EMBO/sd-character-level-ner huggingface dataset:
Figure 4Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2, stained with Spata2 and γ-tubulin antibodies and the nuclear dye DAPI. Scale bar, 10 µm. Confocal immunofluorescence analysis of Spata2 localization in RAW264.7 cells stably expressing GFP-Spata2, stained with γ-tubulin antibody and DAPI. Scale bar, 10 µm. Confocal microscopy of HEK293 cells stably expressing GFP-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) that were stained with γ-tubulin antibody and DAPI. Confocal microscopy of HEK293 cells stably expressing GFP-tagged CYLD along with an empty vector or vectors encoding Flag-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) stained as in (C). Scale bar, 10 µm. Co-immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants (ΔPUB orΔ51-81) in HEK293 cells transfected with the indicated expression vectors. CYLD and Spata2 proteins were detected by immunoblot using anti-HA and anti-Flag antibodies, respectively. Immunoblotting analysis of the active caspase-1 p20 in cell supernatants (Sup) and pro-caspase-1 and Flag-Spata2 proteins in cell extracts (Cell ext) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization (ΔPUB orΔ51-81). Cells were primed with LPS and stimulated with Nigericin for 1 h. Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
Figure 3a) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at the time of tissue collection. Clones connected to hair follicles were more likely to be larger in anagen compared to telogen (Chi square test, *** p=0.0009 at 3 weeks, *** p<0.0001 at 5 weeks, * p=0.0193 at 12 weeks, *** p=0.0001 at 24 weeks). However no difference between the two phases was observed in clones that were not attached to HFs. Clone numbers are indicated on the graph. All data are represented as mean ± s.e.m. b) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post-induction. Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling. Average clone size of attached (circles) and non-attached (squares) clones were plotted over the number of club hair. T-test, **** p<0.0001. c) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at induction. Even at a rate of one cell division per 24h, 7 cycles of divisions results in 128 cells. Therefore the findings reported are not incompatible with our claims as suggested. Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe. Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones. Chi square test, *** p=0.001; ** p=0.01.
a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.f1(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm. (b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody. (c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.
Figure 1(A) Strategy for the screening of neutralizing antibodies. B cells from SARS-CoV-2 convalescent patients were enriched using magnetic-activated cell sorting (MACS) beads coated with biotinylated RBD. Phage display libraries expressing scFv were selected against RBD and the resulting clones were screened using ELISA, fluorescence-detection size exclusion chromatography (FSEC), and neutralization assays. (B, C) Kinetics of the binding between the scFv of FD20 (scFD20) and RBD from SARS-CoV-2 (B) or SARS-CoV (C). Biolayer interferometry (BLI) assay was performed with RBD immobilized and scFD20 as analyte at indicated concentrations (nM). Raw data are shown in color and fitted lines are shown in grey. KD values were calculated using 1:1 global fitting. Note, although the fitting of the binding curves in D gave a KD of 4.8 nM, the accuracy of the kinetic parameters may be compromised owing to the mismatch between the raw data and the fitted curves at the initial dissociation phase. The reason for the abnormal BIL profile is unknown but it may be caused by possible denaturation of the commercial SARS-CoV RBD during storage. It was also noted that the BLI signal in D was ~5 times less than that with SARS-CoV-2 RBD under similar conditions (C). (D) Neutralizing assay of scFD20 against SARS-CoV-2 (black) and SARS-CoV (red) pseudoviruses. (E) Neutralization assays using FD20 against pseudoviruses harboring the wildtype SARS-CoV-2 S (blue), S D614G mutant (red), or SARS-CoV S (black).
Figure 4Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). (A) Schematic representation of the experimental setup. Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C). Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM). Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E). Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cells ILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H). A non-significant increase in T regulatory cell ICOS expression was also noted (I).
PGE2-induced nuclear export of HDAC5 is regulated by both PKD and PAK2 Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours.Figure 6 Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6
Evidence that Rab8A Ser111 is not a direct substrate of PINK1.Figure 7Ubiquitin, Parkin, Rab8A and Rab1A phosphosites adopt two distinct conformations. Ser65 in ubiquitin (blue) and Parkin (magenta) follows a β‐turn, before the start of the 5th β‐strand. Conversely, Ser114 in Rab1A (green) and Ser111 in Rab8A (ochre) lie after a C‐terminal helix cap just before the start of a β‐turn. Representative three‐dimensional structures are superimposed by C‐α positions for the observed phosphosites and their sequence neighbours. Side chains for observed sites are shown as sticks, and ribbons depict backbone and secondary structure. To highlight the local environment, regions more than 3 amino acids away from the phosphosites are made transparent (see Materials and Methods for PDB IDs). In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.
Figure 6In this model an increase in PIF4 protein levels as a consequence of elevated temperatures promotes formation of the PIF4-BES1 complex, and activated expression of the DWF4, CPD, BR6ox2 BR biosynthetic genes. Increased BR levels support plant growth by inactivating BIN2 and promoting accumulation of the active PIF4 and BES1 proteins. In response to light, phyB destabilizes PIF4 and the BES1 dimeric protein is dominant over the PIF4-BES1 complex. Dimeric BES1 represses expression of BR biosynthetic genes and leads to decreased BRs levels. As a consequence, BIN2 is reactivated and phosphorylates PIF4 and BES1, inactivating these factors, which leads to increased transcription of BR biosynthetic genes during the night. Light and temperature cues thus control PIF4 and BES1 levels, the nuclear balance of both factors providing a highly sensitive mechanism for rapid modulation of plant growth and development in response to environmental cues
a) Morphological analysis.f1(a) Morphological analysis. Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h. Drug-treated cells were classified as normal, apoptotic or abnormal, with enlarged and irregular nuclei, with multipolar mitoses or multinucleated. Cultures treated with etoposide (0.25 μM), cisplatin (3 μM), taxol (4 nM) and nocodazole (10 nM) exhibited high levels of cells with abnormal nuclei. None of the treated populations showed a significant fraction of apoptosis (see also Supplementary Fig. S1). Values are means±s.d. for three independent experiments. Right: representative examples of these morphological categories are shown. Scale bar, 10 μM. Similar abnormalities were also seen after treating NB4 and MDA-MA-231 cells with etoposide or cisplatin (Supplementary Fig. S2). (b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.
AosR binds to a stress-inducible auxiliary promoter in conjunction with SigH.Figure 5Alternate model for the transcriptional regulation mediated by AosR. Upon oxidative stress, AosR regulates auxiliary promoter upstream of mec-cysO-cysM resulting in enhanced L-cysteine biosynthesis. The promoter of sigA and mec was fused to luciferase, and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=6) Outline of interactome experiment. Venn diagram illustrating the overlaps among interacting partners found in different conditions. Table listing the proteins that interact with AosR specifically under oxidative stress. BLI sensorgram demonstrating relative binding affinities of 800nM His-AosR to immobilized His-SigH under oxidizing and reducing conditions. Rv::pN and Rv::pN-F-sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-AosR. 50 µg WCLs were probed with α-FLAG,α-AosR, and α-SigA as controls. Expression of CysO, CysM, AosR and GroEL1 (control) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h. SigH dependent promoter is present upstream of mec. The consensus -35 and -10 regions along with TSP are indicated (Sharp et al., 2016). 100 bp upstream of mec was fused to luciferase, and transformed cells were either left untreated or exposed to 50 μM CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=3) 5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.
Figure 6Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. (a Expression of IAV proteins (i.e. NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05). Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. c) Expression of IAV proteins (i.e. PA, PB2, M2, M1, was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM.
Figure 2 (B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').
Figure 4A Levels of bioactive LMs and IL-10 released from monocyte and non-monocyte fractions. B Non-monocyte fractions were pre-incubated with IL-10R inhibitor and PGE2 receptor (EP2 and EP4) antagonists (or DMSO as a control) and incubated with CLys (left upper panel) or CSN (10µg/ml) (left lower panel) in presence or absence of monocytes and Treg induction was analysed after 72h. On the right panel, PGE2 receptors were first blocked on monocytes (2) or non-monocytes (3) or both (4) before addition of non-monocytes (2) or monocytes (3) and incubation with CSN for 72h and analysis of Treg induction compared to control condition (1). C Treg induction by CLys and CSN in presence of monocyte/non-monocyte fractions and naïve CD4+CD45RA+CD45RO- T cells. D Monocyte phenotyping in presence of CVF, CLys and CSN by FACS using CD68, CD80, CD163 and CD206 surface expression. E Levels of LMs and PGE2 released by macrophages in presence of CVF, CLys and CSN Additionally, macrophages were lysed and gene expression of PTGS2, PTGES, ALOX5, ALOX15 and MaR1 quantified by RT-PCR. F Treg induction following naïve CD4+CD45RA+CD45RO- T cells incubation with CLys-, CSN-monocyte conditioned media.
Figure 7A Immunoprecipitation analysis of acetylation of p97 in RAW264.7 cells cultured in media containing reduced concentration of NaCl (-17 and -34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates B Western blot analysis of USP33 in RAW264.7 cells cultured in media containing reduced concentration of NaCl (-17 and -34 mM) for 12 hrs. C Western blot analysis of Viperin in HT1080 cells treated with IFNα (1,000 IU/ml, 12 hrs) in media containing reduced concentration of NaCl (-17, -34 and -51 mM). Data information: Data are representative of at least two biological replicates D RAW264.7 cells were infected with VSV (MOI=1.0, 12 hrs) in media containing reduced concentration of NaCl (-17 and -34 mM). Viral RNA levels were analyzed by RT-qPCR. Data information: Data (D) represent mean and SD of four biological replicates For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test). E Mice (n=3) were fed an NSD (0.45% NaCl) or LSD (0.30% or 0.15% NaCl) for 3 days. Na+ concentration in mouse serum was analyzed by a Micro Blood Sodium Concentration Assay Kit. Data information: Data show mean and SEM of three biological replicates. For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test). F Schematic diagram of analysis of in vivo viral levels in VSV-infected mice fed either an NSD (0.45% NaCl) or LSD (0.15% NaCl). G Western blot analysis of USP33 and Viperin protein levels in the spleen tissues of mice (n=5) fed an NSD (0.45% NaCl) or LSD (0.15% NaCl) for 3 days as indicated. Data information: Data are representative of at least two biological replicates Data show mean and SEM of five biological replicates. H Mice (n=5) were intraperitoneally infected with VSV (1x108 PFU per gram body). 12 hrs post infection, mice were fed an NSD (0.45% NaCl) or LSD (0.15% NaCl) for 3 days. Then VSV RNA levels in mouse spleen tissues were detected by RT-qPCR. I Virus titers in the mouse blood (n=5) from (H) were analyzed by the TCID50 assay. Data information: Data show mean and SEM of five biological replicates. For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test). J Immunofluorescence analysis of the amount of VSV-GFP in liver tissues of mice intraperitoneally infected with VSV-GFP (1x107 PFU per gram body) and then fed an NSD/LSD as (H). Scale bars, 20 µm. K A proposed model of high salt-mediated restriction of host antiviral ability by activating p97 acetylation and subsequent degradation of the deubiquitinase USP33 and antiviral protein Viperin.
CbpC could bind to p62 and act as a decoy for autophagic degradation of Atg14 to suppress autophagic degradation.Figure 3A Lysates from MEFs stably expressing HA-Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. B MEFs/HA-Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (C A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti-Atg14 Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of perinuclear-localizing Atg14 containing cells were quantified. A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and -GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of intact Golgi apparatus-containing cells were quantified. G S. pneumoniae (Sp) invasion-permissive A549 cells (marked with GFP) and Sp invasion-non-permissive (pIgR knocked down) A549 cells were co-cultured at a 1:3 ratio. Atg14-disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 µm. The lines show each cell shape and the arrows show invasion-permissive A549 cells marked with GFP. H The percentages of perinuclear-localizing Atg14 containing cells in (G) were quantified. I Lysates from 293T cells transiently expressing GFP-CbpC or GFP, and HA-Atg14 were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against HA or GFP. J Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP were immunoprecipitated using GST-GFP-Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62-3Myc and HA-Atg14, and bound proteins were analyzed by immunoblotting. K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified. L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62. M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag-Stx17 and Nanoluc-Atg14 in the presence or absence of GFP-CbpC or GFP.
BACE1 inhibition in AppNL-G-F mice by AAV-VHH-B9 delivery significantly reduces amyloid beta load.Figure 3(A) Co-staining with anti-N-Terminal Aβ 82E1 (green) and Thioflavin-S (magenta) reveals Aβ plaques (arrow heads) in regions of interest. Representative images are shown. Scale bar: 50 μm. (B, C) The number of Aβ plaques in regions of interest was significantly reduced by treatment AAV-VHH-B9 (B), as was total plaque area (C). ( (D) Quantification of insoluble Aβ1−40 and Aβ1−42 in the cortex and hippocampus of AppNL-G-F mice measured using ELISA. Each dot represents the mean between duplicate Aβ measurements in each individual mouse. Long-term VHH-B9 expression resulted in a significant reduction in the amyloid load.