EMBO/sd-character-level-ner huggingface dataset:
Figure 9A Mitochondrion in WT Hela cells expressing ATP5I-PAGFP was photoactivated, in the area shown by a blue box, at 0 s. Inset of white box at 0.98 s clearly shows fluorescent crista (red arrow), absent at 0.38 s, 0.59 s & 0.78 s (red arrowhead showing the area enclosed by white dotted lines), confirming merging of non-fluorescent cristae vesicle with the IBM. Threshold images of insets are shown above the whole images. Scale bar 500 nm. B Dual-colour STED super-resolution images of fused mitochondrion in WT Hela cells (Left Panel) showing colocalization of cristae marked with ATP5I-GFP (using anti-GFP antibody) and ATP5I-SNAP (stained with silicone-rhodamine). (Right Panel) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I-GFP (using anti-GFP antibody) or ATP5I-SNAP (stained with silicone-rhodamine). Arrowheads show merged cristae while arrows show cristae that maintain their individual identity. Scale bar 500 nm.D Schematic illustration of the proposed 'Cristae fission and fusion' (CriFF) as a working model and its link to CJ dynamics.
Figure 1A, Photoreceptor transplantation schematic B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time. D, Analysis of Cre-recombinase transfer after transplantation of Crx::Cre donor photoreceptors into Nrl-/- recipients after 14 (n=6), 21 (n=5), 42 (n=4), 90 (n=6) and 150 (n=6) days. The number of acceptor photoreceptors in the recipient retina that activated the Cre reporter gene is quantified. E, Resolution of the transplant-induced retinal detachment in C57BL/6J recipient retinas (n=11), as measured by the maximum detachment length (mm) over time. Fundoscopy (images on the left) was used to locate the region of cell delivery for OCT imaging (green line) shown in the images on the right. Red lines correspond to the callipers used to determine the maximum length of the detachment at each time point. Before injection (B.I.) (n=11), immediately post-injection (I.P.I.) (n=10), 5 days post-injection (5 D.P.I.) (n=11), 9 days post-injection (9 D.P.I.) (n=11), 21 days post-injection (21 D.P.I.) (n=6), F, Co-transplanted P3-4 Crx::Cre and P3-5 ROSAmT/mG donor photoreceptors into C57BL/6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post-transplantation.
Figure 3(A-C) Illustrations of the three simple and fundamental motifs that frequently reoccur in perfect or near-perfect adaptation (A), incoherent feedforward (B) and state-dependent inactivation (C). Here, the input is the concentration of nuclear NICD and the output is target gene promoter activity, i.e. the rate of sim mRNA production (not to be confused with total levels of sim mRNA in the cell). The components (A) and (B) in these motifs are placeholders for single or compound molecular regulators; at this point, no specific assumptions are made in our models about their identity. Note that in (C), Ain stands for the inhibited state of (A), which can no longer be activated. The dashed line back to Aoff indicates recovery at slow time scales relative to the motif's adaptation dynamics. (D-I) Results of elementary mass action ODE model fitting to sim adaptation dynamics. The input (NICD nuclear concentration; dashed blue line) is modelled based on separate experiments elucidating opto-Notch import-export dynamics Sim expression is total MCP::GFP spot intensity, linearly rescaled to match the non-dimensional scale of the ODE models (D,F,H) Despite their simplicity, the models of all three motifs can readily be fitted to sim adaptation dynamics. (E,G,I) Without further fitting, the resulting models also approximate the loss of adaptation during pulsatile activation, although the model predictions are slightly lower and slightly less responsive to the second and third pulse than experimentally measured nuclei, which may be due to the simplifying assumptions made in the equations describing the motifs.
Figure 5Mice lungs were collected from PBS-(mock) vaccinated BALB/c mice at day 5 post-RSV infection, fixed, paraffin embedded, and slides were processed for imaging. Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera (left panels) or rabbit anti-sera against RSV F protein (center panels), or against RSV F-p27 (110-136) peptide (right panels). Scale bar = 100 µm. The percentage of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown. The experiments were performed twice and variation between the two independent expeiremnts was <9%.
Figure 7A Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death. B Anti-DDX3X western blot of B35 cells transfected with either of two independent anti-DDX3X LNAs or a control LNA. C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate. Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates). D, E Transfection of anti-DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)100 (+1) EGFP-expressing neurons (Cox proportional hazard analysis; n=2408-3676 cells/condition). Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).
Figure 5A PBL27 phosphorylates MAPKKK5-C in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. CBB, Coomassie brilliant blue. B BIK1 does not phosphorylate MAPKKK5-C in vitro. RBOHD was used as a positive control. Asterisk indicates artificial bands. C Identification of amino acid residues of MAPKKK5 phosphorylated by PBL27. The C-terminal domain of MAPKKK5 was phosphorylated by PBL27 in vitro and subjected to LS-MS/MS analysis. All MS/MS spectra of phosphorylated peptides were validated manually. The amino acid residues phosphorylated by PBL27 are shown in red. D The S622A mutation strongly reduces phosphorylation by PBL27. Each of phosphorylation sites was substituted by alanine, and subjected to the in vitro phosphorylation assays. E PBL27 does not phosphorylate MAPKKK5-C6xAin vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. F PBL27 interacts with MAPKKK5-C6xA in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 10 mM 3-AT indicated a positive interaction.
GAP TBC1D15 facilitates spatial segregation of late compartments marked by Rab7 and Arl8b.Figure 7Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP. A. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC), two different siRNA oligos targeting TBC1D15 (#1 and #3) or oligo pool targeting TBC1D17 and ectopically expressing GFP-Arl8b (green) in combination with HA-RILP (cyan) and Myc-SKIP (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to depletion of TBC-containing proteins, n≥4 images (3≥ cells per image) analysed per condition from 2 or more independent experiments C. Plot of Rab7-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells analysed per condition from 2 independent experiments. D. Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR, n=3 independent experiments. E. Effect of TBC1D15 depletion on Rab7-dependent MHC-II receptor trafficking to the SKIP-positive compartment, n siC=21, n si1D15=21 cells analysed from 3 independent experiments
Figure 6Simulated kymograph of a less polarized cell with a narrow PI(3,4) P2 enriched region. The white dashed lines correspond to the periphery of the back regions which spanned on average 30% of the cell perimeter. Simulated kymograph of a more polarized cell with broader PI(3,4) P2 enriched region compared (A). Slower vesicle arrival time and reduced decay rate for PI(3,4) P2 was assumed in comparison to parameters used for the simulation in (A). Total intensity profiles (Mean ±SEM) of tPH-CynA-KikGR on the membrane over time show different decay rates for less polarized (green, n = 5) and more polarized (red, n = 8) Dictyostelium cells. The dashed lines show the respective single exponential fit to the data. Mean half time for vegetative cells = 7.5 min; for developed cells = 55 min (from single exponential fit to the data). The boxplot shows the relative width of the back regions in less (green) and more (red) polarized Dictyostelium cells. The square markers of respective colors show the mean relative width of the back regions obtained from the simulated kymographs in (A) and (B). *** P < 0.001 (n = 5). Mann-Whitney-Wilcoxon test was performed. The plots are standard box plots showing medians (central bands) along with the respective first and third quartiles. Schematic representation of our previous study demonstrating the mutual inhibition between Ras activity and PI(3,4) P2 which establishes polarity even in immobilized latrunculin A treated cells in a chemotactic gradient (with stationary back) or in caffeine treatment (with rotating back). In Dictyostelium, caffeine is used to inhibit the synthesis of the chemoattractant cAMP. Schematic representation of the working model in less polarized Dictyostelium cells. PI(3,4) P2 is internalized on macropinosomes and transported into the cytosol. These anterior macropinosomes break up into smaller PI(3,4) P2 vesicles, which dock and fuse at the rear of the cell. Schematic representation of the working model in more polarized Dictyostelium cells. Slow vesicle arrival of the PI(3,4) P2 vesicles and slower decay rates of PI(3,4) P2 created a broader back.
Mass spectrometry identifies ribosome biogenesis factors to be recruited to H2AQ105me.Figure 2A) Schematic of the experimental strategy. B) Volcano plot of quantitative SILAC experiments. Left upper quadrant indicates significantly, 2-fold enriched proteins bound specifically to H2AQ105me. Members of the FACT complex (SPT16/POB3) are highlighted in red. C) STRING network of proteins enriched on H2AQ105me (highlighted in red are proteins involved in ribosome biogenesis). D) Gene ontology enrichment for proteins bound to H2AQ105me. E) Gene set enrichment analysis for the altered transcriptome in an H2AQ105A background compared to wildtype. The normalized enrichment score (NES) is reported as are individual p-values. Vertical black lines on the x-axis represent genes in gene sets. The green line connects points of enrichment score and genes. Enrichment score is the maximum deviation from zero as calculated for each gene going down the ranked list, and represents the degree of over-representation of a gene set at the top or the bottom of the ranked gene list.
Figure 1(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP or NBR1 under the CAG promoter for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test. (B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test. (C) Immunofluorescence microscopy. Wild type hepatocytes were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test. (D) FRAP assay. Wild type hepatocytes cultured in glass-bottom plates were infected with GFP-NBR1 adenovirus for 48 hrs. The signal recovery after photobleaching was measured, quantified from at least three independent biological replicates, and is represented as means ± s.e. Bar: 1 µm.
TNT's formation and function depend on βCaMKII phosphorylation status.Figure 3Representative confocal images of CAD cells expressing the indicated GFP-fusion proteins. Top: Cells showing WGA (white) and DAPI (blue) staining. Bottom: Cells showing phalloidin (red) and DAPI (blue) staining. Note that the expression pattern of βCaMKII WT and the mutants K43R and A303R, is rather punctate than diffused. Percentage of TNT-connected CAD cells 24 h after transfection with indicated GFP-fusion proteins. Percentage of transfection rate of indicated GFP-fusion proteins in CAD cells. Percentage of acceptor cells containing DiD-labeled vesicles when acceptor cells were co-cultured with donor cells expressing the indicated GFP-fusion proteins. Top: Schematic of the co-culture system used to assess vesicle transfer in transfected cells. Donor cell were transfected with the indicated GFP-fusion proteins and then labeled with DiD. Acceptor cells were transfected with a H2B-mCherry plasmid. After 24 h, the cells were detached, replated in 1:1 ratio and cultured for 4 h. Cells were then fixed and stained with HCS CellMask™ Blue. Bottom: Representative confocal images of co-cultured donor and acceptor CAD cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.
Host response analysis of SARS-CoV-2 infection in organoid-derived bronchioalveolar-like and small airway cells.Figure 6A) pSTAT1 translocation in mock and SARS-CoV-2-infected bronchioalveolar-like cells visualized in green. Scale bars indicate 50 μm. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white). B) Heatmaps depicting top 20 enriched genes based on adjusted pvalue in bronchioalveolar-like cells after SARS-CoV-2 infection. Colored bars represent Z-scores of log2 transformed values. C) pSTAT1 translocation in mock and SARS-CoV-2-infected small airway cells visualized in green. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white). Scale bars indicate 50 μm. D) Heatmaps depicting top 25 enriched genes based on adjusted pvalue and log2 fold changes in small airway cells after SARS-CoV-2 infection. Colored bars represent Z-scores of log2 transformed values. E) GO term enrichment analysis for biological processes of the 20 most significantly up-regulated genes upon SARS-CoV-2 infected in bronchioalveolar-like cells. P-values were determined using PANTHER Overrepresentation Test. F) GO term enrichment analysis for biological processes of the 25 most significantly up-regulated genes upon SARS-CoV-2 infected in small airway cells. P-values were determined using PANTHER Overrepresentation Test.
Figure 6Figure 6a. Overlap between down-regulated genes and genes with differential exon usage upon CDK12 inhibition. Venn diagram shows the overlap between significantly differentially expressed genes (identified by DESeq2) and genes with differential exon usage (identified by DEXSeq) in nuclear RNA-seq data (0>log2 fold-change>0, p<0.01, restricted to genes included in the DEXSeq analysis). Figure 6b. Differentially used exons are enriched at gene 3´ends. Graph shows the distribution of the relative genomic position of the exon on the gene (relative exon position: 0 = at gene 5'end, 1 = at gene 3'end) of differentially used exons (0> log2 fold-change >0, p<0.01). Figure 6c. For down-regulated genes with differentially used exons, exons close to the 5´ and 3´end tend to be up- and down-regulated, respectively. Box plots show the log2 fold-change in exon usage after CDK12 inhibition determined by DEXSeq. Exons were grouped into deciles according to their relative exon position. n=3 replicates.
Atomistic and coarse-grained simulations of TDP-43 LCD: Phosphomimicking residues form fewer protein-protein interactions and more protein-solvent interactions.Figure 3TDP-43 LCD phase separates in coarse-grained simulations with explicit solvent. Condensate of TDP-43 Wt LCD is shown, protein colored according to chain identity. Water omitted for clarity. Ions shown in cyan. B Normalized probability of protein-protein contacts by phosphomimicking aspartates in 12D and serines in Wt resolved by amino acid type from coarse-grained simulations. Error bars smaller than symbols. Inset: Distributions of the number of water molecules within 5 Å of side chains of phosphomimicking aspartates of 12D and corresponding serines in Wt from 15 µs of coarse-grained molecular dynamics simulations. C Atomistic simulation setup of 32 TDP-43 LCDs. Different LCD chains shown in different colors in space-filling representation. For one chain (lower left), a transparent surface reveals its atomic structure as sticks. Normalized probability of protein-protein contacts by phosphomimicking aspartates in 12D and serines in Wt resolved by amino acid type from atomistic simulations. Two 1 µs simulations are distinguished by color intensity. Inset: Distributions of the number of water molecules within 5 Å of the side chains of phosphomimicking aspartates of 12D and the corresponding serines in Wt from atomistic simulations. E E Representative snapshots of atomistic simulations showing water within 3 Å of (left) Wt S407, S409, S410 with nearby LCDs in surface representation and (right) 12D D407, D409, and D410. Protein surfaces are colored according to chain identity. F Density profiles in TDP-43 LCD condensates (peak at center) coexisting with dilute solutions for Wt, 12D, 5pS, 12pS, and 12A from coarse-grained simulations with the implicit solvent coarse-grained HPS model. G, H Snapshots of 12D condensate (G) and fragmented 12pS clusters (H) in simulations with the coarse-grained HPS model. Side view on elongated boxes (blue lines).
Figure 3(A) poly-GA aggregates co-localize with p62-positive deposits. (B) Redistribution of nuclear TDP-43 to the cytoplasm in a poly-GA/poly-GR double-positive cell. Note that nuclei of poly-GA/poly-GR double positive cells are frequently disrupted. (C) Knockdown of hnRNPA3 increases repeat RNA expression. n=2 experiments performed in duplicates. ANOVA, Dunnett. (D) Filter trap assay probed with anti-FLAG, anti-myc or anti-HA antibodies reveal abundant DPR production. (E) Relative expression level of poly-GA, poly-GP and poly-GR normalized to GR. n=3 experiments, ANOVA, Tukey. (F) Expression of all the three DPRs is increased upon knockdown of hnRNPA3. Signals from knockdown with siRNA#8 were normalized to 1. n=3 experiments, ANOVA, Dunnett. (G) Co-expression of two/three different DPRs in single cells is increased upon hnRNPA3 knockdown. n=3 replicates, ANOVA, Dunnett. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Scale bars=20μm. (see also Figure EV2 and EV3).
Figure 2Images of Rad51-ssDNA molecules bound by both mCherry-Rdh54 (30 nM; magenta) and GFP-Rad54 (green; 30 nM). Quantification of bound GFP-Rad54 (10 nM) signal intensity for Rad51-ssDNA molecules with and without bound mCherry-Rdh54 (10 nM). Error bars represent s.d. of individual ssDNA molecules (N = 21). Images showing increasing concentrations of GFP-Rad54 (green) only or mCherry-Rdh54 (magenta) only bound separately to Rad51-ssDNA molecules. Images of GFP-Rad54 (green) and mCherry-Rdh54 (magenta) co-bound to the same Rad51-ssDNA molecules. Quantification of GFP-Rad54 binding in the absence and presence of mCherry-Rdh54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43). Quantification of mCherry-Rdh54 binding in the absence or presence of GFP-Rad54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).
Modest effects of transient MPPβ, PARL and AFG3L2 knockdown on mitochondrial integrity.Figure 2(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm. (B) MTR DR/Green ratio from images as in A. N=3; *P0.05 compared with NT siRNA. (C) Mitochondrial interconnectivity from images as in A, N=3. (D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.