EMBO/sd-character-level-ner huggingface dataset:
TFIISmut shows splicing defects consistent with kinetic coupling of transcription and splicingTFIISmut shows upstream splice site selection.Figure 6A TFIISmut shows upstream splice site selection. The splicing was assessed by RT-PCR and capillary electrophoresis in 3‐week‐old soil grown plants. The chart represents the average relative contribution of the mRNA forms found in the total pool of amplified products. The error bars represent ± SD (n = 3). To the right of the charts, the structures of the examined transcripts are shown. The black boxes, white boxes and black lines represent constitutive exons, alternative regions and introns, respectively. The black arrows show the locations of the primers. Representative splicing assays are shown.B Directionality of splice site selection in TFIISmut. For each type of alternative splice event, the black and white boxes show the contributions of opposite direction splicing events. The numbers represent the percentage of splice events supporting the direction of the splice site event change (also shown on horizontal axis). The numbers on the right‐hand panel represent the number of affected splicing events versus total number of splicing events analysed. The white bars represent downstream 3′ and 5′ splice site selection (3′SS/5′SS), exon skipping (ES) and intron retention (IR), while the black bars represent upstream 3′/5′ splice site selection (3′SS/5′SS), exon inclusion (ES) and intron splicing (IR). C TFIISmut and the atntr1 mutant have opposite splicing phenotypes. Heat map with scale representing the absolute difference between each respective mutant and Col‐0 in alternative splice site usage. The colours represent the direction of the events as described on the axis. Each splicing event is labelled with the gene name and the type of alternative splicing event. The 3′SS/5′SS notation signifies upstream/downstream 5′/3′ splice site selection; ES marks exon skipping or exon inclusion; and IR represents intron retention or intron splicing.
Figure 2A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies. C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.
Figure 2A. Schematic of Flag-FoxM1 deletion mutants in a mammalian expression system. FL: full length; NT: NH2-terminal domain; FB: forkhead box domain; CT: COOH-terminal domain. B. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel). C. Sequence analysis of humanFoxM1 identified three putative GSK3β target sites at S228, T309, and S474. D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody. E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody. F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody. G. GSK3βCA or vector and Flag-tagged FoxM1 (WT), mutants S474A or S478A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho-S474 antibody.
Figure 4Figure 4. Helix-7 discourages non-Watson Crick pairing at g6 and g7. A-C) Plots of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 (wild type and Ηhelix-7) concentration for targets with an increasing number of G:U wobble pairs in the seed 3' end. D) Plot of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 concentration for a target with mismatches at positions g6-g8. For A-E, average values from at least three independent experiments ± SD are plotted. E) Bar plots of the Kd of wild type (left) Ηhelix-7 (right) Ago2-miR122 for indicated target RNAs. For A-E, average values from at least three independent experiments ± SD are plotted.
Figure 7A-C, Pre-embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof+/+ (A) and OtofI515T/I515T (B) but not in Otof-/- (C) mice. IHCs are highlighted in beige. Scale bar 100nm. D-G, Magnified synaptic ribbons (R) and postsynaptic afferent boutons of SGNs (Aff), displaying otoferlin immunogold labelling at active zone membranes (pink arrowheads) but not around the synaptic ribbon, using saponin permeabilization (D-F), or Triton-X-100 treatment (G). H, Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle. Scale bar 200nm. I,J, Some otoferlin labelled structures bear clathrin coats (red arrowheads), suggesting that they originate from bulk endocytosis. Scale bar 100nm.K, Otoferlin labelled vesicles were on average larger in OtofI515T/I515T IHCs (OtofI515T/I515T, n=86 vesicles, 18 images; Otof+/I+, n=82 vesicles, 6 images; Wilcoxon-rank-sum-test, as in L-M).L, Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T/I515T (6 images each).M, Distal from the plasma membrane, immunogold labels were reduced in OtofI515T/I515T IHCs. N, Representative electron micrographs of IHCribbon synapses in OtofI515T/I515Tand Otof+/ (conventional embedding without immunogold) after pre-incubation for 10min at the indicated temperature followed by high K+ stimulation or 0 Ca2+ inhibition for 1min 45sec. Scale bars 100nm. O, Illustration depicting criteria for random section analysis (not drawn to scale). Ribbon-associated synaptic vesicles (SVs, green) in the first row ≤80 nm from ribbon (R) and membrane-proximal vesicles (yellow) in ≤25 nm membrane-to-membrane distance from the plasma membrane (blue) and ≤80 nm from each side of the presynaptic density. P-Q, Synaptic vesicles with >45 nm diameter appeared at 39°C in random sections; fraction of large membrane-proximal (P; Kruskal-Wallis test) and ribbon-associated (Q; one way ANOVA followed by Tukey's test) synaptic vesicles (see also Appendix Table S1).
a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP).figf8(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs. (b) We transfected SBMA NPC lines (three different NPC lines per patient) with either the BFP-empty vector or BFP-TFEB expression construct, treated the NPCs with JC-1 dye and counted the percentage of cells with depolarized mitochondria. Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(6) = 3.217. (c) Representative images from transfection of SBMANPCs with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct. BFP-empty vector-expressing SBMANPCs display a preponderance of yellow puncta (autophagosomes), while BFP-TFEB-expressing SBMANPCs exhibit a shift toward almost entirely red puncta (autolysosomes), consistent with rescue of the autophagic flux defect. Scale bars, 20 μm. (d) We transfected SBMANPC lines (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct, counted the number of autophagosomes and autolysosomes in BFP-expressing NPCs and determined the number of autophagic vesicles per cell. TFEB overexpression markedly reduced autophagosome formation in SBMANPCs. ***P 0.001; t-test, t(30) = 4.373. Data in d are presented as mean ± s.e.m. Individual P values are available in the Supplementary Methods Checklist.
NAT1 regulates TORC1 activity via eIF4A.Figure 6(a) Mass spectrometry analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.(a') Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown. (b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates. (c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates. (d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates. (e) TSC2 is epistatic to NAT1 for TORC1 regulation. As in (d), knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown. (f) Schematic diagram showing the signaling relationships between NAT1, eIF4A and TSC2. When amino acids are present, NAT1 binds eIF4A and inhibits it. Upon amino acid removal, NAT1 releases eIF4A, which activates TSC2 to inhibit TORC1.
Figure 7Three CLK-mouse lines were generated and subsequently immunized with eOD-GT8 60mer in adoptive transfer/immunization system (precursor frequency:1 in 104). eOD-GT8-specific splenic IgG1+ or IgM-IgD- B cells were single-cell sorted at day 36 post immunization for single-cell PCRs. (A) Phylogenetic trees of CLK21, CLK09 and CLK19. These trees were generated using paired aminoacidic sequences solely isolated at day 36. Single paired amino acid sequences were joined and aligned using MUSCLE Clonal lineage trees were generated using FastTree and a Jones Taylor Thornton model for AA evolution The length of the branches reflects sequence distance. Three CLK-mouse lines were generated and subsequently immunized with eOD-GT8 60mer in adoptive transfer/immunization system (precursor frequency:1 in 104). eOD-GT8-specific splenic IgG1+ or IgM-IgD- B cells were single-cell sorted at day 36 post immunization for single-cell PCRs. (B) SHM are detectable in both IGHV and IGLV at day 36 post immunization with eOD-GT8 60mer.nt represents "nucleotide", aa represents "amino acid". Three CLK-mouse lines were generated and subsequently immunized with eOD-GT8 60mer in adoptive transfer/immunization system (precursor frequency:1 in 104). eOD-GT8-specific splenic IgG1+ or IgM-IgD- B cells were single-cell sorted at day 36 post immunization for single-cell PCRs. (C) Hotspot analysis. The quality of mutations was assessed via hotspots analysis for both heavy (left) and light (right) antibody chains. Weblogos were generated via publicly available online tools Three CLK-mouse lines were generated and subsequently immunized with eOD-GT8 60mer in adoptive transfer/immunization system (precursor frequency:1 in 104). eOD-GT8-specific splenic IgG1+ or IgM-IgD- B cells were single-cell sorted at day 36 post immunization for single-cell PCRs. (D) Mature bnAbs-like aa VH1-2 mutations in CLK B cells at day 36. The red diagonal line indicates a 100% efficiency of VRC01-class bnAb-type VH1-2 mutations. The black stair step indicates a calculated VH1-2 antigen-agnostic mutation distribution, which might include mutations that improve expression or stabilize Ab structure Shaded area indicates that no mutation is positive in this region.
Figure 7(A) Western blot analysis of Parn expression in control and Parn+/- MEFs. (B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs. (C) Schematic representation of mouse rRNA precursors and position of DNA probes used in northern blot. (D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.
Figure 1A) Representative images of HeLa cells treated with Control, H2A.Z.1 or H2A.Z.2 siRNA for 72 h, fixed, and stained with DAPI. White arrows point at micronuclei. Scale bar: 10 μm. B) Quantification of the percentage of cells with micronuclei from experiment in (A). Three biological replicates were analysed for each condition (Control si N=1271; H2A.Z.1 si N=1091; H2A.Z.2#1 si N=1188; H2A.Z.2#2 si N=1193). The error bars represent the SD. Data sets were statistically analysed using Chi-square test. ***=p<0.001. C) Quantification of the percentage of cells with micronuclei from HeLa cells co-transfected with H2A.Z.2 siRNA and GFP:H2A.Z.2.1WT with either single or double mutations. Error bars represent the SD of three biological replicates (Control si N=1316; H2A.Z.2 si N=1877; H2A.Z.2 si + wt N=1973; H2A.Z.2 si + A14T N=618; H2A.Z.2 si + S38T N=558; H2A.Z.2 si + A127V N=649; H2A.Z.2 si + A14T/S38T N=1090; H2A.Z.2 si + A14T/A127V N=1038; H2A.Z.2 si + S38T/A127V N=1150). Data sets were statistically analysed using Chi-square test. ***=p<0.001; black stars refer to the comparison with the H2A.Z.2si data and blue stars refer to the comparison with the H2A.Z.2si + wt data; no stars = non significant. D) Schemes of the GFP constructs used for the rescue experiments in (E) and (F). Green boxes represent the GFP, brown boxes represent the H2A.Z.2.1 isoform. Solid fill represents the WT construct whereas striped box represents the KR mutant form where the residues indicate the mutations performed. Dots represent the possible post-translational modifications in the mutated amino acids: acetylation (ac, dark blue), methylation (me, pink) and ubiquitination (ub, light blue). E) Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and each of the constructs in D (green). Scale bar: 5μm. F) Quantification of the percentage of cells with micronuclei from experiment (E). The error bars represent the SD of three biological replicates (Control si N=887; H2A.Z.2 si N=1005; H2A.Z.2 si + wt N=760; H2A.Z.2 si + KR N=479). Data sets were statistically analysed using Chi-square test.
Figure 2(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown. (B) MDMs were infected with the KOS or F strains of HSV-1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β-actin were determined by Western blotting. (C) HFFs and MDMs were infected with HSV-1 (KOS) MOI 0.1 and 1.0. Culture medium was replaced 3 hpi, and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates +/- st.dev. (D) THP1 cells were infected with the indicated strains of HSV-1 (MOI 3) for 12 hrs. Cell lysates were isolated and levels of ICP27 and β-actin were determined by Western blotting.
Figure 1(A) Chemical structures of thalidomide, pomalidomide and lenalidomide. (B) Schematic diagram of the thalidomide-dependent in vitro binding assay between CRBN and substrates using AlphaScreen technology. (C) Detection of luminescent signals of thalidomide-dependent interactions between bls-CRBN and FLAG-GST-IKZF1. Dose-dependent signals (DMSO, 2.5, 5, 10, 25, 50 or 100 µM thalidomide) were analysed with an in vitro binding assay using the AlphaScreen technology. (D) Results of in vitro high-throughput screening targeting 1,118 human transcription factors. Green and red spots denote known neosubstrates and candidate clones, respectively. (E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology. (F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).
Figure 9 Figure 9. Model for the molecular switch mechanism. A model outlining the key components required to switch on mitophagy in a high oxidative (ROS) environment where Parkin has a MetO at position 192. Release of MsrB2 upon mitochondrial damage, swelling and rupture, removes the brake leading to formation of the autophagosome at the site of rupture. This is the underlying molecular process observed on the EM in Fig. 1A. where mitophagy is taking place only with the ruptured mitochondria, sparing the adjacent intact mitochondria.
65-day-old SOD1G86R mice have improved performance during endurance exercise.Figure 1A-F (A-C) Exercise performance on a treadmill apparatus using an intense exercise (incremental speed paradigm) that relies solely on anaerobic metabolism. (D-F) Exercise performance on a treadmill apparatus using a moderate exercise (constant sub-maximal speed) that relies on the recruitment of aerobic metabolism and that is representative of endurance exercise. (A, D) Kaplan-Meier curves representing values corresponding to the percentage of mice still running at a given time point. (B, E) Median values of the time that mice ran are represented in the min to max graphical representation; 15.5 min for wild-type (WT) and 14.33 min for SOD1G86R in (B); 59.33 min for WT and 65.43 min for SOD1G86R in (E). (C, F) Mean distance run ± SEM by WT and SOD1G86R mice in (C) and by WT and SOD1G86R mice in (F).
Figure 6(A) DVL3 co-immunoprecipitates KMT2D in MDA-MB-468 cells (upper panel) and MCF7 cells (lower panel). IgG heavy chain (Hc) was blotted for as a control for equal antibody capture for immunoprecipitation and whole cell extracts (WCE) as a positive control. (B) MDA-MB-468 histone extracts from NTC, shA DVL3 and shB DVL3 subjected to immunoblot analysis for H3K4me3 and H3. (C) An assembly of IgG (first row), DVL3 (second row) ChIP-Seq in MDA-MB-468 cells, IgG (third row), and, DVL3 (fourth row) ChIP-Seq data in MCF7 for the KMT2D gene, visualized by IGV. Each column is 20000 bp wide. The fifth row show the location and orientation of the KMT2D gene. (D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H (H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm (I) Sequential ChIP (ChIP-reChIP) to examine occupancy of DVL3 and KMT2D at RFX5, HAUS5, COX14, APOC1P1, WDR74, JUNB, and, HLA-A promoters analyzed by end-point PCR. (J) Sequential ChIP (ChIP-reChIP) to examine occupancy of DVL3 and H3K4me3 at RFX5, APOC1P1, JUNB, and, HLA-A promoters analyzed by end-point PCR.
hTauAT expression causes Tau phosphorylation and mis-sorting in organotypic hippocampal slice cultures.Figure 3A. Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygouse hTauAT (lane 2, (+/-)) or homozygouse hTauAT slices (lane 3, (+/+)) were analyzed after three weeks in culture. Actin served as a loading control.B. Quantification of hTauAT expression levels (n=3-6 slice homogenates). The ratio between hTauAT and endogenous mouse Tau is shown. C. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image). D. Immunohistochemistry against phosphorylated Tau at the PHF1-epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures. E. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft.
Model for TRIM21-mediated Cross Presentation.Figure 5Immune complexes (eg Ab:N protein) are phagocytosed - 'Uptake' - by APCs, before being moved from the endosome to the cytosol - 'Import'. While multiple routes for uptake have been described, the mechanism and regulation of exogenous protein import is not well understood. Once in the cytosol, immune complexes are detected by the high-affinity Fc receptor and E3 ubiquitin ligase TRIM21. TRIM21 catalyses ubiquitination to recruit the proteasome and cause complex degradation. Proteasome-derived peptides can then be loaded onto MHC Class I molecules and exported to the cell surface to activate T cells.