EMBO/sd-character-level-ner huggingface dataset:
Figure 1A HEK293T were transfected with NF-kBluciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (A) NF-kB reporter gene expression. HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (B) IL-8 secretion. Values are the mean of triplicates ± s.e. P-values are compared against empty vector treatment. Significance levels, ns P>0.05, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Unless otherwise stated, p-values are p<0.001. HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (C) Immunoblotting for c-Jun, phospho-c-Jun, JNK, phospho-JNK, p38 and phospho-p38 as indicated. Actin was used as a loading control. Data are representative of two independent experiments.
Figure 5A. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti-LRP6 antibody and blotted with OGT. WCL, whole-cell lysates. B. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates. D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting. E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis. F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.
CAR forms microclusters that are signaling-active.Figure 1A. Domain structure of CAR. B. Schematic of the system for stimulating CAR T cells and imaging CAR dynamics. Supported lipid bilayers (SLBs) were coated with the extracellular domain of CD19 and an integrin ligand ICAM-1. C. TIRF microscopy revealed time courses of the CAR-GFP microcluster formation as CAR T cells spread on the CD19 and ICAM-1-coated SLBs. D. Enrichment of phosphor-tyrosine (pY) at CAR microclusters. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-pan phosphor-tyrosine antibody. No CD19 was included in the control experiment (only ICAM-1). Scale bar: 2 μm.
Framework for including enzymes as metabolites in a genome-scale model.Figure 1Figure 1: Framework for including enzymes as metabolites in a genome-scale model. (A) GECKO uses a genome-scale model and includes enzymes as part of reactions. (B) Expansion of the stoichiometric matrix. M refers to metabolites, E to enzymes, v to metabolic fluxes and e to enzyme usage. Note that 4 submatrices appear inside the new stoichiometric matrix: the upper left submatrix is equivalent to the original stoichiometric matrix, the upper right submatrix has only zeros, the lower left submatrix has the kinetic information, and the lower right submatrix is the identity matrix.
Figure 4(A) Schematic diagram outlining the different domains of human POGZ and the different deletion constructs of POGZ that we generated and analyzed. (B) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, cells were transfected with an empty Flag vector (EV) or a siRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ366-848, 366-848). 24h after plasmid transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated protein was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 100 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001. (C) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001. (D) Representative images used for quantification in (C). Scale bar = 5 μm. (E) Similar as in (C) except that γ-H2AX foci were monitored at the indicated time points by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05. (F) Representative images used for quantification in (E). Scale bar = 5 μm.
During MCMV infection, the TLR9/Myd88-dependent pathway is necessary for pDC cytokine production, irrespective of their IFN-I responsiveness (A) Dot plots represent IFN-I vs GFP staining in 5.Figure 4 (A) Dot plots represent IFN-I vs GFP staining in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-GFP-infected MBMC. One staining representative of 6 animals from 2 independent experiments is shown  (B, C) Percentages of GFP+ cells within 5.2+ pDC (B) or of GFP+ (green) vs GFP- (white) cells within 5.2+ IFN-I-producing pDC (C) isolated from indicated MCMV-GFP-infected MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group  (D) qPCR for the expression of the indicated genes was performed on whole mRNA isolated from the spleen of indicated MCMV-infected MBMC. CTR MBMC (black) and Myd88-/- TST MBMC (dark green) were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group  (E-G) CD86 MFI in 5.2+ pDC of each indicated MBMC type (E) and 5.2/ 5.1 ratio of cytokine-producing pDC (F, G). Data (mean±SEM) are representative of 2 pooled independent experiments each with 2-3 mice per group CD86 MFI and percentages of cytokine-producing cells within pDC of each indicated mouse strain. Black, C57BL6; red, Ifnar1-/-; orange, Ifnar1-/-x Myd88-/- CD86 MFI and percentages of cytokine-producing cells within pDC of each indicated mouse strain. Black, C57BL6 green, Tlr9-/- and pale green, Tlr7-/-. In (K-L) indicated mice were treated with IC (square) or blocking α-IFNAR1 mAbs (triangle). Data depicted (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group.
Figure 3A. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting. B. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (-) or VSVG-LRP6. 1 d after transfection, cells were incubated in serum-free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting. C. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting. D. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated decrease of YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin and LATS1/2. After 1 d, these cells were transfected again with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P< 0.01 and N.S: Non-significant. Student's t-test was used for statistical analysis. E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B. F. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 d were transfected again with HA-Merlin and HA-Merlin∆FERM. Cells were lysed and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA-Merlin, and the arrowhead indicates HA-Merlin∆FERM. G. MST1/2 were necessary for the LRP6-knockdown-mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6 and MST1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting. H. MAP4K4/6/7 were necessary for the LRP6 knockdown mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed and the cell lysates were analyzed by immunoblotting.
Schistosomes target the differentiation toward the Th2 pathway.Figure 1Th cells (CD4+) were purified using magnetic beads from spleen and lymph nodes of 4-5-weeks-old mice. The cells were stimulated with anti-CD3 and anti-CD28 antibodies, beneath a trans-well with 0.4μm pore containing 25-30 worms. The Control wells contained only unused schistosomal-medium. 72h after stimulation with anti-CD3 and anti-CD28, the cells were re-stimulated with P+I for 2h, and the mRNA was subjected to qRT-PCR using the indicated primers for the lineage specifying transcription factors (B) or cytokines (C). The graphs represent an average of at least 4 independent experiments. In each graph, the expression level in the control was set as 100%. P-value was calculated by t-test. * p<0.05, ***p<0.005
Fig.Figure 7Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min. (A) Rab8A and Rab8A pT72 levels were analysed by Western blot. (B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.
Figure 6Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig. 5A and DNA content was analyzed by flow cytometry. Analysis of chromosome mobility by Pulse Field Gel Electrophoresis (PFGE) after staining of DNA with ethidium bromide. In G1, the chromosomes are linear and are sorted according to their length. Upon entry into S phase (30 min), they cannot enter the gel due to the presence of replication intermediates. Quantitation of chromosomes re-entering the gel shows completion of DNA replication in 40% of rad9∆, rad53-11 and rad9∆ rad53-11 mutants, but not in wild type cells and mrc1 mutants as determined for a representative experiment. Error bars indicate SD between the signal intensity determined for the three larger chromosomes (arrowheads). DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin. Representative images are shown. Red: DNA, green: BrdU. Bar is 20 kb. Length distribution of BrdU tracks measured at that time for wild type, rad9∆, rad53-11 and rad53-11 rad9∆ cells. Median lengths are indicated in red. For each sample, at least 200 tracks were measured from two biological replicates.
Figure 3A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant. B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.
Figure 9Figure 9. Model for the role of AMOTL2 ubiquitination in the Hippo pathway.
In vivo nonsense editing of Pten.Figure 3Sanger-sequencing chromatograms showing the target region of sgPtenQ245* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPtenQ245*. EditR was used to calculate the frequency (%) of C-to-T conversion at C4 of the protospacer targeted by sgPtenQ245* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors. Overview of the intraductal injections performed in WB1P-BE3 females with high-titer lentiviruses encoding Myc and either a non-targeting sgRNA (Lenti-sgNT-Myc) or the sgRNA targeting Pten (Lenti-sgPtenQ245*-Myc). Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPtenQ245*-Myc (n=11) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (37 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test). BE Analyzer (Hwang et al., 2018) was used to assess from next-generation sequencing data the fraction of wild-type Pten alleles, base edited alleles or alleles with insertions/deletions (indels) in tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc or Lenti-sgPtenQ245*-Myc. TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P-BE3 mice injected with Lenti-sgPtenQ245*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgPtenQ245* (PCR products were subcloned for clarity). Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by indels at both alleles, while in the upper one by Q245* base editing in one allele and a deletion at the second copy of the gene.
Figure 6Sleep curve for Tβh mutant and control flies in video-based method. Total sleep for Tβh mutant and control flies in video-based method (From left to right mean±SEM: 688.2±32.71, n=48; 123.2±11.68, n=46). Number of Grooming behaviors in 10min during ZT3.5-4.5 (From left to right mean±SEM: 9.571±2.644, N=7; 4.429±1.343, N=7; 7.429±2.170, N=7; 24.29±3.300, N=7).
Figure 6Protocols of HDAC1 activator treatment and behavioral tests. WT or FTLD-TDP Tg mice (6-mon-old) were i.p. injected with HDAC1 activator 5104434 for 2 months (6 mg/kg/day), following which they were subjected to a series of behavioral tests and then sacrificed for further examination. Escape latency of mice in Morris water maze task. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by two-way ANOVA with Bonferroni's multiple comparisons. #P < 0.05, ***P ≤ 0.001 and ****/####P ≤ 0.0001 Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison. The representative searching path of mice in the probe test. Scores of mice with respect to the number of times they crossed the hidden platform, time spent searching in the target quadrant, and the velocity of swimming in the probe test at 24 h after escape training. ** p = 0.009, *** p = 0.0002 by multiple comparison. Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison. Scores of the discrimination index in the novel object recognition test.   Scores of mice in the rotarod test.   Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.
Ubqln1 interacts with Ubqln4 through its UBA domain.Figure 4(A) 293 cells were co‐transfected with GFP‐S‐Ubqln1, GFP‐S‐Ubqln1 ΔUBL (deletion of UBL domain) or GFP‐S‐Ubqln1 ΔUBA (deletion of UBA domain) and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. (B) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1, Ubqln1ΔUBL and Ubqln1ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0041. (C) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBL or HA‐Ubqln4 ΔUBA. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. (D) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0003 and **P‐value=0.0001. (E) 293 cells were co‐transfected with GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBA or HA‐Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. (F) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin‐associated; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.
Figure 2M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. mRNA levels of profibrotic markers, Arg1 (A), CD206 (B) and CD163 (C) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition). M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. protein levels of secreted profibrotic CCL18 (D) and antifibrotic cytokines, CXCL10 (E) and IL-6 (F) (n=3, technical replicates) were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).
EBV‐treated pDCs fail to prime a naïve T‐cell response.Figure 5(A) ) After 24 h treatment with either EBV or CpG, pDCs were co‐cultured with cord blood‐derived allogeneic CD4+ T cells. The proliferative response was evaluated by measuring ( thymidine and is expressed as cpm. Data are shown as mean + SD of triplicate cultures. One representative experiment out of two performed is shown. (B) Supernatants from the T−cell/pDC co−cultures described above were harvested and the production of IFN−γ (left) and IL−2 (right) were measured by CBA. The data are shown as mean + SEM of results pooled from two independent experiments.