Journal: Clinical and Translational Allergy

Article Title: Topical inflammasome inhibition with disulfiram prevents irritant contact dermatitis

doi: 10.1002/clt2.12045

Figure Lengend Snippet: Disulfiram induced a dose‐dependent inhibition of ASC speck formation and cytokine release in cellular assays in vitro . Fluorescence images showing (A) single nuclei of the murine macrophage reporter cell line without activation vs. (B) activated murine macrophage reporter cells upon Adenosine triphosphate (ATP) stimulation with subsequent Apoptosis associated speck‐like protein containing a CARD (ASC) speck formation (green ASC‐mCerulean) as an indicator of inflammasome activity. Inhibition of ATP‐induced ASC speck formation by (C) Disulfiram 5 μM and (D) 10 μM. Nuclei are counterstained with DRAQ5 (red) (Original magnification x20) (E) Dose‐dependent inhibition of ATP‐induced ASC‐speck formation (relative activity in % in relation to maximal ASC‐formation achieved by ATP‐stimulation of controls) by disulfiram (red curve) in the murine macrophage reporter cell line. MCC950 served as reference substance (blue curve). The results show the data of three replicates. Mean and standard deviation (SD) are shown for each value. (F) Disulfiram inhibits ATP‐induced ASC speck formation in human THP1 ASC‐GFP reporter monocytes dose‐dependently (red curve). MCC950 served as reference substance (blue curve). The results show the data of three replicates. Mean and SD are shown for each value. (G) Dose‐dependent inhibition of Interleukin (IL)‐1β release by disulfiram in human monocytes which were stimulated with ATP and Lipopolysaccharides (LPS) (red curve) compared to reference substance MCC950 (blue curve). IL‐1β release shown as the proportion of the LPS‐ and ATP‐induced release of monocytes in absence of Disulfiram. Mean and standard error of the mean are shown for each value. The graphic shows the results of seven independent experiments. (H) Inhibition of IL‐18 release by disulfiram. Culture supernatants of human monocytes stimulated with LPS + ATP in the presence or absence of disulfiram or MCC950 from three different donors were analyzed by cytokine bead arrays. Relative IL‐18 release (%) in presence of compound as compared to positive control (LPS + ATP) is shown. Bars indicate mean values. The concentrations of disulfiram (10 μM) and MCC950 (0.04 μM) for this assay were selected according to the molar range of their respective IC‐50 derived from the IL‐1β assay on monocytes

Article Snippet: IC50 values of hit compounds were also assessed for the human system using THP1 ASC‐GFP reporter monocytes (Invivogen®) analog to the murine reporter cell screening and IC50 delineation process (Supporting Information ).

Techniques: Inhibition, In Vitro, Fluorescence, Activation Assay, Activity Assay, Standard Deviation, Positive Control, Derivative Assay

Journal: FEBS Open Bio

Article Title: Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties

doi: 10.1016/j.fob.2015.07.002

Figure Lengend Snippet: Ubiquitin impact on the CXCR4 signaling cascade. (A) THP-1 cells were incubated with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. cAMP content was analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format for higher sensitivity. Error bars represent SEM of three independent experiments. (B) HEK293 cells expressing CXCR4 were transfected with a pGL4.29 reporter plasmid and stimulated after 48 h with forskolin without (dark grey bar) or in combination with SDF-1 (red bar) or ubiquitin (F45W Scil Proteins, blue bars) in indicated concentrations. Unstimulated cells (light grey bar) served as negative control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from three experiments. (C and D) HEK293 (C) or COS-7 (D) cells were transiently transfected with CXCR4 and the chimeric G-protein G alphaΔ6qi4myr. Afterwards cells were labelled with 0.3 μCi 3 H-myo-inositol for more than 16 h followed by incubation with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. Accumulated IP3 was collected by anion exchange chromatography from cell extracts followed by a beta counter quantification. Error bars represent SEM from three (HEK) or four (COS-7) independent experiments. (E and F) THP-1 (E) or Jurkat (F) cells were seeded in the upper compartment of 96 HTS transwell plate chambers. The lower compartment was filled with PBS containing indicated concentrations of SDF-1 or ubiquitin (Sigma). After 3 h incubation cells in the lower chamber were collected and analyzed by flow cytometry. Chemotactic index was calculated as the ratio of the number of migrated cells at the indicated ligand concentration to the number of cells migrated to buffer. Error bars represent SEM from three independent experiments.

Article Snippet: In vitro cell migration was assessed using 96 HTS transwell plate chambers (Sigma; pore size: 5 μm for Jurkat and 8 μm for THP-1 cells) as described earlier .

Techniques: Incubation, Lysis, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Chromatography, Flow Cytometry, Concentration Assay

Journal: Haematologica

Article Title: SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia

doi: 10.3324/haematol.2023.282704

Figure Lengend Snippet: TAK981 synergizes with 5-azacytidine to induce acute myeloid leukemia cell death in vitro . (A) HL-60 cells were treated with increasing doses of TAK-981 for 24 hours and immunoblotted for SUMO-1, SUMO-2/3 and GAPDH. SUMO conjugates appear as smears. (B) TAK-981 half-maximal inhibitory concentrations (IC 50 ) were determined using HL-60, U937 and THP-1 cells treated with varying drug concentrations. Cell viability was determined using MTS assays after 24 hours of treatment. Concentration response curves were generated by comparing the viability of TAK-981-treated cells with mock-treated controls. Data are shown as mean +/- standard error of the mean (SEM) of replicate samples (N=5). Absolute IC 50 are indicated in the figure. (C) Bone marrow-derived mononuclear cells (BMMC) (N=2) and peripheral blood mononulcear cells (PBMC) (N=3) collected from healthy donors were treated with varying TAK-981 concentrations for 24 hours. Cell viability was determined by flow cytometry after 24 hours of treatment. Concentration response curves were generated by comparing the viability of TAK-981-treated cells with mock-treated controls. Data are shown as mean +/- SEM of replicate samples. (D) HL-60 cells were treated for 24 hours with TAK-981 and either daunorubicin (DNR), cytarabine (ARA-C) or 5-azacytidine (AZA) and viability was assessed by MTS assay (median of 3 independent experiments for each drug). Left panels: heat maps showing the synergy ZIP-score between the 2 drugs, and the “most synergistic areas” (black squares) estimated using the SynergyFinder software v2.0. Right panel: mean of synergy ZIP-scores in most synergistic areas for HL-60 cells. Data are shown as mean +/- standard deviation (SD) of replicate samples (N=3 for each drug). Dotted line at 10% represents the threshold for significant synergy. (E) HL-60, U937 and THP-1 cells were treated with TAK-981 and AZA every day for 3 consecutive days. Viability was analyzed at day 4 and compared to that in mock-treated conditions (median of 3 independent experiments for each cell line). Left panels: heat maps for the corresponding synergy ZIP-score between the TAK-981 and AZA, and the “most synergistic areas” (black squares) estimated using the SynergyFinder software v2.0. Right panel: mean of synergy ZIP-scores in most synergistic areas for the 3 cell lines. Data are shown as mean +/- SD of replicate samples (N=3 for each cell line). Dotted line at 10% represents the threshold for significant synergy. (F, G) Patient (N=17) (F) or healthy donor (N=6) (G) BMMC were treated for 3 consecutive days (day 1, 2, 3) with TAK-981 (10 nM) and/ or AZA (100 nM) and kept in culture. After 8 days, cells were collected and the number of CD45 + cells was analyzed by flow cytometry in each condition and compared to the mock-treated condition. For each group, plain lines represent the median value, and dotted lines are the quartiles. Groups were compared using RM one-way ANOVA test.

Article Snippet: Human AML cell lines (U937, THP-1, HL-60, MOLM14, MV4.11) were obtained by the American Type Culture Collection and regularly tested for Mycoplasma contamination.

Techniques: In Vitro, Concentration Assay, Generated, Derivative Assay, Flow Cytometry, MTS Assay, Software, Standard Deviation

Journal: Haematologica

Article Title: SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia

doi: 10.3324/haematol.2023.282704

Figure Lengend Snippet: TAK-981 and 5-azacytidine combination has a higher anti-leukemic activity than monotherapies in vivo . (A, B) NSG mice were injected with THP-1 cells and treated with TAK-981 (15 mg/kg, intravenously [IV]), 5-azacytidine (AZA) (2 mg/kg, intraperitoneally [IP]) or the combination according to the schedule presented in Online Supplementary Figure S2B (N=5/group). (A) Quantification, as relative luminescence units, of tumor burden evolution monitored by luminescence intensity in mice injected with bioluminescent THP-1 cells. (B) Overall survival after treatment start of mice injected with bioluminescent THP-1 cells was estimated in each group and compared using Kaplan-Meier method and log-rank test. (C, D) NSG mice were injected with THP-1 cells and treated with TAK-981 (15 mg/kg, IV) and AZA (2 mg/kg, IP) or venetoclax (VEN) (50 mg/kg, oral gavage [OG]) according to the schedule presented in Online Supplementary Figure S2D (N=7/group). (C) Quantification (as relative luminescence units) of tumor burden evolution monitored by luminescence intensity in mice injected with bioluminescent THP-1 cells. (D) Overall survival after treatment start of mice injected with bioluminescent THP-1 cells was estimated in each group of treatment and compared using Kaplan-Meier method and log-rank test. (E, F) NSG mice were injected with primary cells from 2 different AML patients. After engraftment, mice were treated with AZA and/or TAK-981 and euthanized at day 9. The total number of human CD45 + cells (hCD45) was estimated by flow cytometry in bone marrow (PDX #1, N=7) and spleen (PDX #1, N=6; PDX #2, N=4-6), and compared to the mean number of cells collected in the mock-treated group of mice. For each group, plain lines represent the median value, and dotted lines are the quartiles. Groups were compared using ordinary one-way ANOVA test.

Article Snippet: Human AML cell lines (U937, THP-1, HL-60, MOLM14, MV4.11) were obtained by the American Type Culture Collection and regularly tested for Mycoplasma contamination.

Techniques: Activity Assay, In Vivo, Injection, Flow Cytometry

Journal: Haematologica

Article Title: SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia

doi: 10.3324/haematol.2023.282704

Figure Lengend Snippet: TAK-981 enhances 5-azacytidine-induced transcriptional reprogramming in U937 acute myeloid leukemia cell line. (A, B, C) Volcano plots showing differentially expressed genes (DEG) in U937 cell line treated each day for 3 days with 5-azacytidine (AZA) (10 nM) (A), TAK-981 (10 nM) (B) or the combination TAK-981+AZA (10 nM each) (C), analyzed at day 4 by RNA-sequencing and compared to mock-treated cells (N=3). Red dotted lines indicate the 2-fold change cutoff (absolute log 2 fold change [abs log 2 FC]=1) and a P value of 0.05 (log 10 =1.3). The total numbers of up- and downregulated DEG are indicated. Orange and violet dots indicate genes whose expression is respectively up- or downregulated more than 2-fold by AZA-alone treatment. (D) Scatter plot displaying the DESeq2 fold change of DEG in TAK-981+AZA treated U937 cells as a function of their fold change in AZA-treated cells. The red line represents the linear regression of the fold changes for the comparison of AZA+TAK-981 versus AZA replicates. As a control, the grey line represents the linear regression from the comparison of AZA versus AZA replicates. P value was calculated using student t test between the two linear regressions. (E) Gene set enrichment analysis was performed using Hallmark datasets on the RNA-sequencing data obtained from U937 cells. All pathways significantly enriched in the AZA+TAK-981 compared to mock-treated cells are shown (absolute normalized enrichment score [abs NES]>1, P <0.05 and false discovery rate [FDR]<0.05). NES and FDR are also indicated for the AZA and TAK-981 versus mock-treated cells.

Article Snippet: Human AML cell lines (U937, THP-1, HL-60, MOLM14, MV4.11) were obtained by the American Type Culture Collection and regularly tested for Mycoplasma contamination.

Techniques: RNA Sequencing, Expressing, Comparison, Control

Journal: Haematologica

Article Title: SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia

doi: 10.3324/haematol.2023.282704

Figure Lengend Snippet: TAK-981 treatment of acute myeloid leukemia cells leads to natural killer cell activation and increased natural killer cell cytotoxicity. (A) Gene set enrichment analysis enrichment plot for genes involved in natural killer (NK) cell-mediated cytotoxicity in TAK-981+5-azacytidine (AZA)-treated U937 cells compared to mock- (upper panel) or AZA- (lower panel) treated cells. (B, C) Expression of ICAM-1 (B) and MICA/B (C) was measured by flow cytometry on THP-1 treated with 10 nM TAK-981 and AZA at the indicated concentrations for 72 hours. Background was subtracted and results were normalized to mock-treated condition (N=10 for ICAM-1, N=5 for MICA/B, mean +/- standard deviation [SD], RM one-way ANOVA test). (D, E) NSG mice (N=7/group) were injected with primary patient cells (PDX #1), and treated according to treatment schedule presented in . Bone marrows were collected at day 9 after treatment start and ICAM-1 and MICA/B expression on the membrane of human CD45 + cells was assessed by flow cytometry. Data were normalized to the mean fluorescence intensity (MFI) of mock-treated mice group. Plain lines represent the median values, and dotted lines are the quartiles. Groups were compared using Kruskall-Wallis test. (F) THP-1 cells were treated each day for 3 consecutive days with 10 nM AZA, 10 nM TAK-981 or the drug combination, and co-cultured at day 8 with healthy donor peripheral blood mononuclear cells (PBMC) at a 1:10 acute myeloid leukemia (AML):PBMC ratio. After 24 hours of co-culture, expression of activation marker CD69 was assessed by flow cytometry on NK cells (CD3 - /CD56 + cells). Data were normalized to the MFI of CD69 expression on NK cells co-cultured with untreated THP-1, (mean +/- SD, N=6, RM oneway ANOVA test). (G, H) Real-time immune cell killing assay. To evaluate NK cells cytotoxicity against AML cells treated with TAK-981-/+AZA, co-culture experiments were performed during 15 hours using an Incucyte device between, on one hand, THP-1-LucZsGreen cells previously treated for 72 hours with 10 nM AZA, 10 nM TAK-981 or the drug combination, and on the other hand, NK cells, purified from healthy donor PBMC. Cells were used at a 1:1 AML:NK ratio. (G) Relative green fluorescence intensities for THP-1-Luc-ZsGreen cells mock-treated without (grey curve) or with NK cells (light blue curve) and treated with TAK-981+AZA without (red curve) or with NK cells (dark blue curve) (N=5). Cytotoxicity of NK cells was calculated by comparing grey- (mock-treated THP-1-/+NK cells) and red areas (AZA+TAK-981 treated THP-1-/+NK cells). (H) NK cells cytotoxicity was determined by calculating areas between the curves for each treatment condition (10 nM AZA, 10 nM TAK-981 or the drug combination) with or without NK. Data were normalized to mock-treated THP-1 cells (N=5, mean +/- SD, RM one-way ANOVA test).

Article Snippet: Human AML cell lines (U937, THP-1, HL-60, MOLM14, MV4.11) were obtained by the American Type Culture Collection and regularly tested for Mycoplasma contamination.

Techniques: Activation Assay, Expressing, Flow Cytometry, Standard Deviation, Injection, Membrane, Fluorescence, Cell Culture, Co-Culture Assay, Marker, Purification

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: PRMT1 activity is required for suppression of the TRAF6-dependent TLR pathway. A, Huh7.5 cells were transfected with HA-ubiquitin-Lys-63 (all lysines were substituted with arginine except Lys-63). Cells were otherwise untreated, pretreated with 10 μm AMI-1, or treated with negative control (NC) or PRMT1-specific siRNA. Lys-63-linked polyubiquitinated proteins were immunoprecipitated (IP) with anti-HA antibody, and the presence of TRAF6 was analyzed by Western blotting (IB). B, nuclear fractions were prepared from Huh7.5 or THP-1 cells treated as indicated with siRNA, the methylation inhibitor AMI-1 (10 μm), or the TLR6/2 ligand FSL-1. Immunoblots for NF-κB p65 are shown. C, NF-κB luciferase reporter assays of Huh7.5 cells expressing negative control (NC) or PRMT1-specific siRNA, expressing either wild type or dominant-negative PRMT1, or cells expressing TRAF6 dominant-negative construct treated with or without 10 μm AMI-1 as indicated. Transfection efficiency was controlled by expression of Renilla luciferase, and data are presented as the mean ± S.D. of relative luminescence. *, p < 0.05; **, p < 0.01. n ≥ 4. D, NF-κB luciferase reporter assays from RAW264.7 cells (control or pretreated with 10 μm AMI-1) or THP-1 cells expressing negative control (NC) or PRMT1-specific siRNA. Data are presented as the mean ± S.D. *p < 0.05. n ≥ 3. E, relative mRNA of NF-κB target genes TNFα, IL6, IRAK3 (IRAK-M), and TNFAIP3 (A20) in Huh7.5 cells, primary mouse hepatocytes, or primary mouse macrophages treated with or without AMI-1 for 16 h. Data are presented as the mean ± S.D. **, p < 0.01; *, p < 0.05. n = 3.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Activity Assay, Transfection, Negative Control, Immunoprecipitation, Western Blot, Methylation, Luciferase, Expressing, Dominant Negative Mutation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: TRAF6 is demethylated and activated by JMJD6. A, Huh7.5 cells were treated with or without FSL-1 (TLR6/2 ligand) for 1 h, and TRAF6 was immunoprecipitated (IP) from cell lysates. Western blot analysis of JMJD6 and TRAF6 is shown. Immunoprecipitation with nonimmune IgG served as the negative control. B, immunoprecipitation with anti-methyl-arginine antibody and immunoblotting for TRAF6 from control cells or cells overexpressing JMJD6 is shown. MeR, proteins immunoprecipitated with anti-methyl-arginine antibody. C, in vitro self-ubiquitinylation of recombinant TRAF6 (0.2 μg per reaction) in the presence of 0.2 μg of JMJD6 with or without its required cofactor, α-ketoglutarate (α-K). Immunoblots are shown for ubiquitin-specific, methyl-arginine-specific, and TRAF6-specific antibodies. D, Huh7.5 cells were transfected with an HA-tagged modified ubiquitin containing Lys-63 as the only lysine with or without JMJD6, immunoprecipitated with anti-HA antibody, and probed for TRAF6 to detect Lys-63-linked poly-ubiquitinated TRAF6. IB, immunoblot. E, left panel, control Huh7.5 cells or cells expressing JMJD6 were co-transfected with wild type or dominant-negative (DN) TRAF6 and NF-κB luciferase reporter assay were performed. Right panel, RNA was isolated from control or JMJD6-transfected cells, and the indicated NF-κB target gene mRNA abundance was determined by RT-qPCR. Data are presented as the mean ± S.D. **, p < 0.01; *, p < 0.05. n = 3. ICAM, intercellular adhesion molecule 1. F, Western blot analysis of NF-κB p65 in nuclear fractions of control Huh7.5 cells and cells transfected with JMJD6. G, NF-κB luciferase reporter assay in THP-1 cells, either control cells (−) or cells expressing JMJD6 (J6) (left). Data are presented as the mean ± S.D. **, p < 0.01. n = 3. H, representative images of proximity ligation assays in THP-1 cells untreated or treated with LPS for 30 min (right). TRAF6 interaction with JMJD6 was detected using anti-JMJD6 and anti-TRAF6 antibodies. Negative control, signal in the absence of primary antibodies.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Immunoprecipitation, Western Blot, Negative Control, In Vitro, Recombinant, Transfection, Modification, Expressing, Dominant Negative Mutation, Luciferase, Reporter Assay, Isolation, Quantitative RT-PCR, Ligation

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: Identification of functional TRAF6 methylation sites. A, upper panel, in vitro methylation assay. Wild type or mutant TRAF6 purified from Huh7.5 cells was methylated in vitro by recombinant PRMT1 in the presence of 50 μm SAM and analyzed using anti-TRAF6 and anti-methyl-arginine antibody. Lower panel, in vitro ubiquitination assay. Untreated wild type or mutant TRAF6 or each TRAF6 methylated in vitro by PRMT1 as above was incubated in vitro with recombinant E1, E2, and NF-κ-B essential modulator (NEMO) added as a ubiquitination substrate. Polyubiquitinated NEMO was detected using specific antibody against polyubiquitin chains. MeR, proteins immunoprecipitated with anti-methyl-arginine antibody. B, NF-κB luciferase reporter assays from control cells or cells expressing wild type or mutant TRAF6 co-transfected with or without wild type PRMT1 where indicated. Data are presented as the mean ± S.D. *, p < 0.05, n = 3. C, NF-κB luciferase reporter assays. Control Huh7.5 cells or cells overexpressing JMJD6 were co-transfected with wild type or mutant constructs of TRAF6. *, methylation at this site was confirmed by mass spectrometry. Data are presented as the mean ± S.D. †, p < 0.05; ††, p < 0.01, n ≥ 3. D, relative levels of TNF-α mRNA in Huh7.5 cells transfected with indicated TRAF6 mutants with or without co-expression of JMJD6 as indicated. **, p < 0.01 compared with control; †, p < 0.05 compared with wild type TRAF6 alone. n = 3.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Functional Assay, Methylation, In Vitro, Mutagenesis, Purification, Recombinant, Ubiquitin Assay, Incubation, Immunoprecipitation, Luciferase, Expressing, Transfection, Construct, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: Impact of TRAF6 arginine methylation on the ligand-induced TLR response. A, THP-1-Lucia NF-κB reporter monocytes were transfected with negative control siRNA or JMJD6-specific siRNA and treated with ligands for different TLR molecules as indicated. Results of luciferase reporter assays are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01 compared with negative control (NC) siRNA. n ≥ 3. B, luciferase reporter assays in Huh7.5 cells (left panel) or THP-1-Lucia NF-κB reporter monocytes (right panel) transfected with negative control siRNA or PRMT1-specific siRNA. Cells were treated with TLR ligands as indicated. Data are presented as the mean ± S.D. †, p < 0.05; ††, p < 0.01 compared with negative control siRNA. n ≥ 3. C, -fold increase in mRNA levels induced by FSL-1 exposure was determined in Huh7.5 cells treated with or without PRMT1 inhibitor AMI-1 for 16 h where indicated. *, p < 0.05; **, p < 0.01 compared with control; †, p < 0.05 compared with FSL-1 only. ICAM, intercellular adhesion molecule 1. D, FSL-1-induced -fold mRNA changes are shown in primary peritoneal macrophages. Cells were transfected with negative control (NC) siRNA and PRMT1-specific or JMJD6-specific siRNA where indicated. *, p < 0.05, compared with control; †, p < 0.05 compared with NC siRNA, n = 3. E, NF-κB luciferase reporter assays in Huh7.5 cells in the presence or absence of TLR6/2 ligand (1 ng/ml of FSL-1) that were transfected with negative control siRNA, PRMT1-specific siRNA, or both PRMT1- and JMJD6-specific siRNA. Data are presented as the mean ± S.D. *, p < 0.05; **, p < 0.01 compared with NC siRNA; ††, p < 0.01 compared with P1 siRNA. n = 3.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Methylation, Transfection, Negative Control, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: TRAF6 methylation correlates with TLR responses in humans. A, cytosolic and nuclear extracts were prepared from normal human liver tissue (liver transplant donor biopsy samples). Western blots were performed for PRMT1 and JMJD6 in cytosolic fractions and for NF-κB p65 in nuclear fractions. Data points represent values for individual liver specimens. n = 20. B, human peripheral blood monocytes were differentiated into macrophages and treated with or without AMI-1 for 16 h. Left panel, relative mRNA of TNFα in individual human macrophage preparations before (C) and after exposure to AMI-1. Right panel, -fold increase in TNFα mRNA 1 h after LPS exposure. Cells were pretreated with AMI-1 as indicated. Individual patient samples are connected by lines, n = 10. C, TRAF6 methylation was measured by ELISA as described under “Experimental Procedures.” Western blots were performed for JMJD6 protein concentration in cytosolic fractions from individual HBDM preparations. PRMT1 was measured using ELISA. n = 26. D, basal mRNA levels of TNFα and IL6 are plotted against relative TRAF6 methylation in individual HBDM preparations. E, -fold increase in IL6 mRNA levels is plotted against relative TLR4 expression or relative TRAF6 methylation in individual HBDM preparations. n = 26. Dependence of LPS-induced mRNA increases on TLR4 expression and TRAF6 methylation (6Tme) were statistically significant by multivariate linear regression, p = 0.0005 and 0.0017, respectively.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Methylation, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: IRAK3 is responsible for ligand desensitization under low methylation conditions. A, Western blot analysis of IRAK-3 levels in Huh7.5 cells treated with 10 μm AMI-1 for 16 h with or without 1 ng/ml FSL-1 for the indicated times. B, analysis of human blood derived macrophages. Basal mRNA level of IRAK3 is plotted against PRMT1/JMJD6 (left) or relative TRAF6 methylation (right). -Fold increase in IL6 mRNA after 1 h of LPS treatment is plotted against IRAK3 protein concentration (bottom). Each point represents an individual HBDM preparation, n = 26. C, NF-κB luciferase reporter assays in THP-1 cells that were either untreated (control) or treated with TLR6/2 (FSL-1), TLR4 (LPS) or TLR1/2 (Pam3) ligands as indicated. Cells were first transfected with negative control (NC) siRNA, PRMT1-specific siRNA, IRAK3-specific siRNA, or both IRAK3 and PRMT1 siRNA. Data are presented as the mean ± S.D. **, p < 0.01. n ≥ 3. ns, not significant.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Methylation, Western Blot, Derivative Assay, Protein Concentration, Luciferase, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling *

doi: 10.1074/jbc.M115.653543

Figure Lengend Snippet: A model of TRAF6 regulation by arginine methylation. In the basal state TRAF6 is associated with PRMT1, methylated, and inactive (State 1). TLR ligands activate TRAF6 and induce PRMT1 degradation. Reduction of PRMT1 levels allows JMJD6 to demethylate TRAF6 and results in the transition to a fully active form (State 2) leading to maximal NF-κB activation. The NF-κB-driven accumulation of inhibitory molecules, such as IRAK-3, eventually produces feedback inhibition reducing pathway activation. This allows the re-accumulation of PRMT1. Restoration of the PRMT1/JMJD6 ratio results in the re-methylation of TRAF6 and the return to the basal, inactive state.

Article Snippet: THP1-Lucia TM NF-κB cells were transfected where indicated with expression plasmids, or siRNA was incubated for 24 h, treated with TLR ligands, and incubated for additional 24 h before luciferase determination using QUANTI-luc reagent (InvivoGen).

Techniques: Methylation, Activation Assay, Inhibition