EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 1A) z-stacks were acquired from FM4-64 labeled vacuoles in wildtype cells (BJ3505), using a spinning disc microscope (step size 200 nm). 3D reconstructions from these stacks reveal a peripherally located fusion pore. Scale bar 2 µm. B) Wildtype (BJ3505) and isogenic pmc1∆/vcx1Δ mutant cells were labeled with FM4-64. The number of vacuoles per cell was determined in 3 independent experiments evaluating 200 cells each. Scale bar: 2 µm. C) Frequency of visible fusion pores. Cells were grown as in (B). The percentage of cells showing visible fusion pore was determined from z-stacks. Means s.d. are shown for 100 stained vacuoles from 3 independent experiments. D) Schematic view of Vph1-GFP and FM4-64 distribution over the vacuolar membranes in the presence of a nanoscopic fusion pore or of an extended hemifusion diaphragm. E) Distribution of Vph1-GFP and FM4-64 in vacuole-vacuole contact sites. Vph1-GFP expressing cells, labeled with FM4-64, were analyzed under a spinning disk confocal microscope equipped with two cameras for the simultaneous acquisition of GFP and FM4-64 fluorescence signals. Separate channels are shown on the right. The entire contact surface, but not the pore, is accessible to both probes. Scale bar: 2 µm. F) Expansion of a vacuolar fusion pore in real time. pmc1∆/vcx1Δ cells were labeled with FM4-64 and immobilized in a 50 µL flow chamber (Ibidi). The osmotic value of the medium was changed by perfusion with water, using a pump with a flow of 30 µL/s. A frame was acquired every 2 s for a total period of the 30 s, using the laser at minimal intensity.
Epg5−/− mice show motor deficits.Figure 1.(A) Scheme for generating Epg5 knockout mice. HSVTK, herpes simplex virus thymidine kinase; Neo, neomycin. (B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5−/− mice. There is an intervening lane between Epg5+/− and Epg5−/− lanes in the Western blot. (C) Body weight curves of Epg5+/− and Epg5−/− males. Means ± SEM of 12 mice are shown. (D) Time spent on a rotarod. The horizontal lines indicate mean times. (E) Epg5−/− males show gradually worsening limb-clasping reflexes. (F) The hindquarters of Epg5−/− mice are completely paralyzed at 10 mo of age. (G) Spinal deformation in an Epg5−/− male at 10 mo of age. (H) Epg5−/− females gradually develop the limb-clasping phenotype.
Figure 7Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.   Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.   Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.   Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.
Figure 1A. The production of Sox1-GFP+ neural-lineage cells during 7 days of neural differentiation, as shown by microscopy. Scale bars, 100 µm. B. qRT-PCR analysis of NDIME expression during 7 days of neural differentiation (D3, P = 0.068; D5, P = 0.0049; D7, P = 0.0003). C. Relative expression level of NDIME mRNA in the Sox1-GFP- population and Sox1-GFP+ population at day 7 of neural differentiation (P = 0.0118). D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm.F. Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation. The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions, respectively. Data are represented as mean, and error bars represent SEM in all panels. n= 3. G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR. Knockout of NDIME strongly inhibited neural differentiation, as shown by microscopy analyses during 7 days of neural differentiation. Scale bars, 100 µm. I. Knockout of NDIME strongly inhibited neural differentiation, as shown by FACS analyses during 7 days of neural differentiation. Scale bars, 100 µm. J. Knockout of NDIME inhibits the expression of neural-lineage genes at day 5 of neural differentiation. K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm. L. NESTIN and GFP immunostaining of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. Scale bars, 100 µm. M. Expression of neural-lineage genes of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).
Liver inflammation in Stat1Y701F and Stat1-/- mice after infection with Listeria monocytogenes.Figure 8A Immunohistochemical analysis of infection and inflammatory infiltrates. Wild type (WT), Stat1Y701F and Stat1-/- mice were infected by intraperitoneal injection of 1x102 viable L. monocytogenes for 48 h. Liver sections were examined by immunohistochemistry with L. monocytogenes or Ly6C/Ly6G specific antibody. The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images.B Quantitative evaluation of inflammatory infiltrates. Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size. C Liver pathology in infected mice. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x105 viable L. monocytogenes for 72 h. Serum was collected and tested for ALT activity. Lines represent the median and asterisks denote statistically significant differences (*, p≤ 0.05; ***, p≤ 0.001); p-values were calculated using unpaired t-test.
Shorter filaments in the RA-SV pool upon stimulation at wild-type and OtofPga/Pga ribbon synapses(A) Representative wild-type virtual section under rest depicts SVs with ribbon-attached filaments (Ra.Figure 4Representative wild-type virtual section under rest depicts SVs with r-attached fil (Ra.fil., red arrowheads) and one interconnector (Intercon., purple arrowhead), around the ribbon (R). Magnification 12,000x; scale bar, 40 nm. Example 3D reconstruction of the RA-SVs with filaments (green) in resting wild-type. Larger average length is found for ribbon-attached filaments (Ra.fil., red) vs. SV interconnectors (Intercon., purple). For clear visualization, the ribbon (R) is transparent and the same SV pair is shown in a front (A'), side (A'') and top view (A'''). Scale bar, 50 nm. Increased mean length of ribbon-attached filaments vs. SV interconnectors in all conditions for both genotypes. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool. The cumulative probability distribution of RA-SVs plotted against the individual filament length. The graph illustrates shorter filaments in wild-type and OtofPga/Pga ribbon synapses upon stimulation. Decreased mean filament length upon stimulation in wild-type and OtofPga/Pga ribbon synapses. Resting OtofPga/Pga exhibit the shortest mean filament length. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.
Figure 5CL levels after PTPIP51 depletion and reconstitution were monitored. A doxycycline-induced shRNA expression system was used to deplete the PTPIP51 protein in HeLa cells. Left, western blot analysis of HeLa cells in which PTPIP51 was depleted and restored. β-ACTIN was used as the loading control. Right, the mitochondrial CL level (nmol/mg) according to PTPIP51 expression. To reconstitute PTPIP51, the full-length PTPIP51 gene was transiently expressed. The data are presented as the mean ± SD of technical triplicate experiments from one representative experiment (n = 3), and p-values calculated using Student's t-test are shown. Quantification of CL in mitochondria using lipidomic analysis after PTPIP51 depletion and reconstitution. Color codes for each sample were presented in the lower right subpanel. Experiments were technically repeated three times, and the results are represented as the means ± SD of technical replicates (n = 3). *p < 0.05; **p < 0.01 (Student's t-test). Quality control (QC) samples, which were pooled identical aliquots of the mitochondria samples, were measured four times throughout the run for data reproducibility. Upper right, western blot analysis of HeLa cells in which PTPIP51 WT or ∆FFAT mutant were depleted and restored. β-Actin was used as a loading control.
Ultrastructural analysis and immuno labeling of cells expressing mitochondrially anchored Vps41.Figure 3(A-H) Indicated strains were grown in YPG for expression of Vps41-GFP-Fis constructs. Two representative images are shown for each strain. Mitochondria (M) are highlighted in red and vesicular structures in teal (unlabeled for VPS41-GFP-Fis) or green (unlabeled for VPS41-GFP-Fis). Cell wall (CW), plasma membrane (PM), and vacuoles (V) are indicated. Scale bars, 250 nm.
Figure 5(A) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm. (B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA). (E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells. (F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells. (G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1. (H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm. (I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).
Antisense SUF suppresses MpFGMYB expression in males A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region.Figure 5A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region. Diagrams illustrate wild-type and mutant MpFGMYB/SUF loci. Folded lines with a ∆ symbol indicate a deletion. B RT-PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms. C-H Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 µm (D), 50 µm (F), 100 µm (H).
Figure 5A. Schematic of the quantitative SSA assay used in panel B. Grey shadow represents the annealing region of 766 bp present on both sides of a DSB. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the restriction sites used to generate DNA fragments analysed by Southern blotting - BspCNI and SmaI for the fragment S1 and BglII for the fragment S2. B. Generation of double-stranded fragments S1 and S2 in SRS2 and srs2Δ during a time-course experiment. Also see Figure S2B-C for blot images. Red two-ended arrow indicates time delay of ~ 0.5 h in fragment S1/S2 formation in srs2 mutants relative to SRS2 cells. Average ± SD (n=4) is shown for each time point. Strains used: NK4691-4693; NK4805-4808. C. Schematic of PCNA loading assay used in D and I. D. Rad51 inhibits PCNA loading. Increasing amount of Rad51 (0.3, 0.6, 1.2, 2.3 µM) was added to phiX174 ssDNA substrate (0.5 nM) pre-incubated with RPA (75 nM). Loading of PCNA was started by the addition of RFC (21 nM) and 32P-PCNA (10 nM). The relative amount of loaded PCNA in each reaction is indicated below. E. Schematic of PCNA loading assay shown in F and H F. Srs2(1-910) overcomes the inhibitory effect of Rad51 on PCNA loading. phiX174 substrate (0.5 nM) was pre-incubated with RPA (75 nM) followed by the addition of Rad51 (2.3 µM). Increasing amounts of Srs2(1-910) (2.7, 5.5, 11, 22 nM) were added to the reaction and PCNA loading was started by the addition of RFC (21 nM) and 32P-PCNA (10 nM).G. Quantitative analysis of PCNA loading as a function of increased Srs2 (1-910) concentration. Average ± SD (n=3) is shown for each Srs2 concentration. H. Pif1 cannot substitute for Srs2 in promoting PCNA loading. Experiments were done as in F, except Pif1 at 2.8, 5.5, 11, and 22 nM was used instead of Srs2 in samples shown in lanes 5-8. Lane 9, control reaction with 22 nM Srs2(1-910). The relative amount of loaded PCNA in each reaction is indicated below. I. Increased concentrations of RPA suppresses the inhibitory effect of Rad51 on PCNA loading. phiX174 substrate (0.5 nM) was pre-incubated with RPA (0.08, 0.3, and 1.4 µM) followed by the addition of Rad51 (2.3 µM). PCNA loading was started by the addition of RFC (21 nM) and 32P-PCNA (10 nM).
Figure 3(A) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± S.D. (N=3). (B) Deuterium uptake differences were mapped onto the VP30 structure (PDB 6E5X), revealing similar binding regions for the peptides. Residue-level uptake difference was achieved by overlapping peptides.
Figure 2In vivo localization of mCherry-PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five-day-old complemented pi4kβ1 pi4kβ2 plants. The mCherry-PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry-PI4Kβ1. Scale bar, 20 µm. Whole-mount immunostaining of five-day-old seedlings expressing mCherry-PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti-tubulin (red) and anti-mCherry (green) antibodies, and DAPI (blue). (І), (ІІ), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 µm. Relative localization of mCherry-PI4Kβ1 and the TGN marker, ARF1, during cytokinesis. Four-day-old seedlings were immunostainned with anti-mCherry (green) and anti-ARF1 (red) antibodies. Scale bars are 10 µm.
Fig.Figure 2A Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database C Functional enrichment analysis of the signature OCRs allocated to three developmental groups by GREAT. Top five unique GO-terms which have the lowest FDR q-val are shown. In parenthesis: number of uniquely enriched GO-terms / total number of enriched GO-terms.
Figure 7A-E) Expression of 3xFLAG::GFP::ZNFX-1 and PGL-1::mTagRFP-T in a wild type (A), pid-2 (B), pid-4;pid-5 (C), pid-4 (D), and pid-5 (E) mutant backgrounds. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown. Arrow heads indicate individual condensates. Scale bar: 25 µm. F) Quantification of the ratio Z / P granules in wild type, pid-2, pid-4/-5, pid-4 and pid-5 mutant backgrounds. The number of granules is indicated in brackets, next to the genotype. Expression of PID-4::mTagRFP-T (G) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm. Expression of PID-4::mTagRFP-T H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm. I) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using a t-test (two-tailed).
The structure of SARS-CoV-2 monomer.Figure 1.The structure of SARS-CoV-2 monomer.
USP17 is required for plasma membrane repair.Figure 3A 48 hours post transfection the cells were washed with Ca2+ and Mg2+ free ice cold HBSS. The different cell populations were split into three aliquots. One aliquot was placed into fresh medium (Left panels). SLO was added to other two aliquots and then the toxin-containing medium was removed and the aliquots were either placed into Ca2+ and Mg2+ free HBSS (Middle panels), or EBSS containing Ca2+ and Mg2+ (Right panels) prior to staining with propidium iodide and analysis by flow cytometry. B The percentage plasma membrane repair (PMR) was calculated according to the following formula: 1 − (% PI-positive cells with Ca2+ / % PI-positive cells without Ca2+) × 100. Three separate experiments were analysed and plotted. Error bars represent standard error, and * indicates a p-values less than 0.05. One way ANOVA used to determine if statistically significant differences between groups.
Figure 6Schematic representation of the experimental design for reinfection experiments. Cells were infected with Shigella-mCherry (primary infection) or mock-treated. At 3 hpi, cells were re-infected with Shigella expressing GFP (secondary infection). Secondary infection was analyzed at 0.5 hpi (i.e. 1 h after addition of bacteria to cells) Representative image and cfu quantification of re-infection assays with Shigella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated Representative image and cfu quantificatio of re-infection assays wit Salmonella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated Representative images (F) and cfu quantification (G) of the re-infection assays with Salmonella ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock-treated Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment. I and J. Representative images (I) and cfu quantification (J) of the secondary infection with Shigella WT, in cells transfected with ASM siRNA or control siRNA Cfu quantification of the secondary infection with Shigella WT in HeLa cells following amitriptyline Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin transfectio with MAPK p38 siRN Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin SB203580 treatmen
Comparison of active and inactive ARM domain structures.Figure 2 A. Ribbon diagram of SPIN90(306-722). Helices within the ARM domain are colored according to their position within the repeat (1-yellow, 2-red, 3-cyan). Structural features that are not part of the armadillo repeat domain are colored grey  B. Cα trace of SPIN90(351-722) superposed onto the structure of SPIN90(306-722). His580 is shown in stick representation, as are residues on the H5-2 helix that contribute to the conserved surface patch. H5-1/2 is flipped away from the C-terminus of the ARM domain in the inactive 351-722 structure  C. Zoomed in view from panel (B) showing the difference in conformation of the H5-1/2 loop in the inactive (351-722, sticks with grey carbon atoms) versus active (306-722, sticks with cyan carbon atoms) SPIN90 structures. Positive (green) or negative (red) Fo-Fc electron density maps are contoured at ±2.5 σ and were generated by replacing H5-1/2 loop residues (577-585) of the 306-722 structure with the same residues from chain A in the 351-722 structure
Figure 3A. Indicated strains were grown on glucose medium before tenfold serial dilutions were dropped on glucose- or galactose-containing plates. B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector. C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm. D. The patterns of the Q97-GFP distribution were quantified after different time of expression, n=100. Cell boundaries are indicated by dashed lines. E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown. F. The GAL-Mia40 strain was much more resistant to Q97-GFP expression than WT cells, indicating that high Mia40 levels can suppress polyQ toxicity.