EMBO/sd-character-level-ner huggingface dataset:
WOX5 can interact with PLTs.Figure 7A-H, Fluorescence Lifetime Imaging (FLIM) results of transiently expressing N. benthamiana leaf epidermal cells. A-H, Representative FLIM images of WOX5-mVenus (donor only) plus the indicated acceptors after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm. I, Fluorescence lifetimes in ns are summarized in combined jitter and box plots. The dashed line represents the fluorescence lifetime mean value of the WOX5-mV co-expressed with free mCh as negative control. The one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test was used to test for statistical significance. Samples with identical letters do not show significant differences (α = 0.01). Number of nuclei analyzed (n) (biological replicates) is indicated and results from 2 to 9 technical replicates. Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, ▪ = mean value. J, Representative FLIM images of WOX5-mVenus (donor only) after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm. K, Individual nuclei showing nuclear bodies during co-expression of WOX5-mV and PLT3-mCh were analysed for WOX5-mV lifetime in the nuclear bodies or nucleoplasm separately (n = 7 (biological replicates), from four technical replicates. mCh = mCherry. mV = mVenus.
Figure 2A) Snapshots of the divergence of the velocity field from a representative movie. Note the areas of positive divergence in the nests, areas of positive and negative divergence in the LECs, and an increase of magnitude on the differences between them over time. Scale bar = 40 µm. B) Frequency distribution of the divergence magnitude in the nests and LECs in the different growth phases. Nests are always shown in red and LECs in blue. C) Color coded radial kymographs of divergence for a representative movie. Low divergences are blue, high are red (see scales). Note the overall positive divergence in the nests and negative in the LECs.
Figure 1A. Domain structure of nucleolin. RRM - RNA recognition motif; GAR - glycine- arginine rich domain. Amino acid sequence of the GAR domain is shown below. R3 and R4: peptides derived from the GAR domain C-terminus, efficiently binding AS1411 (see panel B). A3 designates the R3 peptide with three arginines substituted with alanines, N4 designates the R4 peptide with four arginines substituted with asparagines. B. R3 and R4, but not the A3 and N4 peptides efficiently bind AS1411 in an ELISA assay with biotinylated peptides shown in (A) immobilized to streptavidin-coated plates. n = 4 technical replicates; means ± SEM; **** p < 0.0001 in AS1411 vs. control aptamer, 2-way ANOVA with Sidak's post-test. a.u. - arbitrary units. C. Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm, identified by mass-spectrometry (schematics are based on (Hirokawa et al, 2009). Biotinylated A3 peptide served as a control. Mean spectral counts (normalized plus pseudocounts) ± SEM, n = 3 independent biological repeats. All proteins shown were found to significantly purify with R3, but not A3, with probability > 0.95 and a mean fold change > 2 by SAINTexpress analysis D. Schematic of kinesin pulldown from mouse sciatic nerve axoplasm, using biotinylated GAR peptides bound to streptavidin beads (STRP). E. Automated capillary electrophoresis immunoassay traces of Kif5a pulled down by biotinylated R3 and R4 peptides. Biotinylated A3 and N4 peptides, respectively, served as controls. 4.5% of input used for pulldowns was loaded alongside the pulldown samples. a.u. - arbitrary units. F. Quantification of (E). n = 3 independent biological repeats; means ± SEM; ** p < 0.01, paired Student's t-test.
MPPβ knockdown induces Parkin recruitment and mitophagy.Figure 5(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification). (B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA. (C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP. (D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.
Figure 3(A, B) Quantitative flow cytometry analysis of myeloid cells in the RPE complex (A) and retinas (B) from injured (filled bars) and contralateral noninjured eyes (empty bars), on days 1, 3 and 7 after ONC. Cells were pre-gated on total leukocytes (CD45+), and analyzed for the expression of CD11b. CD11b+ cells were further discriminated based on their expression levels of Ly6C. n = 6-7 per group. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.
Figure 3A. Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP-2xFYVEHrs and MavQ with or without wortmannin treatment. Scale bars, 5 μm. B. Percentage of HeLa cells transfected with the indicated plasmids in which GFP-2xFYVEHrs localizes to vesicle-like structures. At least 100 cells (n≥100) were counted for each sample. Wort, wortmannin. C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity. D. Wortmannin does not affect the production of PtdIns3P by MavQ. Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature. di-C8-Bodipy-FL-PtdIns and ATP were then added to the reaction mixtures. The TLC assay was employed to detect the generation of PtdIns3P by MavQ. The wortamannin-sensitive PI3K p110α was included as a control.
Figure 7A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons. Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c*: color is converted from blue to red for better contrast Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.
Figure 4 (A) Schematic representation of the fluorscence-2-hybrid approach for analysing interaction with endogenous proteins  (B) Example of interaction screening using an α-H3 antibody against tethered EGFP (EGFP-OMP25), tethered H3.1 (H3.1-EGFP-OMP25) and tethered H4 (H4-EGFP-OMP25). Background corrected, single Z-slices of representative cells are shown with 2D histograms displayed on the right. Nucleus and cytoplasm are portioned with a white line. Pearson's coefficients (R) for the cytosolic region of the depicted cells are shown in the histogram inset. Scale bar represents 10 μm  (C) A boxplot of Pearson's coefficients between tethered EGFP, H3.1-EGFP or H4-EGFP and endogenous histone counterparts or known histone chaperones. P-values of <0.001 from a Wilcoxon rank sum test versus the EGFP alone control are indicated by brackets. Whiskers extend to 1.5 times the IQR  (D) Schematic representation of a fluorescence-2-hybrid assay using mCherry-tagged, forced cytosolic chaperone  (E) Example of interaction screening as in B, but using sNASP-dNLS against EGFP (EGFP-OMP25), tethered H3.1 (H3.1-EGFP-OMP25) and tethered H4 (H4-EGFP-OMP25). Scale bar represents 10 μm  (F) A boxplot of Pearson's coefficients between tethered EGFP, H3.1-EGFP or H4-EGFP and cytosolically forced chaperones. P-values of <0.001 from a Wilcoxon rank sum test versus the EGFP control are indicated by brackets. Whiskers extend to 1.5 times the IQR  (G) Crystal structures of RbAp46-H4, HAT1-H4 and ASF1A-H3-H4 complexes. H3 is shown in red, H4 is shown in blue
Endosome formation is induced early during necrosis.Figure 2(A) Confocal images of touch receptor neurons expressing a pmec‐17APS‐2GFP reporter transgene. During early necrosis, the number of APS‐2::GFP puncta, corresponding to clathrin‐coated pits and vesicles, in touch receptor neurons ofmec‐4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc‐57(e1190) mutants, defective for clathrin‐mediated endocytosis. (B) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec‐4(d) animals significantly increases during early necrosis, whereas it declines later. (C) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐11 reporter transgene. The number of GFP::RAB‐11 puncta that correspond to recycling endosomes in touch receptor neurons of mec‐4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).
Mec1-S1991 phosphorylation limits RNAPIII transcription on HU.Figure 4(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent. (B) DNA-RNA hybrids level measured by DRIP-qPCR at several tDNA loci in asynchronous cells of the indicated genotype. As a technical control, samples were treated with RNaseH (+RNaseH, striped columns). (C-F) TFIIIB (Brf1-3HA) and RNAPIII (Rpc82-3HA) ChIP-qPCR was performed in asynchronous (async.) and after 2h in S phase +0.2 M HU. Enrichment was quantified at several tDNA loci. Data is expressed as percentage of input. ETC4 serves as a control locus which does not recruit RNAPIII. (D,F) Graph depicts the mean percentage of Brf1 or Rpc82 kept on chromatin after HU-treatment, calculated as a ratio HU/async in the indicated strain. Occupancy levels are derived from ChIP-qPCR values.
Figure 5 Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization. Both the wild type and the R105W mutant display nuclear, cytoplasmic and plasma membrane localization. However, the MS-microscopy analysis suggests a more prominent endosomal localization with the R105W mutant. (key: the scale bar for the immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-Microscopy tool.  The physical (AP-MS, green) and functional (BioID, red) interactions of the STK38L wild type and the R105W mutant (key: lower right corner). The R105W mutant displays increased interaction with 11 HCIPs, whereas five interactions decreased. The interactions that decreased (<0.6 fold) with the R105W mutant are indicated with green node color and the interactions that increased (>2 fold) are in blue  The homology modelling of the STK38L shows a shift from the positively charged arginine to a non-polar and bulky tryptophan pointing inside towards the ATP-binding site, thereby possibly inhibiting the ATP-binding and kinase activity of STK38L  The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L, whereas the R105W mutant only slightly increased the number of colonies. The error bars designate the standard deviation (SD)
Prion infection causes deacylation and degradation of PIKfyve.Figure 3A: Acyl-rac captured hydroxylamine-cleavable, acylate PIKfyve from mouse brains. Control: TRAPα, a non-acylated protein. Input: 10% of lysate. B: Brain lysates from prion-inoculated mice (60 dpi) precipitated by Acyl-rac. PIKfyve, but not calnexin (Cnx), was deacylated in prion-infected samples. Each lane represents an individual mouse. Lower panel: lysate used for Acyl-rac. C: Autoradiography of Gt1 cells transfected with PIKfyve-GFP or PIKfyveΔacyl (24h), metabolically labeled with 3H-palmitate (2 hours), and immunoprecipitated with anti-GFP antibodies. Lower panel: Western blot on immunoprecipitates using anti-GFP antibodies. Error bars represent s.e.m. (n=3 individual biological replicates). D: Gt1 cells transiently expressing PIKfyve-GFP or PIKfyveΔAcyl (24h) were subjected to a [35S]Met/Cys pulse (20 min, 37 °C) and immediately lysed or chased (60 min). Proteins were immunoprecipitated with anti-GFP antibody and subjected to SDS-PAGE and autoradiography. Lysates from cells treated with siRNA against PIKfyve were also pulsed and immunoprecipitated and subjected to autoradiography to ensure correct band is detected. Error bars represent s.e.m. (n=3 individual biological replicates). E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m. G: Gt1 cells were transfected with siRNA against zDHHC9+21 and stained with LAMP1. Arrows: vacuoles. H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.
Figure 4A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg/ml DQ-BSA for 12 h and chased for 2 h. Cells were fixed and stained with anti-LAMP1 antibodies and analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.B Quantitation of signal intensities in A showing colocalization of DQ-BSA with LAMP1 (means ± s.d.; n > 50 cells from three independent experiments). Baf.A1 was used as a control for inhibition of delivery and degradation of DQ-BSA via the endocytic pathway. **, P < 0.01; n.s., non significant. C N1E-115 cells treated with siControl, siINPP5Es, or siCHMP5 were stimulated with 100 ng/ml EGF for the indicated time periods, and then analyzed by immunoblot using anti-EGFR and anti-α-tubulin antibodies. Levels of CHMP5 mRNA72 h after transfection of N1E-115 cells with siControl or siCHMP5, as analyzed by RT-PCR.D Quantitation of EGFR degradation ratio in C. Results represent means ± s.d. of three independent experiments. **, P < 0.01.
Figure 2A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type. D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min. E. Comparison of the NaCl dose-responses of Hog1 activation by constitutively-active Ste11-Q301P and constitutively-active Pbs2-DD. Phos-tag band-shift assays were independently repeated three times, and the average values were plotted. Data information: (E) Error bars are SEM (n=3). F. A scheme illustrating the step in the Hog1 MAPK cascade at which osmostress acts to enhance Hog1 phosphorylation.
HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting.Figure 1HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody. (B) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6. (C) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin (green) and LC3 (red). Arrows indicate ubiquitin‐positive aggregates that colocalize with LC3‐positive autophagosomes. Scale bar, 10 μm. (D) WT and ATG5 KO MEFs are treated with 2.5 μM MG132 for 1 day and incubated with normal growth media for 18 h. MEFs are immunostained with anti‐LC3 (red) and anti‐ubiquitin antibody (green).
Figure 1Workflow of the study. Relative uc.291 expression quantification during calcium-induced in vitro differentiation of HEKn (3, 6 and 9 days of differentiation, DD). The quantification is relative to uc.291 expression in proliferating HEKn (0 day). Data shown represent the mean ± standard deviation (s.d.); n=4 different human donors; *p<0.05, **p<0.01, Student's t-test. Involucrin (IVL), Keratin 10 (KRT10) and Loricrin (LOR) expression levels evaluated by RT-qPCR are shown as positive control of differentiation. The quantification is relative to gene expression in proliferating HEKn (0 day). Data shown represent the mean ± s.d.; n=3 technical on 1 of the 4 human donors used in panel (B); *p<0.05, Student's t-test. Haematoxylin/Eosin (HE) staining and in situ hybridization of specific (uc.291) or not specific (Negative Control, NC) probes on human skin section. Scale bars denote 50 and 500 µm. RNA FISH for uc.291 (red, Quasar 570) on HEKn at 9 days of differentiation. Nuclei were stained with DAPI. Scale bar: 10 µm. Relative uc.291 quantification after RNA isolation from cytosolic and nuclear cellular compartments. snRNA U6 quantification was used as nuclear fraction positive control, while ACTB as cytosolic fraction positive control. Data shown are mean ± s.d.; n=3 technical on 1 of the 4 human donors used in panel (B); *p<0.05, **p<0.01, Student's t-test. Strand-specific RT-qPCR show that uc.291 is transcribed in genomic sense orientation in HEKn cells; n=2 technical (#1 and #2) on 1 human donor; SS: sense strand transcript, AS: antisense strand transcript. Picture showing uc.291 and LRMDA genomic location. Expression of uc.291 and LRMDA exons 2-3 and exons 6-7 during keratinocyte differentiation. Data shown are mean ± s.d.; n=2 different donors (3 technical/donor); *p<0.05, **p<0.01, Student's t-test. Relative uc.291 quantification after si-uc.291(1) and si-uc.291(2) in HEKn and FaDu cells. Data shown are mean ± s.d.; HEKn n=3 technical on 1 human donor; FaDu n=2 biological replicates (3 technical/biological replicate); *p<0.05, Student's t-test. RNA FISH for uc.291 (red, Quasar 570) on A253 in scramble (SCR) and silenced (si-uc.291(1) and si-uc.291(2)) cells. Nuclei were stained with DAPI. Scale bar: 10 µM. The histogram derives from the RT-qPCR using the leftover cells after removing the slides used for the FISH staining. Data shown are mean ± s.d; n=3 biological replicates *p<0.05, Student's t-test.
Associative learning in GluN2A(N615S) expressing mice.Fig. 8a Odor discrimination. Grin2aS/S learned to discriminate between amyl acetate and ethyl butyrate in a rewarded go/no go-paradigm (250 trials for each odor stimulus, pseudo-randomized, and counterbalanced by stimulus identity across animals). Acquisition was similar for all three genotypes. b Simple visuo-tactile discrimination. Grin2aS/S mutant mice showed normal acquisition in the simple discrimination T-maze task and were able to associate a specific visuo-tactile insert (black foam versus light blue toweling) with a milk reward. c Morris watermaze. In the standard Morris watermaze task (left graph) the path length to reach the hidden platform decreased across training blocks for Grin2a+/+ and Grin2a+/S mice but not for Grin2aS/S mice. Two probe trials were conducted after 12 and 24 training trials (P1 and P2, respectively) during which the platform was removed from the pool. At both P1 and P2 (right bar graphs) the Grin2aS/S mice failed to search for the platform in the target quadrant. AdjL adjacent left, Target fixed location of the hidden escape platform during acquisition, AdjR adjacent right, Opp opposite of target quadrant. Dashed lines indicate chance levels. d Rewarded alternation. (left) In the T-maze rewarded alternation task (right) spatial working memory performance was substantially impaired in Grin2aS/S mice. e Contiguous task. In the contiguous version of the conditional T-maze task (with floor inserts covering the entire T-maze; white Perspex versus gray wire mesh), Grin2a+/+ and Grin2aS/S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm. f Discontiguous task. Separate groups of mice were trained in the discontiguous version of this conditional task, in which the floor insert cues were now limited to the start arm only. Grin2a+/+ mice readily acquired the task, but Grin2aS/S mice failed to learn. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 8).