EMBO/sd-character-level-ner huggingface dataset:
A variant deposition at the S region.Figure 6(A) Schematic illustration of the Sμ and Sα regions showing the position of the ChIP PCR products. (B) ChIP analysis of H2A and its variants using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The antibodies used for each ChIP set are indicated at right. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to the siControl by two-tailed unpaired Student's t-test ( *p ≤ 0.05, ** p ≤0.01). (C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)
Figure 1(A) The schematic of the Drosophila Ash1 protein organization. Ash1 is 2226 amino acid long and contains eight domains (indicated by colored rectangles). The SET domain together with the AWS (Associated With SET) and the post-SET domains are necessary and sufficient for Ash1 histone methyltransferase (HMTase) activity. The functions of the BAH (Bromo Adjacent Homology), PHD (Plant homeodomain) and AT-hook domains are unknown. The positions of ash122 and ash121 point mutations are indicated by arrows. (B) Segmental expression of the Drosophila bithorax complex genes. The three genes of the complex Ubx, abd-A and Abd-B are shown as colored rectangles. The expression of Ubx gives identity to the third thoracic (T3) and the first abdominal (A1) segments, the expression of abd-A defines the second, third and fourth abdominal segments (A2-A4) and the expression of Abd-B gives identity to the rest of the abdominal segments (illustrated with corresponding color code). (C) Adult phenotypes of the ash1 and NSD mutants. In ash122/ash121 mutants (designated as ash1-) the loss of Abd-B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4, which is visible from the partial loss of pigmentation on tergites 5 and 6 (t5 and t6) and appearance of bristles on sternite 6 (s6, marked with black arrows). The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere (H) to wing and third leg (3L) to second leg (2L) transformations. The former is evident from the change in the haltere shape and the appearance of multiple bristles (black arrowheads). The latter is indicated by the apical and pre-apical bristles (red arrows) on the tibia of the third leg of ash1 mutants. These are normally present on 2L but absent on 3L (compare to wild-type). Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1- flies (red arrowheads), which indicate its transformation towards the second thoracic segment. Phenotype of the NSDds46/NSDds46 (NSD-) flies is indistinguishable from wild-type and the phenotype of the double ash122,NSDds46/ ash122,NSDds46 (ash1-,NSD-) flies is no more severe than that of the single ash122/ash121 (ash1-) mutants. (D) Ubx expression in the haltere imaginal discs. The expression was assayed by immunostaining with antibodies against Ubx (red) and acetylated H3K18 (green, positive control). While ash122/ash121 (ash1-) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs (yellow dashed lines), Set2- larvae have uniform expression of Ubx throughout the haltere disc, resembling that in the wild-type larvae. Scale bars indicate 100μm.
L-MICU2 and structural comparison of MICU1 and MICU2.Figure 1A, B Space filling and cartoon representation of two MICU2 (A) and T4L molecules (B). The protein chain with two calcium ions bound is colored in yellow and the protein chain with one calcium ion bound is colored in purple. Calcium ions are shown as red spheres. C The structure of Ca2+-bound MICU2 (molecule 2) is superimposed with Ca2+-bound MICU1 (PDB code: 4NSD). MICU1 is colored in gray; MICU2 is colored in orange, marine and green-cyan for different domains. Calcium ions are shown as blue and red spheres in MICU1 and MICU2, respectively. D, E Comparison of C-lobes of MICU2 (green-cyan) and MICU1 (gray) in the Ca2+-free (D) and Ca2+-bound (E) states. F Comparison of N-lobes of MICU1 (gray) and MICU2 (orange) shows large divergence in the EF-hand 1 (box in red dotted line) and β-sheet (box in black dotted line). Calcium ions are shown as blue and red spheres in MICU1 and MICU2, respectively. G Superimposition of MICU2 EF-hand 2 in Ca2+-bound (green-cyan) form and Ca2+-free form (bright green). Calcium ion is shown as a red sphere. H Superimposition of MICU2 EF-hand 1(with Ca2+-bound) and MICU1 Ca2+-bound EF-hand 1. Calcium ions are shown as blue and red spheres in MICU1 and MICU2, respectively. I Superimposition of MICU2 EF-hand 1 (with Ca2+-bound) and MICU1 Ca2+-free EF-hand 1. The angle (yellow) represents the conformational difference. The calcium ion in MICU2 is shown as a red sphere.
RNAlow neoblasts exhibit TOR-dependent cell cycle entry during regeneration.Figure 5A Schematic of the experimental design. B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment. C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55). D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5-10). E RNAlow neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54). F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.
Chronic oxidative stress reduces total H2AX protein levels and prevents normal accumulation of γ-H2AX.Figure 1A Up, Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD-/- fibroblasts (Left) or in wt and Nfe2l2-/- fibroblasts (Right). Two representative clones, with their corresponding controls, are shown per genotype. H2B was used as negative control. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). n ≥ 3 independent experiments. B Up, Representative western blot from kidney whole cell extracts showing H2AX protein levels in age-matched wt and junD-/- mice. Mice are 2 months old (young) and 18 months old (old) mice, respectively. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 5 mice per age and per genotype. C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype. D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments. F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, following 2 Gy of γ-irradiation (F) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments. G,I Up, Representative γ-H2AX immunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after γ-irradiation (2Gy, 45 min) (G). Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification. F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, after camptothecin (CPT) treatment (H) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments. G,I Up, Representative γ-H2AXimmunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after after camptothecin (CPT) treatment for 1h (I).. Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.
Figure 7A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Sol (Sol.), Mem (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively. B Model of rhomboid-mediated quality control by selectively targeting orphan components of multi-protein respiratory complexes.
Fig.Figure 1(A-C) (A) Schematic of the LoC model for SARS-CoV-2. Maximum intensity projections show that confluent layers of alveolar epithelial cells (ATs) and endothelial cells with strong expression of tight junction markers populate the top (B) and bottom faces (C) of the porous membrane. Actin, CD31, TJP-1 and nuclear labelling are shown in azure, spring green, bright pink, and electric indigo LUTs, respectively. (D) ACE2 levels were characterized using RNAscope. A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air-liquid interface (3C) is shown. The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT. (G) Same as in (D) with infected LoC at 3 dpi (G) shown. (E, F) qRT-PCR characterization of the AT and endothelial layers on-chip for SARS-CoV-2 entry markers (ACE2, TMPRSS2, NRP1) for cells extracted from the apical and vascular channels from uninfected LoCs (n=3 biological replicates) (E) and infected LoCs (n=4 biological replicates) at 1 day-post infection (F). In both cases expression is normalized to GAPDH levels. Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. Scale bar = 20 μm. (H) ACE2 expression decreases between 1 and 3 dpi in the LoC model, quantified on a per-cell basis for n=4 or 5 fields of view (technical replicates) across control and infected chips (n=1 control and n=1 infected chip) at each timepoint. Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. Scale bar = 20 μm. (I, J) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day-post infection (n=4 biological replicates) and 3 days-post infection (n=3 biological replicates) vs. uninfected control LoCs (n=3 biological replicates). Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001. Scale bar = 20 μm.
Figure 4A. DNA binding fractions by dSaCas9 at the fully matched (target) and partially matched DNA target positions, as revealed by the reverse DNA unzipping assays (n = 15, 16, 22, 26 and 46 from left to right). The subscript "MM" represents the mismatches of the DNA sequence with the sgRNAs. B. Positions and strengths of the pre-PAM interactions (n7-22MM = 21, n11-22MM = 41, n15-22MM = 39) and post-PAM interactions (n7-22MM = 21, n11-22MM = 25, n15-22MM = 45) between dSaCas9/sgRNA and mismatched DNA targets. The error bars represent the SD. C. Schematic depiction of the stopped-flow experiment. 2-AP was substituted at position +18 of the nontarget strand. The 2-AP fluorescence signals as a function of time are shown for four sgRNAs. The solid lines are double-exponential fits of the data D. A summary of fitted values of rates. E. Cleavage activities of SaCas9 on DNA targets with sgRNA containing bases mismatched to DNA. The reactions were quenched at five time points (0, 15, 30, 45, and 60 min). The experiment was performed in triplicate, and a representative gel is shown.
Figure 5A-G HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR antibody, DAPI in combination with (A) anti-PCNA-1 antibody, (B) anti-FANCD2 antibody, (C) anti-BRCA1 antibody, (D) anti-PRP19 antibody, (E) anti-Sm(Y12) antibody, (F) anti-HuR antibody and (G) pS2-RPOL2 (H5) antibody. Intensity profiles along the white line in the merged image are presented on the right side; yellow and green arrows mark GMGs.
Predicting gene function from phenotype profiles (see also ).Figure 4Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds. Examples of gene clusters obtained with hierarchical clustering of phenotype profiles composed of the 17 specific phenotype fractions. Phenotypes 1-17: [1] coat: increased patch number; [2] coat: aggregate; [3] vacuole: class E; [4] late endosome: condensed; [5] actin: bright patches; [6] late endosome: membrane; [7] actin: aggregate; [8] coat: decreased patch number; [9] actin: decreased patch number; [10] late endosome: fragmented; [11] coat: depolarized patches; [12] actin: depolarized patches; [13] vacuole: multilobed; [14] vacuole: fragmented; [15] vacuole: enlarged; [16] vacuole: class G; [17] vacuole Interaction network of NNF2 and YER043W (GTA1). Genes with phenotype profiles with a correlation > 0.7 and genetic interaction profiles with a correlation > 0.2, and at least two significant correlations to NNF2 and/or GTA1 were included in the network. Analysis of phenotype profile similarity between mutants in genes encoding proteins in same or different protein complex structures. Box plot indicates distribution of PCCs between pairs of phenotype profiles for genes that encode protein pairs in direct contact in a protein complex experimental structure (Yes - Direct), code for protein pairs in the same protein complex structure but not in direct contact (Yes - Indirect), and code for protein pairs that do not belong to the same protein complex structure (No). The number of pairs evaluated in each set is shown on the x-axis. Significance was determined using one-sided Mann-Whitney U tests. ***p-value < 0.001. Differentiation of functionally related protein complexes and protein complex organization using phenotype profiles. Heatmaps showing PCCs between components of the ESCRT complexes (E) and the SPOTS complex (F). A more intense blue colour indicates a higher PCC (scale bar at the top left of each heat map).
Regulation of Swi6 dynamics and the role of Swi6 in heterochromatin spreading, boundary formation, and repression.Figure 7Regulation of Swi6 dynamics and the role of Swi6 in heterochromatin spreading, boundary formation, and repressionModel for the formation of a distinct heterochromatic domain. The spreading‐competent open conformation of Swi6 dimers is fully dispensable or acts redundantly with RNAi‐mediated spreading mechanisms in the formation of constitutive heterochromatin. The spreading of heterochromatin into neighboring euchromatin in the absence of DNA‐encoded boundary elements, such as TFIIIC binding sites, can be stopped by the spreading‐incompetent closed conformation of Swi6 dimers, RNA‐mediated eviction of spreading‐competent Swi6, or Epe1 activity. Swi6 molecules interact with H3K9me2 only transiently and exchange between free and RNA‐bound forms (2, 1, or 3, respectively; see also C). Removal of RNA from Swi6 constitutes a rate‐limiting step in the Swi6 exchange cycle (blue arrow) and contributes to tight repression of heterochromatin.In the absence of Swi6, H3K9 methylation is mediated by the RITS complex (Ago1, Chp1, Tas3) and can spread beyond natural boundaries via self‐associating Tas3. This might occur independently of siRNAs.In theory, Swi6 can exist in at least three kinetically distinct populations: 1) freely diffusible Swi6, 2) Swi6 bound to H3K9me2, and 3) Swi6 bound to RNA; 3 is the least and 1 the most mobile population. The blue arrow indicates the rate‐limiting step in the Swi6 exchange cycle. Under physiological conditions, most Swi6 molecules exchange between the RNA‐ and H3K9me2‐bound forms.
Figure 7A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm. B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test. D. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test. E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test. Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test. G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm. H. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.
Loss of MpFGMYB function results in sperm formation in genetically female plants A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants.Figure 4A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm. D RT-PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed. E, F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bars, 100 nm.
Figure 6A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right. C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation. E, F. Size and duration of Cm decay during bulk endocytosis. G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.
Figure 5Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm. Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the SVZ of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm. (C) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar-/- mice. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test. (D) RT-qPCR analysis of relative Fancc mRNA levels from whole brains or testes in mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. Mean ± SEM of n=3 biological replicates, *p < 0.05 and ns by Student's t test. (E) RT-qPCR analysis of relative Fancc or Fancl mRNA levels from whole brains from mock- and ZIKV Paraiba infected Ifnar-/- newborn mice. Mean ± SEM of n=3 biological replicates, ***p < 0.001 by Student's t test. (F) Western blot analysis of Fancc protein in brain regions from mock- and ZIKV Paraiba infected Ifnar-/- mice six days post-infection. HP = hippocampus, CT= cortex, ST = striatum and OB= Olfactory Bulb. (G) Bar graph showing the quantification of Fancc proteins, mean ± SEM of n=3 biological replicates, *p<0.05 and ns by Student's t test. (H) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post-infection. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test. (I) qRT-PCR for Ifnab gene expression in wild type and Fancc KO mice, Mean ± SEM of n=3 biological replicates, ns by Student's t test.