EMBO/sd-character-level-ner huggingface dataset:
a) Membrane defects resembling those accumulating with high concentrations of PE are prevalent in vivo at sites of high curvature and can be recapitulated in vitro by forming liposomes of different diameters.figf3(b) To determine the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mol%) as have been used in other Atg8 or LC3 lipidation publications (for example, refs , , ), with liposomes having the highest densities of liposomes observed in mammalian organelle membranes (30 mol%). At physiologically relevant concentrations of liposomes (30 mol%), lipidation becomes membrane curvature dependent. In vitro coupling reactions were performed as in Fig. 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Supplementary Table 1). Top: the lipidation of GL2, GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55% PE. However, when the liposomes is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. Bottom: to confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on Nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient. (c) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm, black squares; sonicated, red circles) and varying molar percentage of DOPE. The extent of lipidation is determined as in Fig. 2. (n = 2 for 30 and 55% PE, n = 4 for all the other points, and error bars represent standard deviation.) P values were determined by comparing sonicated with 400 nm samples at each PE concentration. **P 0.1; *P 0.01. Uncropped versions of all gels are included in Supplementary Fig. 7.
Figure 1A) Mfn2protein expression was measured in gastrocnemius muscle from young (6-month-old) and old (22-month-old) mice (n= 11-13 mice per group, *p<0.05) Data were normalized by tubulin and expressed as relative values of young mice. B) Venn diagram depicting genes differentially expressed in Mfn2-deficient muscle and old muscle compared with muscle from young or old control mice. C) Heatmap of genes differentially expressed in Mfn2-deficient muscle. D) GSEA of genes down and upregulated in Mfn2-deficient muscle in relation to aging in control mice.
Figure 3A. A representative micrograph of the TssA1 complex (left panel) showing discrete ring-shaped particles with a diameter of ~260 Å. The rotationally symmetrised average of the data set (inset panel) presents two distinct rings (numbered), disclosing the layered nature of the TssA1 complex. Representative class averages of the TssA1 complex (right panel) demonstrating the wide range of conformations taken by the ring-shaped structures. Scale bars are 500 Å in the left panel, 130 Å in the inset and 100 Å in the right panels, respectively. B. Double MSA classification: (i) representative orientation class averages show TssA1 rings arranged into four-fold (4x), five-fold (5x) and six-fold (6x) symmetry-like complexes (indicated in red, blue and green, respectively); (ii) sub-class averages obtained from sub-classification of the corresponding orientation class; and (iii) calculated difference images showing some white or black peaks (coloured circles) at the outer ring region. Scale bar is 100 Å. C. Secondary structure prediction based on the amino acid sequence of TssA1 using the web-based prediction server ESPript 3 (Robert Gouet, 2014). The linker region from Gly240 to Ala280 is highlighted in red. The positions of the first and last residues of the TssA11-245 truncated protein are indicated by red arrows. D. SEC-MALS analysis of the purified His6-TssA11-245. The TssA11-245 elution peak is shown and the corresponding mass is indicated. The molar mass and the differential refractive index (RI) are plotted versus the elution volume. Data were analysed using the ASTRA method (Slotboom, Duurkens et al., 2008).
Figure 3C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24-h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT-PCR 24 h after differentiation induction. n = 3, mean ± SD. *p < 0.05. n.s., not significant. Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used. Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control. The expression levels of myogenin and Myoparr evaluated by qRT-PCR in myogenin-depleted C2C12 cells. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test). ChIP-qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. **p < 0.01 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test). Retrieval rate of Myoparr using Myoparr-ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected. ChIRP-qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3´UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. ** p < 0.01 (unpaired two-tailed Welch's t-test). ND, not detected. Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test). The intersection of genes regulated by Myoparr KD and myogenin KD shows a significant (Fisher's exact test) overlap 25.6 times as large as expected by chance alone. Genes significantly differentially expressed in both Myoparr- and myogenin-depleted cells show correlated expression (R = 0.63, log 2 ratio scale). Enrichment of GO categories in genes up-regulated by Myoparr KD (K), down-regulated by Myoparr KD (L), down-regulated by myogenin KD (M), and commonly regulated by both Myoparr KD and myogenin KD (N).
Dot1L ablation affects the cellularity of mature peripheral B cell populations with a strong reduction in Marginal Zone and Germinal Center B cells.Figure 2A-G. Statistical analysis of absolute number of total nucleated splenocytes, splenic B- and T-cells, and indicated mature B cell subsets in WT and KO mice. Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group (A: (WT) n = 16, (KO) n = 17; B (B-cells): (WT) n = 16, (KO) n = 17; B (T-cells): (WT) n = 8, (KO) n = 9; C-E: (WT) n = 9, (KO) n = 9; F: (WT) n = 6, (KO) n = 6; G: (WT) n = 9, (KO) n = 9). H. Representative flow cytometry plots showing gating strategy (left panel) to identify germinal center B cells (PNAhigh, CD95+) from the spleen of unchallenged WT and KO mice and statistical analysis of their absolute number (right panel). Results represent the data pooled from two independent experiments and numbers represent biological replicates for each group (WT; n = 4, KO; n = 6). I. Identification of germinal centers by lectin histochemistry of Peanut agglutinin (PNA) in spleens from WT and KO mice. The scale bar: 20 μm. J. Quantification of germinal centers identified in spleen sections as shown in I. Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).
Figure 4A. Schematic representation of zebrafish Endouc protein and its internal deletion mutants. B. Histograms presented the relative luc activity obtained from zebrafish embryos microinjected simultaneously with puORFchop-luc, phRG-TK, and each one of the expression plasmids as indicated followed by analysis of luc activity at 96 hpf. Embryos microinjected with pCS2 vector during normal condition (non-stress; blank column) served as a control group while the microinjected embryos treated with 40℃ at 72 hpf for 1 hr were the heat-shocked group (solid column). The relative luc activity of microinjected embryos shown on the right was normalized by the amount of RNA in the injected embryos shown on the left. The relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from control group. The luc activity mediated by the huORFchop transcript was measured by dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR. C. Histograms presented the luc activity obtained from HEK 293T cells co-transfected with puORFchop-luc, phRG-TK, and each one of the plasmids as indicated; these were treated with either DMSO (control group) or thapsigargin (TH; stress group) followed by analysis of luc activity. The luc activity of cells transfected with pCS2 vector and kept at normal condition served as a control group. The relative luc activity of transfected cells shown on the right was normalized by the amount of RNA in the transfected cells shown on the left. The relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from pCS2-transfected control group normalized as 1. The luc activity mediated by the huORFchop transcript was measured by the dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR. D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot . Here, α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.
Negatively charged residues within the CCD of ATG16L1 weakens its interaction to lipids.Figure 6ATG16L1 CCD region 164-198 depicted on a heptad repeat with the relevant residues highlighted in red (acidic; D164 and E165), purple (hydrophobic; I171), and blue (basic; K179 and R193). Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein Liposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel). Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.
Figure 1A Schematic representation of the constructs utilized in (B) and (C). The first construct contains 58 (G4C2) repeats outside of the native C9orf72 sequence and GFP in the GP frame (polyGP-GFP). The start codon of GFP was removed. The second construct AUG-RFP is used as positive control of canonical translation. B HTS. HEK293T cells were co-transfected with the constructs in (A) and, negative (DMSO, orange dots) and positive (CHX 5 µM, purple dots) controls added 3 h after reverse co-transfection with compound-libraries (5 µM). Images and data acquisition collected after about 30 h of treatment. Scatter plot shows distribution of compounds. On the Y-axis reported the Z-score values of cells expressing AUG-RFP and on the X-axis the Z-score values of cells expressing polyGP-GFP. Grid lines represent the thresholds arbitrarily set up around DMSO distribution (polyGP-GFP ±1.5 on the X-axis and ±1.5 for AUG-RFP on the Y-axis) to select compounds for the confirmatory screening and eliminate the ones without effect (orange square). C Confirmatory screening performed as described above. Schematic distribution of compounds based on Z-score values of cells expressing polyGP-GFP and AUG-RFP (above) and on Z-score values of the fluorescent intensity of the two reporters per each compound (below). Data are from four technical replicates, boxes are the Z-score mean value and whiskers represent ±SD. Baseline of Z-score = 0 indicates that Z-score is identical to the mean score. Asterisk (*) represents the selected compounds: 1 Forskolin (FSK), 2 Erysolin (ERY), 3 Geldanamycin (GELD), 4 Helenin (HLN) and 5 Spironolactone (SPL). Number sign (#) represents cellular stress inducers: #1 Thapsigargin and #2 Tunicamycin. D, E Dose-response analysis of ERY, HLN, SPL, FSK and GELD. Cells were co-transfected with AUG-RFP and polyGP-GFP plasmids and treated with two concentration ranges 0.5, 1, 5 and 10 µM (D) and 20, 40 and 60 µM (E) for 24 h. CHX used only low dosages. Data are mean ± SD from three biological replicates. 1-way ANOVA followed by Dunnett's multiple comparison tests: *P<0.5; **P<0.01; ***P<0.001
Figure 4(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies. (B) Representative immunoblots of co-immunoprecipitations between hnRNP L and the indicated VP30 mutants which were previously described to exhibit varying degrees of NP binding activity. (C) Co-immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6. VP30 was co-expressed with FLAG-tagged PPxPxY containing host proteins and either a 5-fold or 10-fold excess of RBBP6 plasmid. IP was performed using anti-FLAG beads followed by Western blotting using the indicated antibodies. (D) Densitometric analysis of the Western blots shown in panel C. The graph represents the relative amount of VP30 present in the eluates compared to the input. The data represent the mean ± S.D. from two independent experiments (n=2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. **p<0.005.
Fig.Figure 5(A) FACS plots from EdU pulse-chase experiment. Cells were DIA or mock-treated for four hours and then labelled with EdU for one hour. The EdU was then washed out and samples were taken every three hours for 12 hours and analysed by EdU/PI staining and FACS. The top left panel shows the gates that were used for analysis. The quantifications shown in the panels to the right are from three independent repeats of this experiment. (B) Quantification of cells progressing into Gate 1 (Top panel, new S-phase cells) and Gate 2 (Bottom panel, G2 cells) over time. The lines represent the mean of three experiments, the coloured areas indicate the standard deviation. (C) Relative changes in the percentage of EdU positive cells in gates 3,4 and 5 (G1/early-S, mid S and late S/G2 phase). The bars indicate the relative mean values from three independent experiments, the error bars represent the standard deviation. (D) Cyclin A2dd cells with inducible CycB1-YFP (B1-WT) and CycB1-YFP-NLS (B1-NLS) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2 antibodies (E) Quantifications of EdU pulse chase in DIA treated cyclin A2 cells expressing B1-WT, B1-NLS or YFP as indicated. (F) Accumulative mitotic index measurement of cyclin A2 cells following cyclin A2 depletions and induction of indicated cDNAs. The lines represent the mean of three experiments, the coloured areas indicate the standard deviation. (G) Quantification of metaphase duration in A2dd cells treated with DIA following induction of indicated proteins (H) FACS plots from EdU pulse-chase experiment in A2dd cells following induction of indicated genes. (I) Immunoprecipitation of YFP from extract from A2dd following induction of YFP (Y), CycB1-YFP (BY) and CycB1-YFP-NLS (BYN). The samples in the odd lanes (IAA/Asv-) were treated with 1µg/ml doxycycline alone, while samples in even lanes (IAA/Asv+) were treated with Auxin and Asv to induce cyclin A2 depletion. Total cell extract and immune-precipitates were probed by immunoblotting with the indicated antisera.
Cartoon depicting astrocyte-derived PTX3 effects on PNN remodeling and on AMPA receptors at the synapse.Figure 7PTX3, released from astrocytes, binds to hyaluronic acid through TSG6 and activates β1-integrin. β1-integrin controls AMPAR trafficking through downstream MAP Kinase activation. Indeed the PTX3-induced AMPAR enhancement is prevented by 1) genetic removal of the key HA-binding factor, TSG6; 2) neuron exposure to hyaluronidase (HAse, which destroys the extracellular matrix; 3) Inhibition of β1-integrin activation; 4) pharmacological blockade of MAPK activation (PD98059)
Figure 6(A) Double immunofluorescence staining with anti-GA (green) and anti-hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 #1) more poly-GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co-localization of poly-GA and hnRNPA3 aggregates. (B) Granular layer neurons of a C9orf72 mutation case with (white arrows) or without (orange arrows) nuclear clearance of hnRNPA3. (C) poly-GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean value. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (p = 0.0086) after removal of the highest outlier in the low nuclear A3 group. Two-tailed t-test. Scale bar = 10 μm.
INQ formation does not involve ubiquitinationS.Figure 3A S. cerevisiae wt cells expressing GFP‐VHL (green) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132. Ubiquitin (red) was stained by immunofluorescence microscopy using specific antibodies. DNA was stained by DAPI (blue). Changes in protein localizations were recorded. Scale bars, 2 μm. B tGnd1‐GFP is stabilized in ubr1Δ san1Δ cells. S. cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1‐GFP were grown at 30°C. Cycloheximide was added, and protein levels were determined at the indicated time points by Western blot using GFP‐specific antibodies. Zwf1 levels are given as a loading control. C Ubiquitination status of tGnd1‐GFP expressed in S. cerevisiae wt and ubr1Δ san1Δ cells was determined by GFPpull‐down and Western blot analysis using ubiquitin‐specific antibodies. Levels of isolated tGnd1‐GFP after GFPpull‐down were determined by Western blot using GFP‐specific antibodies and are given as a control. D S. cerevisisae wt, ubr1Δ san1Δ, and ubr1Δ san1Δ btn2Δ cells expressing tGnd1‐GFP (green) were grown at 30°C. DNA and nuclear envelopes were visualized by coexpressing Htb1‐mCherry or Nic96‐mCherry, respectively (red). The fraction of cells showing specific tGnd1‐GFP foci numbers per cell was determined. INQ formation in cells containing tGnd1‐GFP foci was determined (% cells with INQ) based on close vicinity of tGnd1‐GFP foci to Htb1‐mCherry or localization inside Nic96‐mCherry signals, yielding comparable results. Scale bars, 2 μm.
UPF3A KOs show light NMD-independent transcriptome alterations A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1).Figure 2A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands. B Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1). C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method. D,E Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method F nVenn Diagram showing the overlap of up- or downregulated genes in the UPF3A KO cell lines 14 and 20. Log2 fold change <1 (downregulated) or >1 (upregulated) and adjusted p-value (padj) < 0.05. DGE = Differential Gene Expression.
Figure 1(A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235). (B) Box-and-whisker plot showing quantification of mono-phosphorylated p-tau231 in control (n=10) and AD (n=10) TBS-soluble fractions using a targeted IP-MS method (P >0.05, non-significant, ns, Mann-Whitney U test). (C) Box-and-whisker plot showing the quantification of double phosphorylated p-tau(231+235) in control (n=10) and AD (n=10) TBS-soluble fractions using a targeted IP-MS method. Data information: Box-and-whisker plots show the median and the 25th and 75th percentiles. Group differences determined using Mann-Whitney U test (****P <0.0001). All samples were run in singlicates.