EMBO/sd-character-level-ner huggingface dataset:
DCA treatment had protective effects on muscle strength and prevented the expression denervation markers.Figure 8Grip strength is represented as mean of percent from T0 for each experimental group ± SEM. ##P = 0.0045 and ***P = 0.0003 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test). Relative mRNA levels of muscular atrophy markers Murf1 and Atg-1 were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.0079 and ***P = 0.0018 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test). Relative mRNA levels of denervation markers AChRα, AChRγ, and MuSK were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.015 for AChRα, #P = 0.028 and **P = 0.074 for AChRγ, #P = 0.042 and **P = 0.0029 for MuSK (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).
Figure 7A List of KEGG pathways significantly enriched in the genes targeted by miRs up- or down-regulated upon Omomyc induction (Table 2). The number of affected genes and the number of miRs targeting them are indicated for each pathway together with merged p-values computed as in [48], denoting the probability that the examined pathway is not enriched with gene targets of at least one selected microRNA. B Immunoblots of EGFR, ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with (+) or without (-) Dox. Representative images are shown. C RT-qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence (+) or absence (-) of Dox (n = 3; mean ± SD). Expression levels were normalized using U6RNA as control. Expression levels in non-induced cells were set as 1. D EGFR, ZEB1, and GAPDHimmunoblots in the same cells as in panel C. Representative images are shown. E EGFR, ZEB1, and GAPDHimmunoblots in uninduced U87FO cells (-) and in cells transfected with LNA oligos targeting miR-200a-200b-429 (LNA 200) and miR-146b (LNA 146b), or with a non-targeting LNA (C), and induced with Dox for 48 h (+Dox). Representative images are shown. F MTS cell proliferation assay of uninduced or Dox induced U87FO cells, transfected with either a non-targeting LNA (LNA C), or LNA oligos targeting the miR-200a-200b-429 cluster (LNA 200), miR-146b (LNA 146b), or both (LNAmix) and analysed at 24, 48 and 72h (n = 3; mean ± SD). G Migration assay of U87FO cells transfected with non-targeting control LNA oligos (LNA C) or with LNA oligos targeting miR-200a-200b-429 (LNA 200); cells were either untreated (-) or treated with Dox for 24 h (+Dox); (n = 3; mean ± SD).
Loss of UPF3B does not affect NMD efficiency, only in combination with KD of UPF3A A Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1).Figure 3A Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1). UPF3A and UPF3B (AK-141) protein levels were detected, Tubulin serves as control. Protein levels of UPF3A were quantified, normalized to tubulin expression, and shown as datapoints and mean. Fold-changes of relevant conditions are shown. The asterisk indicates unspecific bands. B Quantitative RT-PCR of the indicated cell lines with the indicated knockdowns. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC, n=3). 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 transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj. p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj. p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method.
Figure 1A. Western blot of PTEN variants in WT (lane 1), PTENα-/-, β-/- (lane 2), and PTEN-/- (lane 3) Hela cells with a polyclonal antibody against full-length PTEN. Except for PTENα, PTENβ, and canonical PTEN, immunoblotting revealed four additional bands with molecular weights lower than PTENβ in WT and PTENα-/-, β-/- Hela cells. Hela PTEN-/- cells have no expression of PTEN variants or canonical PTEN. The asterisks shown here and in other immunoblot images throughout this manuscript indicate the corresponding protein band of PTENε. B. Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C-terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control. C. Immunoprecipitation of PTEN variants with a homemade PTENα antibody before immunoblotting with a PTEN monoclonal antibody in Hela and HepG2 cells D. Western blot of PTEN variants in WT (lane 1) and N-terminal extended PTEN isoforms-/- (lane 2) Hela cells with a PTEN monoclonal antibody
Figure 6A) Receiver operating characteristic (ROC) curve for the XGBoost-based model to classify PD+ vs. PD- individuals. Random performance is indicated by the dotted diagonal line. The gray area represents the standard deviation from the mean ROC curve. The blue lines show the values for a total of 15 repeats with four stratified train-test splits. B) Confusion matrix showing the model performance for classifying PD+ vs. PD- individuals. Numbers represent the mean number from 15 repeats of cross-validation with four stratified train-test splits. C) ROC curve for the XGBoost-based model when trained on one cohort and tested on the other cohort. Random performance is indicated by the dotted diagonal line. D) classification of LRRK2 G2019S vs. LRRK2 WT carriers. E) classification of LRRK2 G2019S vs. LRRK2 WT carriers. F) classification of LRRK2 G2019S vs. LRRK2 WT carriers. G) classification of PD+ vs. PD- in LRRK2 G2019S carriers. H) classification of PD+ vs. PD- in LRRK2 G2019S carriers. I) classification of PD+ vs. PD- in LRRK2 G2019S carriers.
GRINA Knockdown or Overexpression Inhibits Endosome-to-Golgi Retrieval.Figure 5(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom). (B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1). (C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35. (D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells. (E-G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell's TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.
Figure 1A Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses. B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species. C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.
a) Immunoblot for the indicated proteins in homogenates (Hom) and lysosomes (Lys) isolated from cells untreated (None) or treated for 12 h with 20 μM of the indicated compounds.figf7(a) Immunoblot for the indicated proteins in homogenates (Hom) and lysosomes (Lys) isolated from cells untreated (None) or treated for 12 h with 20 μM of the indicated compounds. (b) mRNA levels of LAMP-2A in control mouse fibroblasts (left) or RARα(−) cells (right) treated with AR7 or paraquat (PQ) as in a; n = 4-5. Values are presented as fold change in mRNA relative to untreated cells. (c) Cellular viability of control (left) or LAMP-2A(−) (right) fibroblasts exposed to 2 mM or 0.5 mM paraquat, respectively, and treated with the indicated compounds for 12 h before or after the paraquat treatment; n = 3. (d) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α-synuclein and left untreated (None) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7; n = 3. In b-d, all values are mean ± s.e.m. Differences with cells that are untreated (marked with asterisk) or those treated only with paraquat (§) were significant for P 0.001. Full-length blots are shown in Supplementary Figure 21. (e) Immunoblot for α-synuclein (α-syn) in the same cells as in d. Top, higher-exposure blot to highlight oligomeric (oligo) species. Asterisk denotes nonspecific band. M, monomer; MW, molecular weight.
Figure 3Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac. Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. F. Odds ratios as determined by logistic regression showing associations between cell frequencies and the risk of symptomatic P. falciparum infection. Symbols represent the odds ratio estimated using 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, and vertical lines depict the 95% confidence interval. * p<0.05, **p<0.01, ***p<0.001.
CUL3Δ403–459 is unable to ubiquitylate WNK1 or WNK4 kinases in an in vitro system, and this deficiency cannot be rescued by the presence of CUL3WT.Figure 3A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitatedWNK1 in (A). In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3Δ403-459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3WT, CUL3Δ403-459 and an equimolar solution CUL3WT:CUL3Δ403-459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitatedFLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403-459 on the ubiquitylation activity of CUL3WT. Notably, the presence of CUL3Δ403-459 does not inhibit WNK ubiquitylation by CUL3WT.In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.
to buffer endogenous dsRNA levels and regulate growth.Figure 6A, Summary of mass-spectrometric analysis of AGO1x (n=5) or IgG (n=2) IP from control cells. P-values were calculated using Fisher's exact test and corrected for multiple testing using the Benjamini-Hochberg method. B, Western blot analysis to validate the interaction of AGO1x with PNPT1 and DHX9 in control MDA-MB-231cells. AGO1x pulldowns were compared to IgG pulldowns to verify the relative levels of PNPT1 and DHX9 from four independent biological replicates. The immunoglobulin heavy chain band served as a loading control. Immunoprecipitation of PNPT1 from control and mutant MDA-MB-231 cells and western blotting to validate the specificity of PNPT1 antibody in comparison to IgG control (C). Validation of AGO1x-PNPT1 interaction by reciprocal pulldown. Presence of AGO1x and Fibrillarin in PNPT1 immunoprecipitates. Three independent biological replicates were used, and the eluted proteins were analyzed by western blotting (D). E, in vitro interaction assay of isolated nuclear extracts from control MDA-MB-231 cells with FLAG-tagged AGO1 or FLAG-tagged AGO1x immunoprecipitated from cells stably expressing the constructs. Table indicates the peptide counts from a second round of immunoprecipitation with anti-FLAG antibody and subsequent analysis by LC-MS/MS. P-values are from Fisher's exact test, corrected for multiple testing using the Benjamini-Hochberg method. Western blot analysis to verify the depletion of PNPT1 upon siRNA treatment (F) a representative immunofluorescence image of siPNPT1/siControl-treated control and W6A mutant MDA-MB-231 cells stained with J2 antibody (red). DAPI was used to mark the nucleus (blue) (G). The right-most panels show a magnification of the cells enclosed by the white boxes in the middle panels. H, Model of AGO1x function. A complex of AGO1x with PNPT1 interacts with nuclear RNAs to prevent the accumulation of structured dsRNAs, supporting proliferation of breast cancer cells. Depletion of AGO1x leads to deleterious accumulation of these RNAs, which in turn lead to activation of interferon (IFN) response and apoptosis.
USP42 deubiquitinates ZNRF3 and inhibits Wnt/β-catenin signalling A, Scheme of the R-spondin complex with ZNRF3 and LGR, which promotes auto-ubiquitination and clearance of ZNRF3.Figure 1A, Scheme of the R-spondin complex with ZNRF3 and LGR, which promotes auto-ubiquitination and clearance of ZNRF3. The deubiquitinase (DUB) that stabilises ZNRF3 remains unknown. B, Example of a TOPflash reporter assay used to screen for DUBs regulating Wnt signalling. Cells were transfected in triplicates with the single siRNAs against the indicated DUBs or the positive control ZNRF3, and stimulated with control or Wnt3a conditioned media. C, Scheme showing the predicted protein organization of USP42, including the catalytic cysteine of the ubiquitin-specific protease (USP) domain, and the predicted monopartite nuclear localisation signals (NLS) as displayed by NLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). D, Representative immunofluorescence microscopy images showing endogenous USP42 (green) in HEK293T cells transfected with the indicated siRNAs from n = 3 independent experiments. Scale bar = 10 μm. , TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs. In (E), 3 independent siRNAs or a siRNA pool against USP42 were used. In (F), Parental wt HEK293T and ZNRF3-/-/RNF43-/- HEK293T cells were used. TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs. I,J, Ubiquitination of ZNRF3 in HEK293T cells transfected with ubiquitin-HA (Ub-HA). Cells were co-transfected with siUSP42 (I) or GFP-USP42 (J). Cells were treated for 6h with 10 μM MG132 and 5 nM Bafilomycin A1 to prevent ZNRF3-Ub degradation. Ubiquitinated ZNRF3 was isolated by immunoprecipitation (IP) under denaturing conditions and analysed in Western blots with anti-HA antibodies. K, Representative co-immunoprecipitation experiments in HEK293T cells that were transfected with GFP-USP42 or the indicated GFP-tagged control, together with the stated Wnt signalling component (N = 3 independent experiments). IgG heavy chain is indicated with an asterisk. Dvl1 functions as a positive control that binds both ZNRF3 and Axin1. L,M, TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. HEK293T, Parental HEK293T and USP42-/- HEK293T cells were transfected with wt USP42, the catalytically inactive mutant USP42C120A, ZNRF3 or ZNRF3/RNF43.
Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal.Figure 11 Figure 11 Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal. Cells were fixed with PFA or glyoxal for 10 minutes, and were stained for endogenous vimentin, or for expressed alpha-synuclein. A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha-synuclein, but that PFA was superior for endogenous vimentin (leftmost graph). The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal; the latter allowed more mOrange2 fluorescence to be detected (rightmost graph). N = 29 - 81 cell regions per condition. * p < 0.05, *** p < 0.001 Scale bar: 10 µm.
Complex-centric profiling of the HEK293 proteome by targeted query of CORUM, BioPlex and StringDB.Figure 4Schematic overview of the targeted, complex-centric analysis of the protein-level co-fractionation map recorded in SEC-SWATH-MS via CCprofiler. The three-tiered analysis is centered on complex hypotheses (i.e. groups of proteins queried for co-elution in the SEC data) obtained from CORUM or formulated from BioPlex and StringDB. At complex hypothesis FDR controlled to 5 % via the decoy-based error model, co-elution evidence is confidently detected for 621, 1052 and 1795 (representing 35.4, 13.8 and 19.2 %) of the queried CORUM-, BioPlex- and StringDB-derived hypotheses, respectively. Heterogeneity and redundancy within and across the different hypothesis sets translates to the co-elution signals retrieved, which, pieced together by collapsing on composition and SEC elution fraction, identify 462 distinct, chromatographically resolved co-elution groups representative of distinct complexes or equisized families of complexes (also see Appendix Figure S5). For a list of all detected complex signals, see Dataset EV4.
Fig.Figure 4A: Top hits of ELDR interacting proteins analyzed from the ELDR sense vs. antisense RNA pull-down assay followed by mass spectrometry in lysates from JHU022 and Cal27 cells. B: ELDR interacting proteins identified by mass spectrometry classified by GENEONTOLOGY PANTHER classification system based on "Molecular Function". C: Cell lysates from Cal27 and JHU022 cells were incubated with biotinylated ELDR sense and antisense RNA, pulled down and subjected to Western blot analysis for the ILF3 using specific antibodies. Bottom panel shows Western blot analysis of PUM-1 (an unrelated RNA binding protein). D: Cal27 and JHU022 cell lysates were immunoprecipitated against control or ILF3 antibody and RNA was isolated from the precipitates. Relative expression of ELDR (left penal) and PCAT1 (right panel) was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD, E: Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows the quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD, F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.