Journal: Frontiers in Immunology

Article Title: Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

doi: 10.3389/fimmu.2019.01169

Figure Lengend Snippet: Endothelial subpopulations have distinct mRNA expression levels of LPS signaling pathway components and inflammatory genes. (A) Experimental design for EC sorting using FACS. (B) mRNA expression levels of E-selectin, VCAM-1, TLR4, RIG-I, and MAVS in sorted EC subpopulations as determined by RT-qPCR using GAPDH as the housekeeping gene. Bars represent the mean ± SD of 5 independent experiments. p < 0.05 is considered statistically significant. (C) Heatmap displaying pro-inflammatory adhesion molecule, cytokine, and chemokine mRNA levels in sorted EC subpopulations compared to unsorted LPS control EC. The data shown is from four independent experiments.

Article Snippet: Quantitative (q)PCR was performed in a ViiA 7 PCR System (Applied Biosystems Nieuwerkerk aan den IJssel, The Netherlands) using the following assay-on-Demand primers (Applied Biosystems,): GAPDH (assay ID Hs99999905_m1), E-selectin (assay ID Hs00174057_m1), VCAM-1 (assay ID Hs00365486_m1), TLR4 (assay ID Hs00152939_m1), RIG-I (assay ID Hs00204833_m1), MAVS (assay ID Hs00920075), IL-6 (assay ID Hs00174131_m1), IL-8 (assay ID Hs00174103_m1), MCP-1 (assay ID Hs00234140_m1), CXCL10 (assay ID Hs01124251_g1), and CXCL6 (assay ID Hs00605742_g1).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Immunology

Article Title: Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

doi: 10.3389/fimmu.2019.01169

Figure Lengend Snippet: TLR4 and RIG-I-MAVS differentially regulate LPS-mediated E-selectin/VCAM-1 subpopulation formation. (A) The effect of small interfering RNA (siRNA)-based RIG-I and TLR4 knockdown on the formation of LPS-induced EC subpopulations based on E-selectin and VCAM-1 expression after 4 h of LPS exposure compared to scramble siRNA (siScr) controls. Bars represent the mean ± SD of 4 independent experiments. (B) The consequence of MAVS knockdown on the formation of the EC subpopulations. Bars represent the mean ± SD of 3 independent experiments. p < 0.05 is considered statistically significant.

Article Snippet: Quantitative (q)PCR was performed in a ViiA 7 PCR System (Applied Biosystems Nieuwerkerk aan den IJssel, The Netherlands) using the following assay-on-Demand primers (Applied Biosystems,): GAPDH (assay ID Hs99999905_m1), E-selectin (assay ID Hs00174057_m1), VCAM-1 (assay ID Hs00365486_m1), TLR4 (assay ID Hs00152939_m1), RIG-I (assay ID Hs00204833_m1), MAVS (assay ID Hs00920075), IL-6 (assay ID Hs00174131_m1), IL-8 (assay ID Hs00174103_m1), MCP-1 (assay ID Hs00234140_m1), CXCL10 (assay ID Hs01124251_g1), and CXCL6 (assay ID Hs00605742_g1).

Techniques: Small Interfering RNA, Knockdown, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA-mediated mitochondrial fragmentation is independent on the fission machinery proteins DLP1 and MFF. (A) Immunofluorescence analyses of MEFs MAVS-MITO cells: (a) control cells, (b) DLP1 silenced cells, (c) MFF silenced cells: (a–c) anti-TIM23; (d–f) overexpression of vMIA-Myc: (d) anti-TIM23, (e) anti-Myc, (f) merge image of d and e; (g–i) overexpression of vMIA-Myc in DLP1 silenced cells: (g) anti-TIM23, (h) anti-Myc, (i) merge image of g and h; (j–l) overexpression of vMIA-Myc in MFF silenced cells: (j) anti-TIM23, (k) anti-Myc, (l) merge image of j and k. Bars represent 10 µm. (B,C) Statistical analysis of mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS-MITO cells in the absence of DLP1 or MFF, respectively. Approximately 600 cells were analysed per condition. Data represents the means ± SEM of three independent experiments analysed using two-way ANOVA with Bonferroni’s multi comparations test (ns = non-significant, ****– p < 0.0001). Error bars represent SEM.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Immunofluorescence, Control, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA-induced peroxisomal and mitochondrial fragmentation is independent of MAVS. vMIA does not disrupt STING-MAVS interaction at peroxisomes. (A) Immunofluorescence analyses of MEFs MAVS KO cells: (a, b) peroxisomal and mitochondrial morphologies in control cells: (a) anti-PMP70, (b) anti-TIM23; (c–e) peroxisomal morphology upon overexpression of vMIA-Myc: (c) anti-PMP70, (d) anti-Myc, (e) merge image of c and d; (f, h) mitochondrial morphology upon overexpression of vMIA-Myc: (f) anti-TIM23, (g) anti-Myc, (h) merge image of f and g. Bars represent 10 µm. (B,C) Statistical analysis of peroxisomal or mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS KO cells, respectively. Approximately 600 cells were analysed per condition. (D) Co-immunoprecipitation analysis of the interaction between overexpressed STING-FLAG and vMIA-Myc in MEFs MAVS-PEX cells. The pull-down was performed using an antibody against MAVS. Western blot was performed with antibodies against FLAG and Myc. IN represents total cell lysate (input), IP represents immunoprecipitation and OUT represents the cell lysate extracted after incubation with the antibody (output). (E) Quantification of the ratio between IP and IN, in the presence or absence of vMIA. Data represents the means ± SEM of three independent experiments, analysed using unpaired T test (ns - non-significant; ***– p < 0.001, ****– p < 0.0001).

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Immunofluorescence, Control, Over Expression, Immunoprecipitation, Western Blot, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA inhibits MAVS oligomerization at peroxisomes and mitochondria. MFF is essential for the vMIA-mediated inhibition of MAVS oligomerization at peroxisomes but not at mitochondria. (A,B) HEK293T cells infected with SeV in the presence or absence of vMIA. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (A) Peroxisome-enriched fraction, (B) Mitochondria-enriched fraction. (C,D) HEK293T cells infected with SeV in the presence or absence of vMIA and in the absence of MFF. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (C) Peroxisome-enriched fraction, (D) Mitochondria-enriched fraction. (A–D) Immunoblots were performed with antibodies against MAVS, Myc-tag, COXIV, PEX14 and PMP70. (E) Whole cell lysates were resolved by SDS-PAGE. SeV infection, and consequential activation of MAVS downstream signalling, was confirmed using anti-p-STAT1. vMIA-Myc overexpression and MFF silencing were also confirmed using anti-Myc and anti-MFF, respectively. Antibody against Actin was used as loading control.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Inhibition, Infection, Isolation, Western Blot, SDS Page, Activation Assay, Over Expression, Control

Journal: Neurogenetics

Article Title: Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse

doi: 10.1007/s10048-021-00657-2

Figure Lengend Snippet: Innate immunity activation due to mutations in mitochondrial factors is mediated in the cytosol via the cGAS/STING pathway (when accumulated and possibly fragmented mtDNA is extruded to the cytosol) or the DDX58/IFIH1/MAVS sensors in the pattern recognition RLR pathway (when accumulated mitochondrial double-strand RNA is extruded). They stimulate the nuclear induction of interferon type I signaling. It is unclear if these established mechanisms also are prominent in ClpP-deficient cells, or additional pattern recognition receptors (TLR, NLR) and other cytosolic sensors play a relevant role. MRG, mitochondrial ribosomal granule; VDAC, PORIN; DDX58, RIG-I; IFIH1, MDA-5; STING, STING1, also known as TMEM173; ISRE, interferon-stimulated response element in the promoter of nuclear genes; other symbols can be retrieved in the GeneCards database

Article Snippet: The following TaqMan assays (Thermo Fisher Scientific) were employed to quantify the individual messenger RNA (mRNA) levels: Aim2 - Mm01295719_m1, ClpP - Mm00489940_m1, Ddx58 - Mm01216853_m1, Dnaja3 - Mm00469723_m1, Eif2ak2 - Mm01235643_m1, Gbp3 - Mm00497606_m1, Ifi204 - Mm00492602_m1, Ifi205b (=Mnda) - Mm04204353_mH, Ifi35 - Mm00510329_m1, Ifi44 - Mm00505670_m1, Ifih1 - Mm00459183_m1, Ifit1 (= Isg56 )- Mm00515153_m1, Ifit3 - Mm01704846_s1, Ifna1 - Mm03030145_gH, Ifnb1 - Mm00439552_s1, Irf3 - Mm00516784_m1, Mavs (= Ips-1 )- Mm00523170_m1, Mb21d1 (=cGas) - Mm01147496_m1, Nfkb1 - Mm00476361_m1, Nlrp3 - m00840904_m1, Nlrx1 - Mm00617978_m1, Oas1b - Mm00449297_m1, Oasl2 - Mm00496187_m1, Rsad2 - Mm00491265_m1, Stat1 - Mm00439531_m1, Stat2 - Mm00490880_m1, Tbp - Mm00446973_m1, Tlr3 - Mm01207404_m1, Tlr9 - Mm00446193_m1, Tmem173 - Mm01158117_m1, Trim25 - Mm01304226_m1, Trim30a - Mm00493346_m1, Trim56 - Mm01207494_m1, and Tspan6 - Mm00451045_m1.

Techniques: Activation Assay

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Effect of LacCer and cPLA2 inhibition on MAVS oligomerization, analyzed by SDD-AGE and western blot. (unpaired T test, compared to all, intensity normalized to vehicle Naïve condition) (B) ROS levels in activated murine and data are shown relative to ROS levels in resting astrocytes. (unpaired T test, compared to all) (C) LacCer induces cPLA2 recruitment to mitochondria and co-localization with MAVS. Astrocytes were treated with 10 μM LacCer or vehicle for 4 hours then stained and analyzed by confocal microscopy. Bar plots depict the ratio of total cPLA2 co-localization with mitochondria, the ratio of total MAVS co-localization with mitochondria, and the ratio of cPLA2-MAVS co-localization in mitochondria. (n≥25 cells per group, unpaired T test, compared to vehicle) (D) MAVS-cPLA2 interaction in astrocyte is analyzed by CoIP assay with MAVS-specific antibody and western blot. (unpaired T test, compared to all, intensity normalized to vehicle naive condition) (E,F) Domain binding analysis for cPLA2-MAVS interaction. Full-length human cPLA2 together with flag-tagged human MAVS (Full length, W56A mutant, G67A/W68A/V69A mutant (AAA), M2–6L mutant or M2 isoform) (E); or human cPLA2 (full-length, phospholipid binding domain deficient or C2 domain deficient) together with full-length flag-tagged human MAVS (F) were co-expressed in HEK293 cells, protein complexes were pulled down with anti-Flag antibodies and analyzed by western blot. (G) The effect of cPLA2 overexpression in trigger MAVS oligomerization. cPLA2 (full-length and C2 domain only) and MAVS were co-expressed in HEK293 cells, mitochondria were isolated 24 hours later and analyzed by SDD-AGE and western blot. (H) The effect of cPLA2 overexpression in triggering MAVS-mediated NF-κB activation, analyzed by luciferase assay. (unpaired T test, compared to all) (I) Effect of MAVS on NF-κB activation in WT and Mavs knockout astrocytes. The level of nucleus p65 level is analyzed by western blot. (unpaired T test, compared to corresponding condition between WT and MAVS−/− astrocytes) (J) mRNA expression determined by qPCR in activated WT and MAVS−/− astrocytes. (unpaired T test, compared to corresponding condition between WT and MAVS−/− astrocytes) (K) Recruitment of NF-κB to Ccl2, Csf2 and Nos2 promoters in murine WT and MAVS−/− astrocytes in culture determined by ChIP assay as in Figure 2O. (unpaired T test, compared to all) (L) EAE development in NOD mice treated with lentiviral constructs expressing shRNAs targeting Mavs or control in astrocytes 37 and 43 days after EAE induction. (n≥7, N=3, Regression slope T test, compared to shControl). (M,N) CNS samples were harvested 50 days after EAE induction from mice treated with lentivirus-delivered shRNAs shown in Figure 3L. (unpaired T test, compared to shControl) (M) The number of CNS-infiltrating inflammatory monocytes. (N) Inflammatory genes expression in astrocytes from control and Mavs knockdown mice was detected by nCounter analysis system (n=4). (O, P) Astrocyte-conditioned medium was prepared from activated WT or MAVS−/− astrocytes for test in in vitro monocyte migration. (O, unpaired T test, compared to all) and neurotoxicity assays (P, unpaired T test, compared to all). Migrating monocytes and neuronal death in the resting shControl-treated group were set as 100%.

Article Snippet: Primary antibodies used in this study were: anti-GFAP mouse mAb (#MAB360, Millipore, 1:500), anti-COX4-I1 goat polyclonal Ab (#AF5814, R&D Systems, 1:100), anti-NF-κB p65 (acetyl-Lys310) rabbit polyclonal Ab (#SAB4502616, Sigma-Aldrich, 1:100), anti-mouse c3d goat polyclonal antibody (#AF2655, R&D Systems, 1:100), anti-MAVS rabbit mAb (#4983, Cell Signaling, 1:100), and anti-cPLA2 mouse mAB (#sc-454, Santa Cruz Biotechnology, 1:100).

Techniques: Inhibition, Western Blot, Staining, Confocal Microscopy, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis, Over Expression, Isolation, Activation Assay, Luciferase, Knock-Out, Expressing, Construct, In Vitro, Migration

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Level change of glucose metabolism in activated astrocytes measured by metabolomic profiling. Green color indicates metabolites decreased by cPLA2i treatment, red color indicates increased metabolites, black color indicates metabolites for which no data were collected. (B, C) Effect of cPLA2 (cPLA2i) or MPCs (UK-5099) inhibition in astrocyte on lactate release (B, unpaired T test, compared to activated vehicle condition, normalized to naive vehicle condition) and pyruvate level in mitochondria (C, unpaired T test, compared to activated vehicle condition, normalized to naive vehicle condition). (D,E) Metabolomic profiling analysis of activated astrocytes in the presence of cPLA2i. (D) Effect of cPLA2 inhibition on saturated and unsaturated fatty acids level in activated astrocyte, shown relative to their levels on resting astrocytes. Each dot indicates a different fatty acid. (unpaired T test, compared to activated vehicle condition) (E) Metabolite categories altered by cPLA2i treatment. (F) Effect of methylglyoxal supply on lactate release by activated astrocytes. (unpaired T test, compared to activated control condition) (G) Effect of cPLA2 or MPCs inhibition in mitochondrial function of astrocyte analyzed by Mito Stress test assay. (unpaired T test, compared to all) (H,I) Effect of gene targeted siRNA knockdown in mitochondrial function of LacCer loaded astrocytes analyzed by Mito Stress test assay (H, unpaired T test, compared to all) and lactate release by astrocytes (I, unpaired T test, compared to naive siControl condition). (J) Effect of LacCer loading or cytokines stimulation on the interaction of MAVS with HK2 and cPLA2 in astrocyte, evaluated by pull-down assay and western blot. (K) Mitochondrial HK enzymatic activity in activated astrocytes. (unpaired T test, compared to vehicle condition) (L) MAVS binding domain analysis. Flag-tagged human MAVS (Full length, M2–6L, M2, AAA or W56A) was over-expressed in HEK293 cell and 24 hours later MAVS protein complexes were pulled down and analyzed by western blot. (M) Human cPLA2 (cPLA2-GFP or C2 domain-GFP respectively) were over-expressed in HEK293 cell and 24 hours later MAVS protein complexes were pulled down and analyzed by western blot. (N) Effect of cPLA2 or cPLA2 C2 domain overexpression in HEK293 cells on mitochondrial HK enzymatic activity, lactate release and mitochondrial pyruvate levels. (unpaired T test, compared to Control condition) (O) Effect of 2-DG treatment on lactate release and mitochondrial levels of pyruvate in astrocyte. (unpaired T test) (P) Lactate release by Bsg knockdown or control astrocytes. (unpaired T test) (Q) EAE development in C57BL/6 or NOD mice that received lentiviral constructs to knockdown Bsg in astrocytes at the time points indicated by the arrows. (n≥7, N = 2, Regression slope T test). (R) Axonal loss and demyelination in spinal cords from NOD EAE mice. (unpaired T test)

Article Snippet: Primary antibodies used in this study were: anti-GFAP mouse mAb (#MAB360, Millipore, 1:500), anti-COX4-I1 goat polyclonal Ab (#AF5814, R&D Systems, 1:100), anti-NF-κB p65 (acetyl-Lys310) rabbit polyclonal Ab (#SAB4502616, Sigma-Aldrich, 1:100), anti-mouse c3d goat polyclonal antibody (#AF2655, R&D Systems, 1:100), anti-MAVS rabbit mAb (#4983, Cell Signaling, 1:100), and anti-cPLA2 mouse mAB (#sc-454, Santa Cruz Biotechnology, 1:100).

Techniques: Inhibition, Pull Down Assay, Western Blot, Activity Assay, Binding Assay, Over Expression, Construct

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Miglustat levels in the CNS after oral administration of 600 mg/kg Miglustat. (unpaired T test) (B) EAE development in NOD mice treated with Miglustat (600 mg/kg administered orally before initiation of progressive phase, n≥6, N=3, Regression slope T test). (C-G) CNS samples were harvested 41 days after EAE induction from mice treated with Miglustat or vehicle as shown in Figure 6b. The number of CNS-infiltrating inflammatory monocytes (C, unpaired T test). Whole genome expression in astrocytes isolated from Miglustat treated NOD EAE mice (D; n = 6). Axonal loss and demyelination in spinal cord (E, unpaired T test). Immunoflourescence analysis of C3+GFAP+ astrocytes. Bar plots depict the number of C3+GFAP+ astrocyte within the observation field (F, unpaired T test, compared to EAE condition). Immunofluorescence analysis of NF-κB activation among MAVS+cPLA2+GFAP+ astrocytes (G, unpaired T test, compared to EAE condition). Bar plots depict the number of MAVS+cPLA2+acetyl-p65+GFAP+ astrocyte within the observation field. (H) mRNA expression determined by qPCR in activated human and mouse astrocytes in the presence of Miglustat. Data are shown relative to resting astrocytes. (unpaired T test) (I) Human and mouse astrocyte conditioned medium were analyzed using in vitro monocyte migration and neurotoxicity assays. Migrating monocytes and neuronal death in the resting vehicle-treated group were set as 100%. (unpaired T test) (J) mRNA expression in polarized microglia was determined by qPCR. Microglia was co-cultured with activated astrocytes. (unpaired T test) (K) Lactate release from activated astrocytes in the presence of Miglustat. (unpaired T test, compared to activated vehicle condition) (L) Effect of Miglustat treatment on cPLA2 enzymatic activity in astrocytes, evaluated by cPLA2 activity assay. (unpaired T test, compared to all)

Article Snippet: Primary antibodies used in this study were: anti-GFAP mouse mAb (#MAB360, Millipore, 1:500), anti-COX4-I1 goat polyclonal Ab (#AF5814, R&D Systems, 1:100), anti-NF-κB p65 (acetyl-Lys310) rabbit polyclonal Ab (#SAB4502616, Sigma-Aldrich, 1:100), anti-mouse c3d goat polyclonal antibody (#AF2655, R&D Systems, 1:100), anti-MAVS rabbit mAb (#4983, Cell Signaling, 1:100), and anti-cPLA2 mouse mAB (#sc-454, Santa Cruz Biotechnology, 1:100).

Techniques: Expressing, Isolation, Immunofluorescence, Activation Assay, In Vitro, Migration, Cell Culture, Activity Assay

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Immunostaining analysis of the co-localization (white arrowheads) of cPLA2 and mitochondria marker Tom20 in GFAP+ astrocytes in MS tissue. GFAP+ astrocytes in lesion, NAWM near lesion (20–50 μm from lesion) and NAWM far from lesion (>500 μm) (B) Immunostaining analysis of the co-localization (white arrowheads) of cPLA2 and MAVS in MS or HC tissue GFAP+ astrocytes. (C) Quantification of cPLA2+MAVS+ co-localization signal in GFAP+ and GFAP− fields. (n = 30–54 cells per condition, unpaired T test, compared to all) HC = healthy control, WM = white matter, GM = gray matter, NAWM = normally appearing white matter, NAGM = normally appearing gray matter.

Article Snippet: Primary antibodies used in this study were: anti-GFAP mouse mAb (#MAB360, Millipore, 1:500), anti-COX4-I1 goat polyclonal Ab (#AF5814, R&D Systems, 1:100), anti-NF-κB p65 (acetyl-Lys310) rabbit polyclonal Ab (#SAB4502616, Sigma-Aldrich, 1:100), anti-mouse c3d goat polyclonal antibody (#AF2655, R&D Systems, 1:100), anti-MAVS rabbit mAb (#4983, Cell Signaling, 1:100), and anti-cPLA2 mouse mAB (#sc-454, Santa Cruz Biotechnology, 1:100).

Techniques: Immunostaining, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Human myeloid dendritic cells (mDCs) were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells induced or not to realtime PCR. The profile of TRIM29 expression in different cells is indicated. The relative expression of TRIM29 was compared by plotting the values extracted from the gene expression database. A value < 1 indicated the absence of gene expression. (B) Immunoblot analysis of TRIM29 and MABVS in human mDCs treated with control shRNA with a scrambled sequence (sh-Ctrl), shRNA targeting mRNA encoding TRIM29 (two shRNAs: Trim29#1 and Trim29#2) or shRNA targeting mRNA encoding MAVS. GAPDH serves as a loading control throughout. (C) ELISA of IFN-β in human primary mDCs treated with scrambled shRNA (sh-Ctrl) and left unstimulated (Mock) or treated with shRNA as above and then stimulated for 16h with long poly I:C (LPIC, 20 μg/ml) delivered by Lipofectamine 3000. *P<0.05, **P<0.01, ***P<0.001 (unpaired t test). N-STM, cells without stimulation. Data are representative of three independent experiments with similar results (mean + s.d.).

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Purification, Isolation, Expressing, Gene Expression, Western Blot, Control, shRNA, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Immunoblot analysis of endogenous proteins of TRIM29 and MAVS precipitated with anti-MAVS, anti-TRIM29 or control immunoglobulin G (control IgG) from whole-cell lysates of BMDCs from wild type mice stimulated without (No) or with poly I:C (25 μg/ml) for 8h, and then with MG132 treatment for 3h. Input, 20% of the BMDCs lysate. (B) Full-length MAVS and serial truncations of MAVS with deletion of various domains (top). Below, immunoblot analysis of purified HA-tagged TRIM29 with anti-HA (bottom blot), and immunoblot analysis of purified Myc-tagged full-length MAVS and MAVS truncation mutants alone with anti-Myc (top blot) or after incubation with HA-tagged full-length TRIM29 and immunoprecipitation with anti-HA (middle blot). (C) Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains (left margin); numbers at ends indicate amino acid positions (top). Below, immunoblot analysis of purified Myc-tagged MAVS with anti-Myc (bottom blot), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone (top blot) or after incubation with Myc-tagged MAVS and immunoprecipitation with anti-Myc (middle blot). (D) Activation of the IFN-β promoter in human HEK293T cells transfected with an IFN-β promoter luciferase reporter, plus expression vector (each 100 ng) for wild-type MAVS or expression vector for wild-type TRIM29 (T29-a) or TRIM29 mutants T29-b and T29-g; results are presented relative to those of renilla luciferase (cotransfected as an internal control). (E) Colocalization of endogenous TRIM29 and MAVS in BMDCs. Confocal microscopy of BMDCs infected with reovirus for 4h. MAVS was stained with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (green), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Scale bars represent 20 μm. Data are representative of three independent experiments.

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Western Blot, Control, Purification, Incubation, Immunoprecipitation, Activation Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Confocal Microscopy, Infection, Staining, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Immunoblot analysis of Myc-tagged MAVS (top blot), HA-tagged TRIM29 (middle blot) and β-actin (bottom blot) in HEK293T cells cotransfected with an expression vector for Myc-tagged MAVS and empty vector or expression vector for HA-tagged TRIM29, with or without treatment of 5 μM MG132. (B) Immunoblot analysis of TRIM29 (top blot), MAVS (second blot), phosphorylated IRF3 (third blot), phosphorylated p65 (fourth blot) and β-actin (bottom blot) in WT and TRIM29 KO BMDCs stimulated for various times (above lanes) with poly I:C (10 μg/ml). (C) Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot) and K11-linked ubiquitination (third blot) of Myc-tagged MAVS in HEK293T cells transfected with empty vector or expression vector for HA-tagged TRIM29, truncation T29-g (losing binding site of MAVS), and stimulated for 4 h with poly I:C (20 μg/ml), assessed after immunoprecipitation with anti-Myc; and immunoblot analysis whole-cell lysates with anti-HA (fifth blot) and anti-β-actin (bottom). (D) Immunoblot analysis of TRIM29 in WT and KO BMDCs (top), and of the abundance (second blot), total ubiquitination (third blot) and K11-mediated ubiquitination (bottom blot) of MAVS in those cells, stimulated for 4 h with poly I:C (10 μg/ml), assessed after immunoprecipitation with anti-MAVS. Data are representative of three experiments.

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Western Blot, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Transfection, Binding Assay, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TRIM29 negatively regulates the type I interferon production in response to RNA virus

doi: 10.4049/jimmunol.1701569

Figure Lengend Snippet: (A) Immunoblot analysis of Myc-tagged MAVS and its mutations (top blot), HA-tagged TRIM29 (middle blot) and β-actin (bottom blot) in HEK293T cells cotransfected with an expression vector for HA-tagged TRIM29 and expression vectors for Myc-tagged wild-type full-length MAVS and its mutations. (B) Immunoblot analysis of HA-tagged TRIM29 (top blot), Myc-tagged MAVS and its mutations (second blot), β-actin (third blot) and K11-linked ubiquitination (bottom blot) in HEK293T cells cotransfected with expression vector for Myc-tagged MAVS or its mutations (above lanes) and expression vector for HA-tagged TRIM29, with treatment of 5 μM MG132 (above lanes). (C,D) Activation of the IFN-β promoter (C) or NF-κB promoter (D) in human HEK293T cells transfected with an IFN-β promoter luciferase reporter (C) or NF-κB promoter luciferase reporter (D), plus expression vector (each 100 ng) for wild-type MAVS or various MAVS mutants alone or expression vector for TRIM29 plus wild-type MAVS or MAVS mutants; results are presented relative to those of renilla luciferase (cotransfected as an internal control). Data are representative of three independent experiments with similar results (mean + s.d.)

Article Snippet: BMDCs isolated from the WT mice were infected with reovirus for 4h and were then fixed in 4% paraformaldehyde and permeabilized with 0.1% triton-100, then blocked for 30 min with 5% BSA, incubated with Rabbit anti-MAVS polyclonal antibody (Cat: 14341-1-AP, proteintech) and mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz) for 2 h, followed by Alexa Fluor 594 goat anti-rabbit secondary antibody and Alexa Fluor 488 goat anti-mouse secondary antibody for 1h and then examined with confocal microscopy.

Techniques: Western Blot, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Activation Assay, Transfection, Luciferase, Control