EMBO/sd-character-level-ner huggingface dataset:
Figure 1TRIP6 promotes YAP activity by inhibiting LATS1/2 (A) Full length (WT), the amino-terminal half (1-277), or the carboxy-terminal half (278-476) of TRIP6 were tested for binding to LATS2 by immunoprecipitation. FLAG-TRIP6 variants were co-expressed with LATS2-GFP in HEK293 cells, anti-FLAG or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS2-GFP and FLAG-TRIP6. Schematic diagram depicts TRIP6 domains (NES: Nuclear Export Signal; LIM: LIM domain; PDZ: PDZ domain binding motif). (B) Lysates from MCF10A cells were subjected to immunoprecipitation using anti-TRIP6 or control (IgG) antibodies, and immune complexes and lysates were probed for TRIP6 and LATS1. (C) FLAG-TRIP6 was tested for binding to various LATS2-GFP deletion mutants as described in part (A). Schematic diagram of LATS2 shows MOB1 binding domain, and the autophosphorylation (S872) and MST1/2 phosphorylation sites (T1041) in the kinase domain. The regions marked in green depict TRIP6 binding sites on LATS2 (D) Lysates from HEK293A cells transfected with control or FLAG-TRIP6 plasmid were analyzed by western blotting using the indicated antibodies (quantification is shown in (F)). (E) Lysates from control (WT) or CRISPR generated TRIP6 null (TRIP6-KO) HEK293A cells were analyzed by western blotting using the indicated antibodies (quantification shown in (G)). (F) The relative levels of LATS1 activating phosphorylation (pLATS1-1079, 909) and YAP S127 inhibitory phosphorylation from (D) were measured relative to LATS1 and YAP levels respectively. (Mean ± SD; n=3; **P≤0.01, ***P≤0.001, T-test) (G) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part (E) were quantified (Mean ± SD; n=3; *P≤0.05, T-test). (H) GFP-MST2 was expressed with or without FLAG-TRIP6 in HEK293 cells and the levels of MST2, MST2 activating phosphorylation (pMST2-T180), and FLAG-TRIP6 were measured by western blotting with the indicated antibodies. (Mean ± SD; n=3; n.s.≥0.05, T-test). (I) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6, and YAP target gene expression was analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ****P≤0.0001, T-test). (J) The levels of YAP target gene expression was analyzed using RT-qPCR in control (WT) and TRIP6-KO HEK293A cells. (Mean ± SD; n=3; ***P≤0.001, T-test) (K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.
Figure 2A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time. B Overlap between UACC257 and A2058 datasets. From top to bottom: Identified proteins, identified phosphopeptides, EDN regulated phosphopeptides at q < 0.1, and q < 0.1 & FC > 1.5 up or down. C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.
Formation of Lewy body-like α-synuclein inclusions in cultured cells.Figure 1A) Purified His-α-synuclein protein was agitated for 0-2 weeks (lane 1, 0weeks; lanes 2 and 3, 1week; lanes 4 and 5, 2weeks). Aliquots from each sample were separated into supernatant (lanes 2 and 4) and pellet (lanes3 and 5) fractions. These fractions were subjected to immunoblotting with anti-α-synuclein antibody. Numbers at left are molecular weight markers (MW) in kilodaltons. (B) Fibril formation of α-synuclein was measured with ProteoStat, an aggregation detection dye. a.u., arbitrary units. (C) AFM images of α-synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode. A high-magnification view of the boxed area is shown in the lower panel. The scale on the right represents the height of pixels in the image. Scale bars, 100 nm. (D) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.
Impaired Nup35 recruitment can explain the abnormal membrane channels caused by excess PA to DAG conversion A) The NPC is comprised of several subcomplexes.Figure 8The NPC is comprised of several subcomplexes. Confocal imaging of 5DO fat body nuclei expressing UAS transgenes and labeled by anti-Megator/Tpr, Nup214, Nup35, Confocal imaging of 5DO fat body nuclei expressing UAS transgenes and labeled by mAb414, or expressing Nup107RFP. mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1-/- animal. *** and **** indicate significant difference compared to control mGFPSec61b expressing cells. Chi-square test followed by individual post hoc Chi-square tests. *** p < 0.001 and **** p < 0.0001. NE ultrastructure of 5DO fat body cells from a (I) control animal, (J) homozygous Nup35BG01311 and (K) homozygous Nup35MB02683 animals. Nuc, nucleus; Cyto, cytosol. Model of the relationship between nucleocytoplasmic channels, PA metabolism, and NPC maturation.
Figure 5(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group. (B) A visualization of selected differentially expressed pathways comparing the virus+remdesivir group to the control group using gene set enrichment analysis (GSEA). Y-axis represents normalized enrichment score (NES), positive value means higher expression in the virus+remdesivir group compared to control, vice versa. The dashed line corresponds to NES=0 (i.e. separating positive and negative enrichment). Pathways on the X-axis are ordered by their NES values, all pathways displayed have adjusted P<0.05; pathways with names in red are those that are not significantly different (adjusted P>0.2) when comparing the virus group to the control group using GSEA, i.e. changes in these pathways may arise from remdesivir-specific effects. The dot color corresponds to the negative log10 adjusted GSEA P value, and dot size corresponds to the number of enriched genes (i.e. "leading edge" genes in GSEA). (C) PCA plot of the average metabolic flux profile computed using the iMAT algorithm (Shlomi et al. 2008; Methods) representative of each of the experimental groups, the labels are the same as (A). (D) A visualization of metabolic pathway enrichment results of the differential metabolic fluxes in the virus+remdesivir group vs the control group, using Fisher's exact tests (Methods). Y-axis represents the odds ratio of enrichment, the horizontal dashed line corresponds to odds ratio of 1. The dot color corresponds to the negative log10 adjusted one-sided P value from Fisher's exact tests, and dot size corresponds to the number of enriched target reactions. The datasets are ordered by P values, all pathways displayed have FDR<0.1. (E) The robust metabolic transformation algorithm (rMTA, Valcárcel et al. 2019) was used to predict metabolic reactions whose knock-out can further transform the metabolic state of the remdesivir-treated SARS-CoV-2-infected cells back to the normal control state, using the Vero E6 cell samples (Methods). The significant metabolic pathways (FDR<0.1) enriched by the top 10% MTA-predicted targets are shown. Axes and the meanings of dot color and size are similar to (D).
Figure 3A Super-resolution SIM image of CHAF1A in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibody was used to label endogenous CHAF1A which was colored into green. B Super-resolution cSTORM image of CHAF1A in HEK293T cells. Two degrees of amplification were performed to show CAF-1 bodies. C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα. E RFP-tagged CHAF1A and GFP-tagged PML body component SP100 were co-overexpressed in HEK293T cells and imaged by SIM. F GFP-tagged CHAF1A and RFP-tagged BRD4 were co-overexpressed in HEK293T cells and imaged by SIM. SUV39H1, HP1α, DNMT1, MBD1, RING1B, EZH2 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination. SUMO4 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination. N-P GFP-tagged CHAF1A was overexpressed in HEK293T cells. CF568-tagged antibodies against H3K9me3, H3K27me3 and H3K4me3 were used to treat IF samples respectively. CHAF1A and each histone modification were imaged by SIM.
Striatal infusion of the high dose of cholesterol rescues synaptic activity and ultrastructure of inhibitory synapses in MSNs of HD mice.Figure 3(A) Spontaneous IPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of 0 mV. (B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001). (D-E) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (D) Representative FIB-SEM segmentation and reconstruction of inhibitory synapses (green) in 200 serial sections of striatum for a total volume of 500 µm3 in wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (E) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 mice/ group). Data information: The data E) are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of synapses counted /µm3 in different blocks of tissue for each group of animals (N=3 animals/group) (E). Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).
Figure 3A) Fft2 and Fft3 are both enriched at LTRs, while Fft2 is also enriched at the tf2 coding region. Fft2 LTR enrichment increases in fft3Δ cells, while Fft3 LTR enrichment decreases slightly in fft2Δ cells. Top: Fft2 -myc enrichment in WT (solid line) and in fft3Δ (dashed line). Bottom: Fft3 -myc enrichment in WT (solid line) and in fft2Δ (dashed line). ChIP-chip signal is normalized against no-epitope control arrays. B) Nucleosome occupancy over the LTR U3 is significantly reduced (average reduction ~50%; average p-values at each position <0.001, Poisson test) in fft2Δ fft3Δ cells. MNase-seq data aligned at the start of tf25'LTRs is shown for WT (black line), and fft2Δ fft3Δ (dark gray line). Light gray line indicates significance of difference between WT and mutant signals: amplitude reflects significance (p-value) and sign reflects the sign of occupancy change in mutant vs. WT cells. Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements. LTR and tf2ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements, while the green dot indicates the TSS in fft2Δ fft3Δ.
Figure 6Representative images of MAP2 positive WT and Mecp2 null neurons (DIV7). At DIV5 neurons were treated with tubacin (TUB, 1µM for 48 h), Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h) or left untreated and morphologically analyzed by Sholl analysis plug-in. Scale bar 20 µm. The graph reports the mean ± S.E. of the number of intersections measured 85 to 145 nm from the soma calculated by Sholl analysis for WT (n=39 neurons) and Mecp2 null neurons, when left untreated (n=57 neurons) or treated with tubacin (n=53 neurons) or TC-S 7010 (n=38 neurons). Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, **p<0.01). Representative images of WT and Mecp2 null neurons (DIV14) immunostained for MAP2 (white), Synapsin1/2 (green) and Shank2 (red). Mecp2 null cells were treated at DIV12 with tubacin (TUB, 1µM for 48 h) or Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h). Scale bar 15 µm, and 5 µm in the enlarged image. The graphs represent the mean ± S.E. of the Synapsin1/2 and the Shank2 puncta density in WT (n=67 neurons) and Mecp2 null neurons, when untreated (n=51 neurons) or treated with tubacin (n=59 neurons) or TC-S 7010 (n=55 neurons). Neurons derived from at least 6 different biological replicates. Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01, ***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, ***p<0.00­­1). The graph depicts the mean ± S.E. of the number of co-localized pre- and post-synaptic puncta (n=20 neurons deriving from 3 different biological samples). Data from WT and null neurons were compared by Mann-Whitney test (***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05).
LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4‐mCherry (top) or IBIS1‐mCherry (bottom) were fixed 22 h or 48 h post‐infection, respectively, and stained with anti‐LC3 to label autophagic membranes.Figure 9LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVNHepatoma cells infected with Plasmodium berghei expressing UIS4‐mCherry (top) or IBIS1‐mCherry (bottom) were fixed 22 h or 48 h post‐infection, respectively, and stained with anti‐LC3 to label autophagic membranes. In the right panels, fluorescence is merged, and selected areas of interest are shown enlarged. Scale bars, 10 µm.
Figure 8A. Diagram representing interhomolog strand invasion between red and blue homologous chromosomes during meiotic prophase I. The resulting displacement loop contains a mismatch (bulge), which is recognized by a MSH2 MutS heterodimers. This causes recruitment or activation of the Class I (ZMM) repair pathway and increases the chance of crossover repair at, or in proximity to, the mismatched intermediate. B. Diagram representing the same scenario as in A., but here MSH2 dependent recognition of the mismatch inhibits progression of recombination. This triggers feedback signaling that recruits SPO11-1 complexes to this region and increases meiotic DSB and crossover frequency. C. Comparison of the budding yeast and Arabidopsis physical maps (blue) and genetic maps (red) (Serra et al, 2018b; Mancera et al, 2008), shown in megabases and centiMorgans, respectively. D. Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col×Ler and budding yeast S288c×SK1 crosses (Cooper et al, 2018). Telomere positions are shown by vertical lines, and centromeres by vertical dashed lines. Centromeres are also labeled above the plots in red, as 'CEN' or 'C'.
a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells.figf8(a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells. A549 cells stably expressing GFP-LC3, with Rubicon or control shRNA, were incubated in nutrient-rich medium with or without the protease inhibitors (PI) E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) treatment for 4 h. The cells were stained with an anti-Lamp1 antibody (upper panels) and colocalization efficiency between GFP-LC3 and Lamp1 was counted (lower panel). The data are mean ± s.d. of 30 cells. (b). Electron microscopy analysis of Rubicon knockdown cells. Control (luciferase) or Rubicon shRNA A549 cells grown in nutrient-rich medium was subjected to electron microscopy analysis (left panels). The arrows indicate autophagosomes and the arrowheads indicate autolysosomes. Scale bar, 1 μm. Autophagosomes and autolysosomes were counted in 21 cells, and the mean ± s.d. are shown (right panel). (c) EGFR degradation in Rubicon knockdown cells. Control or Rubicon shRNA A549 cells were treated with EGF (200 ng ml−1) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated (left panel). The band intensity was measured in three independent experiments and the mean ± s.d. are shown (right panel). (d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies. (e) Model of the function of three Beclin 1 containing complexes. The Atg14L complex functions in autophagosome formation. The UVRAG complex functions in autophagosome and endosome maturation. The Rubicon-UVRAG complex suppresses autophagosome and endosome maturation. Enhanced autophagosome maturation and/or endocytosis may lead to enhanced autophagosome formation. Full scans of the blots in c and d are available in Supplementary Information, Fig. S6. (d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies.
Figure 1(A-B) Illustration depicting the optogenetic system employed to control NICD nuclear translocation (opto-Notch). NICD (purple) with its endogenous NLS deleted was tagged with the AsLOV2 domain (turquoise) from Avena Sativa is such a way that its Jα helix masks an engineered NLS in the dark (A). To ensure maximal nuclear exclusion, Zdark (blue dashed line on the mitochondrion), a protein domain which binds specifically to the dark conformation of AsLOV2, was localised to mitochondria through a TOM20 targeting motif (not depicted). Blue light illumination triggers a conformational change in the LOV2 domain, which results in the simultaneous unbinding from mitochondria and exposure of the engineered NLS, thus allowing NICD nuclear translocation and target gene (sim) expression, appearing as a green dot in the nucleus (B). (C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm. (E) Wild-type sim expression profile in a Drosophila embryo at the onset of gastrulation as revealed by in situ hybridization. Scale bar, 50 μm (F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.
Figure 4(A) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. (B) Transition density plot of idealized ATP FRET states obtained in (A). (C) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve). (E) Comparison of high and low FRET state occupancy in ADP•Pi and ADP•Vi for the clamp and THF. (F) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (G) As in (F), but with high FRET (1773 high FRET states). (H) Comparison of dwell times of the high FRET states for the two-helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two sample Kolmogorov-Smirnov tests with a 1% threshold. n.s.P = 0.012 (left), n.s.P = 0.681 (right); *P < 1*10-35 (between each pair of THF/clamp mutants). 809C/394C: 1656 high FRET states; 809C/103C: 1349 states; 233C/103C: 1773 states; 233C/336C: 1778 states.
Figure 1(A) Schematic representation of the mintbody. H3K27me3-specific single-chain variable fragment (scFv) is genetically fused with a fluorescent protein (FP). (B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm. (C) Time-lapse imaging of a dividing MC12 cell stably expressing H3K27me3-mintbody (sfGFP). Projection images of 7 confocal sections with 2μm intervals are shown with elapsed time (hh:mm). Scale bar = 10 μm.
Figure 5(A) Verification of COUP-TFII loss- and gain-of-function cell lines generated by CRISPR-Cas9 and an inducible lentiviral construct in pericyte-like cell line C3H/10T1/2 in vitro (n=6), ****p<0.0001 by paired t-test. (B&C) COUP-TFII KO had no effect on cell viability, although decreased proliferation rate compared to naïve C3H/10T1/2 cells (WT) (n=6), ****p<0.0001 by two-way ANOVA. (D) COUP-TFII-OE cells, in the absence of TGFβ1 stimulation, displayed an elongated fibroblast shape with increased αSMA expression, similar to WT cells treated with TGFβ1. Quantification by confocal micrographs in 400x hpf, *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD. (E) Overexpression of COUP-TFII alone without TGFβ1 induces collagen1 production (n=3). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA, mean ± SD.