EMBO/sd-character-level-ner huggingface dataset:
Enhanced binding of OTUD1 C320A to K63 diubiquitin in vitro and K63 polyubiquitin chains in cells.Figure 4(A) Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently-labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown. (B) Whole cell lysates of HEK293 cells expressing HA-tagged OTUD1 WT, C320R, and C320A were immunoblotted with indicated antibodies. One representative experiment of three is shown.
Figure 2(A) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR-122 and miR-21 were done by quantitative RT-PCR (mean +/- s.e.m., n=3). GFP used as negative control to confer the specificity of the anti-HuR3A2 antibody in immunoprecipitating HuR. The miR-122 content in the GFP-antibody immunoprecipitated materials was too low to get detected reliably using the qRT-PCR. (B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). In siHuR treated cells, depletion of HuR was monitored by western blotting (mean+/- s.e.m., n=4) (B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT-PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon) treated set were taken as unit. (C). CAT-1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT-PCR and normalized against GAPDH mRNA. (D) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells. Relative levels were estimated by real time qPCR, normalized with respect to U6 snRNA and plotted in the left panel. Levels of miR-122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT-PCR and plotted in the right panel (mean+/- s.e.m., n=3). (E) Level of eIF-2a and its phosphorylated form, estimated by western blot using specific antibodies, in siCon or siHuR treated and starved Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT-1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level. Values of siCon treated samples were taken as unit (mean+/- s.e.m., n=3). In the right panel the relative quantity of phosphorylated eIF-2a measured by densitometry were plotted. Levels of p38 and phosphor-p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot. (F) Western blot analysis to detect the level of phosphorylated eIF-2a and eIF-4E-BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant-let-7a or anti-miR-122 oligos. miR-122 inactivation was monitored by measuring cat-1 level by qRT-PCR using GAPDH as control (mean+/- s.e.m., n=3). * denotes the phosphorylated eIF-4E-BP1. b-Actin was used as loading control. Relative levels of phosphorylated eIF-2a were measured by densitometric estimation of three independent measurements.
De novo DNA methyltransferases control neuronal differentiation from adult hippocampal NPCs in vitro.Figure 2A, Schematic representation of the genomic area of Dnmt3a and Dnmt3b in wildtype (WT) mice and conditional knock-out (KO) mice. WT mice were Nestin::Cre-ERT2(‑/‑)/ Dnmt3afl/fl/Dnmt3bfl/fl, while KO mice were Nestin::Cre-ERT2(‑/+)/Dnmt3afl/fl/Dnmt3bfl/fl. B, Dentate gyri from WT and KO mice were dissociated 10 days after tamoxifen administration and plated for neurosphere formation. NPC monolayer cultures were generated from single neurospheres. Representative pictures of neurosphere (left) and neurosphere-derived adherent monolayer culture (right). Scale bars: 50 µm. C, Representative images of polymerase chain reaction used for genotyping of NPC lines. D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM). E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM. F-G, NPCs from WT and KO mice differentiated into astrocytes (GFAP-positive) and neurons (Tubb3-positive). Increased numbers of both neurons and astrocytes were generated from KO NPCs. Depicted are data points for every sample with genotype means ± SEM. Scale bar: 100 µm. H, High magnification fluorescent image of differentiated neurons. Scale bar: 20 µm. I, Sholl analysis of Tubb3-labeled WT and KO neurons (n = 29 neurons, WT; n = 30 neurons, KO). Reported p-value corresponds to genotype effect from two-way ANOVA. Asterisks highlight data points with p < 0.05 after multiple testing adjustment (Holm method) of repeated t-tests. Depicted are means ± SEM. J, High magnification fluorescent image of differentiated astrocytes. Scale bar: 20 µm. K, Astrocytes from KO cultures covered a larger area than WT astrocytes. Data points represent means of individual cultures (n > 30 astrocytes per culture). Depicted are data points per sample with genotype means ± SEM.
Figure 5A. Comparison of LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages. Data information, , data are shown as the mean of three technical replicates. Error bars denote SEM. B. Ponatinib inhibits LPS-mediated cytokine release in THP1 macrophages in vitro. Data information, data are shown as the mean of three technical replicates. Error bars denote SEM. C. A schematic showing the overall design of in vivo study evaluating the efficacy of ponatinib in LPS-induced lung inflammation mouse model. D. Representative H&E images showing Ponatinib alleviates LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower). E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex. Data information, data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.
TBL1 reverses and prevents tumor growth in vivo and sensitizes toward chemotherapy.Figure 4A-D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6/N mice. Six days later, tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA. (A-C): Data plotted as mean ± SEM; n = 4 animals in control (shNC) group and n = 3 animals in knockdown (shTBL1) group; significantly different (P < 0.05) between control (shNC) and knockdown (shTBL1) *. (A) Tumor volume determined by transcutaneous measurement with digital calipers; P-values: 9.21 × 10−3 at day 15, 6.02 × 10−4 at day 18, 2.49 × 10−4 at day 20; two-way ANOVA with Bonferroni post-test. (B) Luminescence of tumors after intraperitoneal injection of firefly d-luciferin; P = 0.0133 at day 20; two-way ANOVA with Bonferroni post-test. (C) Tumor mass at necropsy; P = 0.0299, one-sided Welch's t-test. (D) Luminescence images of one representative mouse in each group. E-G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection; n = 4 animals in shNC+GEM group, n = 5 animals in all other groups; significantly different (P < 0.05) between groups: shNC vs. shNC+GEM (*), shNC vs. shTBL1 (#), shNC vs. shTBL1+GEM ($), shNC+GEM vs. shTBL1 (%), shNC+GEM vs. shTBL+GEM (†); shTBL1 vs. shTBL1+GEM (‡); two-way ANOVA with Bonferroni post-test. (E) Tumor volume determined by transcutaneous measurement with digital calipers. P-values: day 17: 8.48 × 10−3 (#), 1.07 × 10−3 ($), day 20: 3.07 × 10−3 (*), 9.37 × 10−6 (#), 1.56 × 10−8 ($), 0.0239 (†). (F) Tumor volume relative to the volume at day 7 post-implantation; P-values: day 17: 3.94 × 10−3 ($), day 20: 1.09 × 10−3 (*), 1.39 × 10−7 ($), 0.01195 (%), 3.12 × 10−6 (‡). (G) Area under curve for relative tumor volume; P = 0.0195 (‡).
Figure 2Ultra-pure antigen (Ag, TNF-α) mixed with therapy-grade mAb (infliximab, Ifx) was used to generate sICs. X-Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR-specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL-2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL-2 was measured via anti-IL-2 ELISA (A450nm) and IL-2 concentrations were calculated from an IL-2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. MFI were normalized to untreated cells (ctrl.) and are presented as fold-change increase.
Human HCC tumors frequently upregulate CASP2 and PIDD1 expression (A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b.Figure 5(A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b. PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, "C" and "CC" indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. Densitometric quantification of these blots is shown in Fig. EV4A. (B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc analysis. The central band represents the median, the boxes indicate the 25th and 75th percentile, the whiskers are the 5th and 95th percentiles, statistical significance was defined as * p≤0.05, ** p≤0.01. (C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.
Figure 1Schematic representation of FAM134 protein structures and sequence of the LC3 interacting region (LIR) domain of human (h) and mouse (m) proteins (red square). Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm. Structural models of reticulon homology domains (RHDs) of FAM134 proteins were built and coarse-grained before embedding in POPC bilayers and simulated using MD simulations. The overall shape of the RHD along with its membrane footprint was monitored by measuring the radius of gyration Rg of the protein (grey: TM segments; yellow: AH segments; red, green, blue: cytosolic loop of FAM134A, FAM134B, FAM134C). Sequence alignment of human FAM134 proteins based on hydrophobicity profiles computed using AlignME. The helical structural elements characteristic of the RHD are denoted in red below along with a consensus sequence. Probability density of Rg for FAM134-RHDs, indicating compact (small Rg value) or widespread (high Rg value) conformations. Distinct conformational states of the RHD of FAM134 proteins were obtained from conformational sampling (total n=10.000) using coarse-grained molecular dynamic simulations (10 μs) and subsequent clustering. Representative conformations corresponding to the top 3 populated clusters are shown for each system. Conformation states in light colours represent the most populated states in the reported simulation. Snapshots showing the curvature of bicelles containing DMPC (gray) and short-chain DHPC lipids (red) together with FAM134-RHDs over time. Curvature time-traces (smoothed running averages over 11 ns widows) from individual replicates (n=20) quantify the bicelle shape transformation process during simulations. Black lines (mean value) and shaded region (s.e.m) denote the average behaviour of the system. From the 20 replicates, data on waiting times (t) were collected to compute rates and acceleration factors (Table EV3). Vesicle formation is marked by bilayer-disc curvature, |H| = 0.14 nm−1. Cumulative distribution functions (CDF) of recorded waiting times for formation of vesicle (filled circles: simulations; lines: Single Poisson process with additional lag time).
Dopamine expression during lymph gland growth.Figure 1(A-D) Immunostaining with dopamine antibody at late 3rd instar (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine levels (C') in them. (E-L) Similar expression analysis of dopamine at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L). (D, H and L) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity. (M) Quantifications of dopamine levels in DomeGFP+ progenitors (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL). Data information: DNA is stained with Hoechst in blue, intracellular Dopamine (iDopamine) in red, domeMESO expression in green (representative of progenitors in the medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied is One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.
Fig.Figure 7(A, B) MST analysis of direct binding of NOD1 (A) or NOD2 (B) with ADP in the presence of S1P (2 µM). (C, D) MST analysis of direct binding of NOD1 (C) or NOD2 (D) with S1P in the presence of ADP (2 µM). (E, F) MST analysis of direct binding of S1P (E) or ADP (F) with NOD1 WT or NOD1 H517A.
Figure 2(a) GST-tagged BRLF1-expressing plasmid or control vector was transfected into A549 cells. After HSV-1 infection (MOI=1) for 12 h, the cells were collected, and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads. The immunoprecipitated complexes were then separated by SDS-PAGE, and the image obtained from silver staining is shown. (b) Scramble (sc) or shRNAs against POLR3F and POLR3G with empty control vector (ctr) or BRLF1-expressing plasmid were transfected into EBV-ΔBRLF1-harboring HNE1 cells and A549 cells, followed by HSV-1 infection. Thirty-six hours after the transfection of the HNE1 cells, and 12 h after HSV-1 infection (MOI=1) of the A549 cells, the cell pellets were collected and then the cleavage of caspase-1 and IL-1β were measured as indicated. The release of mature IL-1β and IL-18 from HNE-1 and A549 cells were measured by enzyme-linked immunosorbent assay (ELISA) after ultrafiltration and concentration with Amicon® Ultra-0.5 centrifugal filter devices. (c) GFP-tagged BRLF1 wild type, BRLF1 L578A or empty vector was transfected into 293T cells with GST-tagged POLR3F or POLR3G expressing plasmids for 48 h. Cells were collected and immunoprecipitation with GST-affinity beads was performed, and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated. (d) GFP-tagged empty vector or GFP-BRLF1- or GFP-BRLF1 L578A-expressing plasmids were transfected into A549 cells, after which the cells were then infected with HSV-1, or transfected into HNE-1-EBV-ΔBRLF1 cells. The cells were then collected and lysed. Immunoprecipitation with GST-affinity beads was subsequently performed, and then whole-cell lysates and immunoprecipitated complexes were analyzed as indicated. (e) P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated. (f) 3D structure of BRLF1, the BRLF1 TA domain and the BRLF1 NLS572-582 peptide were predicted using I-TASSER. Receptor-ligand interaction mode of the BRLF1 TA domain and the BRLF1 NLS572-582 peptide with RNA Polymerase III open complex (PDB ID 6F40) was conducted using CDOCKER. The yellow sequence in top figure indicates the TA domain in BRLF1, and the red sequence represents the NLS572-582 peptide. The yellow sequence in the middle figure represents the BRLF1 TA domain, and the red helix in middle and bottom figures represents the DNA double helix.
Interactions between Atg30, Atg8 and Atg11.Figure 2(A1) Atg30 sequences from aa 68-122 of wild‐type (Atg30), the S71A mutant (Atg30S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30WDILSSS and Atg30WSILSSS). (A2) Pexophagy experiments of Δatg30 cells complemented with appropriate wild‐type or mutant Atg30 proteins described in A1. (A3) Two‐hybrid assays between Atg30 wild‐type or mutants (described in A1) and Atg8. Phosphomimic S71E was included to detect the interaction with Atg8. (B1) Schematic of the two Atg30 molecules, one with an Atg8‐binding site mutated and a second with an Atg11‐binding site (A11‐BS) mutated, used to complement Δatg30 cells by co‐expression. P: indicates phosphorylation in vivo. (B2) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1. (C1) Schematic of the Atg30 mutations, S71A and S112A that impair Atg8 and Atg11 binding, respectively. (C2) Pexophagy assays of Δatg30 cells complemented with Atg30 wild‐type and mutants. aa, amino acids; AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; HA, haemagglutinin.
Figure 6(A) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3.FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments. (B) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3 kinase domain. KD represents a truncation of SUMM3 with only the kinase domain (amino acid 201-510).FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.
Figure 11(A) Distribution of exogenous 3Flag-MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were subjected to fractionation and detected using anti‐Tom20, anti‐PMP70, and anti‐Flag antibodies. Tom20 and PMP70 were used as mitochondrial and peroxisomal markers, respectively. The 3,000 g and 100,000 g pellets represent the mitochondria‐rich and peroxisomes-rich fractions. (B) The recovery ratio of 3Flag-MITOL between the mitochondria-enriched (3,000 g pellet) and peroxisome-enriched fraction (100,000 g pellet) following valinomycin treatment for the indicated times. The ratio of peroxisome-localized 3Flag-MITOL to mitochondria-localized 3Flag-MITOL increased with valinomycin treatment for 3 hours. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of 3Flag-MITOL collected in the mitochondria-enriched fractions, and red dots are the ratio of 3Flag-MITOL collected in the peroxisome-enriched fractions. Mean values are also shown. Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria-enriched fraction, but concomitantly increased endogenous MITOL in the peroxisomes-enriched fraction. Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of MITOL-3Flag collected in mitochondria-enriched fractions, and red dots are the ratio of MITOL-3Flag collected in the peroxisome-enriched fractions. Mean values are also shown.
Figure 7qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation (n=3 biological replicates for each group). qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group). qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group). Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat, control shRNA lentivirus, WNT8B shRNA lentivirus. qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus (n=3 biological replicates for each group). qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group). A model describing the role of a unique function for PAX6D isoform in regulating retinal specification. retinal fate choice versus forebrain is controlled by PAX6D by regulating a set of neural genes and retinal genes.