EMBO/sd-character-level-ner huggingface dataset:
TDP-43 seeding depends on specific aggregate conformation(s) A) Strategy to explore the prion-like properties of TDP-43 seeds.Figure 2A) Strategy to explore the prion-like properties of TDP-43 seeds. B) Bar graph showing the seeding effect at day 5 and day 6 post-seeding in untreated, temperature denaturation or autoclaving denaturation conditions. While seeding in untreated condition showed a significant increase in neoaggregates (One-way ANOVA, with post-hoc Fisher's LSD test, Day 5: F=(5,12)=6.713, p=0.0033, non-neurodegenerative control vs FTLD-TDP-A p=0.0016, Day 6: F(5,12)=4.271, p=0.0183, non-neurodegenerative control vs FTLD-TDP-A p=0.0397), this effect was abolished in denaturing conditions (N=3 biological replicates for non-neurodegenerative control, FTLD-TDP-A and FTLD-TDP-C, bar graph showing mean ± SEM). C) TOP: Aggregation kinetics of full length (fl) recombinant human TDP-43 (rec-hTDP-43) measured as an increase in absorbance. The addition of TEV protease induces the cleavage of the MBP tag and subsequent aggregation of rec-hTDP-43. Rec-hTDP-43 aggregates used for seeding were collected 18 hours after the addition of TEV protease. BOTTOM LEFT: rec-hTDP-43 aggregates stained by Thioflavin T (ThT). BOTTOM RIGHT: Transmission Electron Microscopy (TEM) image of the rec-hTDP-43 aggregates. All images are presented at the same scale of 1 μm. D) Rec-hTDP-43 aggregates were transfected on mitotically arrested cells and fixed after 5 or 6 days post-transfection. Surface masks generated with Imaris depict DAPI (blue), NuclTDP-43 (cyan) and pTDP-43 (magenta) for non-transfected AraC control and 1 μg rec-hTDP-43. All images are presented at the same scale, 20 μm as indicated in AraC panel. E) Quantification of neoaggregates (left y-axis) and pTDP-43 (right y-axis) at day 5 and day 6 post-transfection for increasing concentrations of rec-hTDP-43. 2-way ANOVA repeated measure, with post-hoc Fisher's LSD, F(7,80)=5.886, p<0.0001, Day 5: AraC vs 50 ng p=0.0497, AraC vs 500 ng p=0.0002, AraC vs 5 µg p=0.0161. 50 ng vs 100 ng p=0.0208, 50 ng vs 500 ng p=0.0169, 50 ng vs 2.5 µg p=0.0272, 100 ng vs 1 µg p<0.0001, 100 ng vs 5 µg p=0.006, 250 ng vs 1 μg p=0.0005, 250 ng vs 5 µg p=0.034, 500 ng vs 1 µg p<0.0001, 500 ng vs 5 µg p=0.0047, 1 µg vs 2.5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0081, Day 6: AraC vs 250 ng p=0.0385, AraC vs 1 µg p=0.0006, 50 ng vs 1 µg p=0.0016, 100 ng vs 1 µg p=0.0101, 100 ng vs 5 µg p=0.0173, 250 ng vs 5 µg p=0.0006, 500 ng vs 1 µg p=0.0099, 500 ng vs 5 µg p=0.0176, 1 µg vs 2.5 µg p=0.0144, 1 µg vs 5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0121. To reduce complexity, only comparisons with AraC are marked in the graph. N=6 biological replicates for each concentration; bar graph showing mean ± SEM.
GPS cleavage of PC1 is not necessary for channel activity of the PC1/PC2_AA channel.Figure 7A. Transmembrane topology of PC1 and PC2 proteins, showing the cleavage at the N-terminal GPS site (in circles), the "HLT" tripeptide where the cleavage happens, and the two mutations, L3040H and T3041V, that abolish GPS cleavage. Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test). Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test). D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.
Figure 5(A) T489 and S495, within Mig1C, are predicted to be the phosphorylation sites of SNF1 by global phosphorylation sites. (B) Mig1 T489 and S495 are the main phosphorylation sites of SNF1 in response to HU. The SNF1 kinase complex was partially purified from HU-treated Snf1-5FLAG or snf1-KD-5FLAG yeast cells. Recombinant Mig1 or its mutant proteins (mig1-2A, T489AS495A) were purified from E. coli. In vitro kinase assays were performed as described in Methods and Materials. Proteins were immunoblotted using the indiated antibodies. The anti-AMPK substrate antibodies were used to probe the phosphorylation of the substrate Mig1. Less signals were observed when Mig1 was replaced by mig1-2A. Phosphorylation of Snf1 Thr 172 indicates the activation status of the Snf1 kinase. Phospho-mimetic mig1-2D bypasses the role of SNF1 in HU resistance (C) Phospho-mimetic mig1-2D bypasses the role of SNF1 in RPA recruitment (D). (E) The diagram of the strategy to reinforce the Mig1-Ssn6 interaction via a GFP and GBP pair (left panel). The persistent Mig1-Ssn6 association abolishes the phospho-mimicking effect of mig1-2D (right panel).
The Mif2 N-terminus is prevented from high affinity Mtw1c binding in the full-length molecule.Figure 5Co-purification of Strep-Mtw1c through Flag-Mif2 after co-expression in insect cells. Proteins were detected by western blotting using their respective tags. Western blotting of elution fractions from an SEC experiment of Mif2-wt or Mif2Ame1-N swap in the absence (upper panel) or presence (lower panel) of Mtw1c. ITC analysis of Mif21-41 or Ame11-20 binding to MN. Experiments were performed 3 or 4 times, respectively. One representative binding curve is shown, whereas the KD value represents the average of all experiments. Cross-link (XL) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross-linker Bis[sulfosuccinimidyl] glutarate. Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration. The corresponding Coomassie-stained SDS-PAGE gels of Mif2-wt alone or in combination with Cse4n are shown. Fractions used for cross-linking are highlighted. Topological map of Mif2 based on the identified intra-molecular cross-links. Common cross links that are found in both samples are shown in red, whereas unique crosslinks that can be only found in one of the samples are highlighted in blue. Conserved regions and their described functions are indicated. A list of cross-links that link N-terminal residues to C-terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2.
Trf1 deficiency leads to G2 arrest and mitotic defects.Figure 4Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel). Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells. Percentage of giant nuclei in the carcinomas of the indicated genotype.Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.
Figure 3Schematic diagram of the in vitro CDC50A-mCherry expression experiment. After 6 days in vitro (DIV), cortical neurons were transfected with CAG-GFP and CAG-mCherry or CAG-GFP and CAG-Cdc50a-mCherry. Representative confocal z-stack images of the rER (Nissl staining, cyan) with GFP (green) and mCherry/CDC50A-mCherry (red). The dashed line represents where the high magnified insert was taken from. Scale bars, 50 (left), 20 (middle), and 5 μm (right). Schematic diagram of in vivo CDC50A-mCherry expression experiment. Neonatal (P0) wild-type mice were injected with AAV9-CAG-Cdc50a-mCherry. Representative confocal z-stack images of the rER (Nissl staining, green) and CDC50A-mCherry (red) in the CTX (cortex). The dashed line represents where the high magnified insert was taken from. Scale bars, 20 μm (upper) and 5 μm (lower). Schematic diagram of the experiment in which PS exposure was detected in control and Cdc50a cKO neurons in vitro. After 6 DIV, cortical Cdc50afl/fl neurons were transfected with either hSyn-mCherry or hSyn-Cre-p2a-dTomato to generate control and Cdc50a cKO neurons, respectively. Representative live confocal z-stack images showing PS exposure (pSIVA, green) in mCherry/dTomato (red)-expressing neurons. Scale bar, 20 μm. The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice. Schematic diagram of the method in which PS exposure was detected in vivo using secreted Annexin V (secA5) fused with mCherry (red). Representative confocal z-stack images of secA5-mCherry (red) expression in the brains of wild-type mice using AAV9-GFAP-secA5-mCherry injection. The white circles indicate PS exposure detected by secA5. Scale bar, 10 μm. Schematic diagram of the method in which PS exposure was detected in the brains of control and Cdc50a cKO mice in vivo after AAV9-GFAP-secA5-mCherry was injected in the neonatal stage (P0). Representative confocal single-plane images showing PS exposure (secA5-mCherry, red) in the somas of Thy1-CreERT2, -EYFP-expressing HP (hippocampus) CA1 neurons or IC (inferior colliculus) neurons (green) from control and Cdc50a cKO mice. Scale bar, 5 μm. Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).
Figure 3In vitro pull-down of PHYA-C1 (A) and PHYB-C1 (B) with CBF1. The His-tagged PHYA/B-N (photosensory domain), PHYA/B-C1 (PAS-related domain), and PHYA/B-C2 (histidine kinase-related domain) proteins pulled down with GST-tagged CBF1 or GST were detected by anti-His antibody. Input, 6% of the His-tagged purified target proteins used in pull-down assays. LCI assays showing that CBF1 interacted with phyA in living plant cells. Scale bar = 1 cm. D, E Co-IP assays showing that CBF1 interacted with phyA (D) and phyB (E) in vivo. (D) homozygous CBF1-myc seedlings were first grown at 22°C in continuous FR light for 4 d, then the total proteins were extracted and subsequently treated with 5-min R light, or 5-min R light followed by 5-min FR light (R+FR) before immunoprecipitation. (E) homozygous CBF1-myc seedlings were first grown at 22°C in continuous R light for 4 d, then the total proteins were extracted and subsequently treated with 5-min FR light, or 5-min FR light followed by 5-min R light (FR+R) before immunoprecipitation. After light treatments, total proteins were incubated with anti-c-myc Affinity Gel (Sigma-Aldrich). The total and precipitated proteins were examined by immunoblotting using antibodies against myc, phyA, phyB, and RPN6, respectively. The numbers below anti-phyA and anti-phyB blots indicate the relative band intensities of co-precipitated phyA or phyB normalized to those of precipitated CBF1-myc, respectively. The ratio of the first clear band was set to 100 for each blot.
Cathepsin L is required for the entry of 2019-nCoV.Figure 1.The sequence alignment based on the consensus S protein sequences of SARS-CoV and 2019-nCoV. Alignment was performed by using ClustalW method. The amino acid sequence logos generated by using WebLogo was the graphical representation of the multiple alignment of the sequences of SARS-CoV and 2019-nCoV. The overall height of the stack indicated the sequence conservation at that position, while the height of symbols within the stack indicated the relative frequency (Y-axis) of each amino acid at that position (X-axis). The multiple alignment created based on the region containg in the cleavage site of cathepsin L (SIIAYTMSLGA) on the S protein of 2019-nCoV, including 9 from Wuhan, 2 from Shenzhen and 5 from USA. The accession number of each sequence was showed in the strain name. The identity/similarity shading with the color was referred to the chemistry of each amino acid at that position. HEK293T cells were transfected with 200 nM siRNAs per well. After 24 h, the cells were infected with HIV-luc/2019-nCoV pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.
functions as a negative regulator in ova-induced airway inflammation (A-B) Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13, IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin.Figure 4Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13 in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates. Quantitative RT-PCR analysis of mRNA encoding various cytokines IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. (C) ELISA analysis of IL-33 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates. (D-G) ELISA analysis of IL-4, IL-13, IL-1β and IL-18 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates. (H) Cell number and category of BALF samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates. (I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.
Figure 7A. Association of IDAP2 with hyper-DMR loci. The DT loci are shared hyper-DMRs between idap2-1 and idm1-1. ChIP was performed in the wild-type and IDAP2-3Flag transgenic plants with an anti-Flag antibody. B. Association of IDAP1 with IDM1 targeted hyper-DMRs. ChIP was performed in the wild-type and IDAP1-3Myc transgenic plants with an anti-Myc antibody. C. The effect of the IDAP2 mutation on the recruitment of IDM1 to its target loci. ChIP was performed in the wild-type, IDM1-HA-YFP/idm1-3 and IDM1-HA-YFP/idap2-1 transgenic plants with an anti-HA antibody. The transgene was introduced into idap2-1 by crossing. D. Detection of the levels of acetylated H3K18 at the 35S promoter and ACTIN 7 in the wild-type, idm1-14, idap1-2 and idap2-2 plants. ChIP was performed using an antibody against acetylated H3K18. E. The effect of the IDAP2 mutation on the levels of acetylated H3K18. Acetylated H3K18 levels at the DMRs and control regions in the wild-type, idm1-1 and idap2-1 plants were determined by ChIP with an antibody against acetylated H3K18. In (A-E), the ChIP signal was quantified relative to input DNA. Standard deviations were calculated from three biological replications, *P < 0.05 (Student's t-test).
Figure 5(A) ChIP assays with -H2A, -H4 and -H1. Precipitated DNA was analysed by PCR for the presence of sequences corresponding to the BCAS1, KRT23 and MMTV genes. * P-value < 0.05. (B) Left panel: ChIP-seq experiment using H1.2 specific antibody in untreated and cells exposed to hormone. The ratio between +R5020/T0 around the TSS (-475 +400) of the up, down and non-regulated genes is shown. Right panel: down-regulated genes presented an inverse trend for RNA pol II signal around the TSS (-475 +400) after hormone compared with Up and down-regulated genes. (*) P-value < 0.05; (***) P-value < 0.001. (C) ChIP-seq experiments in T47D-MTVL cells expressing HA-tagged versions of the different H1 isoforms in untreated and cells exposed to hormone. The median corrected ratio between +R5020/T0 around the TSS (-475 +400) of the up, down and non-regulated genes is shown. A significant increase of the signal for H1.2 after hormone exposure was found in down-regulated genes compared with up and non-regulated genes. The median-corrected ratio represents the R5020/T0 ratio corrected by the median values obtained from the TSS of non-regulated genes for each isoform. (***) P-values for H1.2 DOWN vs NON: 2.5x10-68, P-value: DOWN vs UP: 8.3x10-72, UP vs NON: 2.7x10-6. (D) Hormone-dependent deposition of H1.2 was quantified in down-regulated genes, which presented PRBs (PRBs+) or not (PRBs-) in their promoter region. (*) P-value < 0.05. (E) T47D-MTVL/H1.2 and H1.3 knock-down cell lines (Sancho et al., 2008) treated or not for 6 days with DOX to induce specific H1 depletion, were incubated for 6h with 10nM R5020 as indicated; cDNA was generated and used as template for real time PCR with gene-specific primers. The extent of knockdown was analyzed by western blotting using specific antibodies (lower panel). (*) P-value < 0.05.
Figure 5A Schematic description of the domain organization of human TOP2A. B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins, and then exposed to nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm. HeLa cells were transfected and treated Example images are shown (C). Scale bars, 10 μm. HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells. Data information: Means and error bars representing S.D. are shown unpaired t-test). HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained Example images are shown (G). Scale bars, 10 μm. HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 250 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test). U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Example images are shown (I). Scale bars, 10 μm. U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t-test). U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Cell lysates were immunoblotted (K).
ER whorls arise from ER stacks and undergo microautophagy.Figure 2A Individual frames from time-lapse imaging of a cell expressing GAL promoter-driven GFP-Pho8 and stained with the vacuole membrane dye FM4-64. Numbers indicate the time in minutes after the start of the image sequence. Scale bar: 2 µm. B Electron micrograph of a cell expressing GPD promoter-driven GFP-Pho8. Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination. Black arrowheads point to vacuole membrane that encases the bent stack and the whorl. C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks. n = 43. The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl. Scale bar: 2 µm.
N-antibodies inhibit SARS-CoV-2 replication intracellularly.Figure 2(A&B) Vero cells OE ACE2 and TMPRSS2 were infected with SARS-CoV-2 in the presence of IgG or anti-N antibodies added directly into media or electroporated into cells. Viral replication was then determined by RT-qPCR (A) or plaque assay (B). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication (*** = p < 0.0002). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way ANOVA. (C&D) As with A&B, except with a titration of electroporated anti-N antibodies. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way ANOVA. (E) Western-blot of 293T cells and 293T ACE2 OE/TRIM21 KOs alone or reconstituted with empty vector (EV) or an endogenous-promoter driven TRIM21 vector (T21)(Zeng et al., 2019). (F&G) 293T ACE2 OE/TRIM21 KO cells reconstituted with EV or TRIM21, electroporated with IgG or anti-N antibodies, and infected with SARS-CoV-2. Viral replication was then determined by RT-qPCR (F) or plaque assay (G). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication only in TRIM21-reconstituted cells (* = p < 0.05). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using two-way ANOVA.