EMBO/sd-character-level-ner huggingface dataset:
Reinstating FAM83D at the endogenous locus in FAM83D knockout cells: A: Schematic illustrating the CRISPR-based strategy used to reintroduce FAM83D into the FAM83D-/- knockout background.Figure 3A: Schematic illustrating the CRISPR-based strategy used to reintroduce FAM83D into the FAM83D-/- knockout background. B: FAM83D-/- (KO), wild-type (WT), and two independent clones from a CRISPR/Cas9-mediated knockin rescue of FAM83D cDNA into FAM83D-/- cells (c. 6 and c. 11), were synchronized in mitosis (M) with STLC. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing FAM83D (C) Representative images of mitotic cells are included. Scale bars, 20 µm. The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing CK1⍺ (D). Representative images of mitotic cells are included. Scale bars, 20 µm. E: Quantification of CK1⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=1. F: The cell lines were STLC-synchronized in mitosis (M) or left AS, lysed and subjected to immunoprecipitation (IP) with anti-FAM83D-coupled sepharose beads, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. G: Asynchronous (AS) FAM83DGFP/GFP/ mCherry/mCherryCSNK1A1 knockin U2OS cells were fixed and imaged. Representative images from the indicated cell cycle stages are included. Scale bars, 10 µm.
Lam6 Is an Uncharacterized Binding Partner of the ERMES Complex.Figure 1(A-D) Pull-down of HA-tagged ERMES components (Mdm10, A; Mdm12, B; Mdm34, C; and Mmm1, D) uncovered that all ERMES components interact with the same uncharacterized protein, Lam6. Lam6 and members of the ERMES complex are marked in green. (E-F) Reciprocal pull-down demonstrated that endogenously expressed C-terminally tagged Lam6 (E) and overexpressed N-terminally tagged Lam6 (F) have the same strong binding partners that include the ERMES complex members (G). GFP-tagged ERMES complex proteins retain their characteristic punctate structure in Δlam6, suggesting that Lam6 is not an essential complex member. Scale bar represents 5 μm.
Figure 5A. HuH7 cells were transfected with control siRNAs (siCtrl) or siRNAs that target RIG-I (siRIG-I), 48h later cells were infected with a moi 0.3 of HCV and evaluated at 48hpi. B. HuH7.5 cells were mock-infected or infected with moi 0.3 of HCV for 72h. C. HuH7 cells were transfected with control siRNAs (siCtrl) or siRNAs that target PKR (siPKR), 48h later cells were infected with a moi 0.3 of HCV and evaluated at 48hpi. D. HuH7 cells were transfected with increasing amounts (0.03, 0.06, 0.125, 0.25, or 0.5 μg per M6 plate) of a plasmid expressing PKR (pPKR) and collected 48h post-transfection. E. HuH7 cells were mock transfected (-) or transfected with 0.5 μg per M6 dish of pPKR (+) and 48h later, cells were transfected with or without 10 μg of pIC or infected with a moi 0.3 of HCV. Cells were collected 8h post-transfection with pIC or 48hpi with HCV. F. Schematic representation of RIG-I and PKR pathways. Indicated is PKR inhibition of translation and induction of NF-κB and IRF3 by RIG-I and PKR. NF-κB response to TNFα and activation of EGOT is also indicated. G and H. HuH7 cells were mock transfected or transfected with plasmids expressing a dominant negative of IRF3 (G; DNIRF3), or a superrepressor of NF-κB (H; pIKB(SA)). One day after transfection, cells were infected with HCV and collected at 48 hpi. (I) Cells were treated with 20 ng/ml of TNFα for 6 or 12h. Where indicated, the relative levels of EGOT were evaluated by qRT-PCR in RNAs isolated from these samples. GAPDH expression was evaluated in all cases and used as a reference to calculate the relative levels of each transcript. Fold increase of treated versus control cells was also calculated.
4D7 blocks mutant CALR-dependent proliferation in vivo and prolongs survival.Figure 5A. Illustration showing bone marrow NSG engraftment model with TPO-independent TF-1 TpoR CALRdel61 cells treated with 4D7 or IgG control twice weekly, starting 7 days after engraftment via intraperitoneal injection and final measurements taken from euthanized mice. B. Pharmacokinetic measurements of serum levels of 4D7 in mice after intraperitoneal injection at 0, 1, 24, 48, 72 and 110 hours since administration. C. Percentage of TF-1 TpoR CALRdel61 human CD33+ cells measured in peripheral blood at 3 weeks post-tail vein engraftment (n=5 mice per treatment). Data information: a student's unpaired t-test was used to determine statistical significance. D. Kaplan-Meier survival curve of bone marrow engraftment model of TF-1 TpoR CALRdel61 cells showing improved survival of mice treated with 4D7 compared with IgG control commencing 1 week after tail vein injection (n=5 mice per treatment). Data information: For all survival curves the log-rank Mantel-Cox test P value is shown. E. Mean tumour volume at 21 days after sub-cutaneous injection of TF-1 TpoR CALRdel61 cells following treatment with 4D7 or IgG. Cells were pre-treated with 4D7 or IgG control for 1 hour prior to injection and treatment continued twice weekly at 12.5 mg/kg until euthanasia (n=3 mice per treatment) Data information: a student's unpaired t-test was used to determine statistical significance. F. Kaplan-Meier survival curve of TF-1 TpoR CALRdel61 chloroma mice treated with 4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration. (n=3 mice per treatment) G. Kaplan-Meier survival curve of NSG mice engrafted with ruxolitinib-resistant TF-1 TpoR CALRdel61 treated with 12.5 mg/kg 4D7 or IgG twice weekly (n=5 and 6 mice for IgG and 4D7 respectively). Data information For all survival curves the log-rank Mantel-Cox test P value is shown.
Figure 3(A) Immunoblots of cell lysates were performed to analyze p-ERK1/2, p-JNK, p-P38 and p-P65 by THP-1 cells stimulated with AG (1 μg/ml) for indicated times, and GADPH as a loading control. Data information: Data are representative of n=3 independent experiments. (B) Immunoblots of cell lysates were performed to analyze p-ERK1/2, p-JNK, p-P38 and p-P65 by mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times left untreated or pretreated with AG aptamer (1 μg/ml), and GADPH as a loading control. Data information: Data are representative of n=3 independent experiments. qPCR analysis of Mmps including Mmp9 (C), Mmp10 (D) from THP-1 cells stimulated with AG (1 μg/ml) for 24h in absence or presence of different inhibitors targeting NF-κB (PDTC), ERK (PD98059), JNK (SP600125) and p38 (SB203580) at the concentration of 10 μM. Data information: Data are means + SD averaged from n=3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ****, p<0.0001. qPCR analysis of Mmp12 (E) from THP-1 cells stimulated with AG (1 μg/ml) for 24h in absence or presence of different inhibitors targeting NF-κB (PDTC), ERK (PD98059), JNK (SP600125) and p38 (SB203580) at the concentration of 10 μM. Data information: Data are means + SD averaged from n=3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ****, p<0.0001. (F) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control. (G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.
Figure 2(A) PS1/2-/- MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A. Antibody 5E12 was used for the detection of the PS1 CTF. (B) Stably transduced PS1/2-/- MEF cells of A were transiently transfected with APPsw-6myc and conditioned media were analyzed for Aβ species by Tris-Bicine urea SDS-PAGE. Individual Aβ species were verified by co-migration with Aβ standards (left panel) and quantified (right panel). Data represent mean ± s.e.m. of n=3 independently performed transfections. Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis. (C) Secreted Aβ from cells in B were quantified by ELISA. Data represent mean ± s.e.m. of n=5 (PS1 WT) or 6 (PS1 L435F) independently performed transfections.
Figure 6Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group). Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group). Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm. Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group). Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).
Figure 1A SARS-CoV-2 genome annotation, which predicts 29 virus gene products. The 16 non-structure proteins (NSPs) are cleaved products of the large polyprotein open reading frame (ORF) 1ab or ORF1a. These polyproteins are cleaved into small function fragments or NSPs after translation. B Workflow for the comprehensive virus-host interactome analysis. Two different strategies, SFB-TAP and BioID2 proximity labeling, were applied in the study. Samples were analyzed by Q Exactive HF mass spectrometry (MS). C Summary of the datasets obtained from SFB-TAP and BioID2 results, including the number of high-confidence interacting proteins (HCIPs). BDFR, Bayesian false discovery rate. D Pearson correlation coefficient among three independent biological replicates of the SFB-TAP results and the BioID2 labeling experiments. Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values. E-G GO analysis. GO enrichment was performed using Ingenuity Pathway Analysis. Protein localization (E), molecular function (F), and biological function (G) are plotted in a single panel. H HCIPs identified in the purification of each SARS-CoV-2 gene. I Correlation between peptide-spectrum matches (PSMs) of identified proteins and their fold change calculated by SAINTexpress.
Figure 5Representative photomicrographs of cultured hippocampal neurons immunostained for TDP-43 (green), FLAG (red) and counterstained with DAPI (blue) in control conditions (untransduced) or infected with lentivirus expressing FLAG-tagged human TDP-43 12xQN construct. TDP-43 intensity in cell nuclei of untransduced or TDP-43 12xQN-infected hippocampal neurons normalized to mean nuclear intensity in untransduced neurons. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). Student's t-test; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using Student's t-test (B) Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to levels in untransduced neurons (N=3 independent experiments; n=3 wells per condition in each experiment). 1-way ANOVA with Tukey's post-hoc; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis BDNF secretion induced by KCl treatment as assessed by in-situ BDNF ELISA in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis BDNF secretion in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis
Figure 4A. In vitro ubiquitination assays with GST-HDAC1, FLAG-TRIM46, FLAG-TRIM46ΔRING, or FLAG-TRIM46ΔC in the presence or absence of ubiquitin and E2. HDAC1 was detected using western blotting.   In vivo ubiquitination assays performed in MCF-7 cells. (B) Stable shCTR- or shTRIM46-expressing MCF7 cells were transfected with His-Ub for 40 h. Cells were then treated with MG132 (10 μM) for 12 h before Ni-NTA bead precipitation followed by IB with anti-HDAC1. (C) and (D) MCF-7 cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni-NTA bead precipitation followed by IB with anti-HDAC1. E. MCF-7 cells were co-transfected with Myc-TRIM46 and FLAG-tagged wild-type HDAC1 or HDAC1 deletion mutants for 48 h. Cellular proteins were extracted for western blotting analysis. F. MCF-7 cells were co-transfected with Myc-TRIM46, HA-ubiquitin, and FLAG-tagged wild-type HDAC1 or HDAC1 mutants for 40 h. Cells were treated with MG132 for 12 h before cellular extracts were prepared for immunoprecipitation assays with anti-FLAG followed by immunoblotting with anti-ubiquitin. G. MCF-7 cells were co-transfected with FLAG-HDAC1-K123R, K126R, K143R, or K144R along with indicated plasmids for 40 h. Cells were treated with MG132 (10 μM) for 10 h before cellular extracts were prepared for Ni-NTA bead precipitation. H. MCF-7 cells were co-transfected with Myc-TRIM46 and wild-type HDAC1 or indicated HDAC1 mutants for 48 h. Cellular proteins were extracted for western blotting analysis.
Figure 2A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data. B. IC50 values [µM] for all compounds towards the yeast, human and mouse β5 subunits.
Figure 3(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection. (B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.
Figure 6(A) Schematic representation of PCR-based analysis of Xbp1 splicing. During conditions of ER stress, a 26 base pair fragment is spliced from Xbp1 mRNA. Forward and reverse PCR primers (shown in blue) were designed to span the splice site. PCR amplification distinguishes between unspliced (Xbp1-u, upper band) and spliced (Xbp1-s, lower band) Xbp1 mRNA. To quantify ER stress, a ratio of spliced: unspliced Xbp1 was calculated. (B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin (Tg) is shown as positive control. (C) Schematic representation depicting cell-cell fusion between a B16 melanoma cell and mouse embryonic fibroblasts (MEF). (D) Xbp1 splicing in B16 melanoma cells and eight B16:MEF fused clonal cell lines. (E) Detection of mRNA expression (by RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine (Rv) at 4µM (DLD1 cells) or 0.5µM (SKOV3 cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD. (F) Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control. (G) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.
PNRC1 reduces DCP1α/DCP2-dependent rRNA processing and snoRNA decapping A.Figure 6A. Confocal images of a Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. B. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1W300A or GFP control. The average of 3 independent experiments ± SD is reported. C. Quantification of 5'-ETS RNA levels in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average of 2 independent experiments ± SD is reported. Levels of U3 (D) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. Values represents the average of two biological replicates with three technical replicates and are Levels of U8 (E) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. F. Levels of U3 snoRNA immunoprecipitated by the anti-m3G cap antibody in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average ± SD of 2 independent experiments with three technical replicates is reported. G. Western blot analysis of input and Flag-IP fractions of RIP experiment. H. RNA levels of U3, U8 and U1 co-immunoprecipitating with GFP, PNRC1WT or PNRC1W300A. Results are shown as the average ± SD of two biological replicates.
Figure 2A. Diagram of the molecular elements within ER-TGN membrane contact sites. B Representative western blot for OSBP in control and U18-treated tsA201 cells. Protein levels were determined by densitometry with β-actin normalization. Each point (n=3) represents protein levels from U18-treated cells normalized to control bands. C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39. E. Confocal images of control and U18-treated hippocampal neurons fixed and stained for OSBP. Dashed black lines represent the outline of each neuron. F. Quantification of OSBP in perinuclear region of control and U18-treated neurons. Control: n=10; U18: n=14. G. Confocal images of control and U18-treated tsA201 cells fixed and stained for VAPA (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of VAPA and TGN46 regions. H. Quantification of VAPA signal in TGN46 regions (VAPATGN46). Control: n=44; U18: n=44. I. Representative western blot of SAC1 in control and U18-treated tsA201 cells. Protein levels were normalized to β-actin, n=4. J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.
Figure 1Region details and sequence selection. Common residues to TrkB-FL and TrkB-T1 are in light blue and include a KFG sequence completely conserved in Trk proteins (filled black box). In the heatmap representation, dark red, white and green represent respectively strong, weak or no effect of point mutations while black corresponds to wild type residues. Black arrows denote theoretical calpain cleavage sites while the red arrow points an experimentally established site. Sequences included in CPPs are emphasized by black rectangles. TM, transmembrane; TK, tyrosine kinase. CPP design. Peptides contain Tat aa 47-57 (italic) followed by the indicated rat TrkB-FL (green) or c-Myc (black) sequences. Confirmation of peptide entry into neurons. Cultures were incubated with Bio-TMyc (25 µM, 1 h) (panel b) or left untreated (panel a). Arrowheads highlight peptide permeability, detected by fluorescein Avidin D (green), into neurons labeled with neuronal-specific antibody NeuN (red). Peptide is not detected in some neurons (arrow) and non-neuronal cells (asterisk). Confocal microscopy images correspond to single sections and are representative of five independent experiments. Scale bar, 10 μm. Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.