterial c.f.u. (d) (means ± s.d., n = 3-5 per group, **P  0.009 by Student's t-test) and fly survival (e) were determined (n = 59, **P  0.001 by log-rank test). f, Survival of flies infected with M. marinum (n = 37, **P  0.0045 by log-rank test).