Journal: PLoS Pathogens

Article Title: PKACs attenuate innate antiviral response by phosphorylating VISA and priming it for MARCH5-mediated degradation

doi: 10.1371/journal.ppat.1006648

Figure Lengend Snippet: (A) Left panels: Knockdown efficiencies of PKACa and PKACb by RNAi. For the upper three panels, HEK293 cells were transfected with Flag-PKACα/β, HA-β-actin, and the indicated RNAi plasmids for 36 h, and then analyzed by immunoblots with anti-Flag or anti-HA antibodies. For the lower two panels, HEK293 cells were transfected with the indicated RNAi plasmids for 48 h before immunoblot analysis with the indicated antibodies. Right histographs: Effects of PKACα and PKACβ knockdown on SeV-induced signaling. HEK293 cells were transfected with the indicated reporter and RNAi plasmids for 36 h, then infected with SeV (MOI = 1) or left untreated for 12 h before luciferase assays were performed. (B) Effects of PKACα and PKACβ knockdown on SeV-induced transcription of downstream genes. HEK293 cells were transfected with the indicated RNAi plasmids for 12 h. The cells were then selected with puromycin for 24 h and then infected with SeV (MOI = 1) for the indicated times before qPCR was performed. (C) Effects of simultaneous knockdown of PKACα and PKACβ on SeV-induced signaling. The experiments were similarly performed as in A by using RNAi plasmids simultaneous target PKACα and PKACβ. (D) Effects of simultaneous knockdown of PKACα and PKACβ on SeV-induced transcription of downstream genes. The experiments were similarly performed as in B by using RNAi plasmids simultaneous target PKACα and PKACβ. (E) Effects of knockdown of PKACα and PKACβ on VSV-induced transcription of downstream genes. THP1 cells were transfected with the indicated siRNA for 36 h and then infected with VSV (MOI = 1) for the indicated times before qPCR was performed. (F) Knockdown of PKACs potentiates cytoplasmic poly(I:C)-triggered activation of the IFN-β promoter. HEK293 cells were transfected with the IFN-β promoter luciferase plasmid and the indicated RNAi plasmids for 36 h, then transfected with low molecular weight (LMW) or high molecular weight (HMW) poly(I:C) for 12 h before luciferase assays were performed. (G) Knockdown of PKACs inhibits SeV-induced phosphorylation of TBK1, IRF3 and IκBα. HEK293 cells were transfected with the indicated RNAi plasmids and selected with puromycin, then infected with SeV (MOI = 1) for the indicated times. Cell lysates were analyzed by immunoblots with the indicated antibodies. (H) Knockdown of PKACs inhibits VSV-induced phosphorylation of TBK1, IRF3 and IκBα. THP1 cells were transfected with the indicated siRNA for 36 h and then infected with VSV (MOI = 1) for the indicated times. Cell lysates were analyzed by immunoblots with the indicated antibodies. (I) Effects of PKACs knockdown on IFN-α-induced STAT1/2 and IFN-γ-induced IRF1 promoter activation. HEK293 cells were transfected with the indicated reporter and PKACs RNAi plasmids for 36 h, then treated with the indicated cytokines or left untreated for 12 h before luciferase assays were performed.

Article Snippet: Lipofectamine 2000 (Invitrogen); RNase inhibitor (Thermo); SYBR (Bio-Rad); mouse monoclonal antibodies against Flag, HA, and β-actin (Sigma), TBK1, phospho-TBK1 (Ser172) and phospho-Ser/Thr (Abcam), phospho-PKA substrate and phospho-IRF3 (Ser396) (Cell Signaling Technology), rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology) and rabbit polyclonal antibodies against VISA (Bethyl) were purchased from the indicated manufacturers; mouse anti- PKACα/β antisera were raised against recombinant human full-length PKACβ; SeV, EMCV and HSV-1 were previously described [ , ]; HEK293 and THP1 cells (ATCC) were purchased from the indicated manufactures; HEK293T cells were originally provided by Dr. Gary Johnson (National Jewish Health).

Techniques: Knockdown, Transfection, Western Blot, Infection, Luciferase, Activation Assay, Plasmid Preparation, Molecular Weight, High Molecular Weight, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: PKACs attenuate innate antiviral response by phosphorylating VISA and priming it for MARCH5-mediated degradation

doi: 10.1371/journal.ppat.1006648

Figure Lengend Snippet: (A) Effects of PKACs and their kinase inactive mutants on VISA modification. HEK293 cells were transfected with HA-VISA and increased amounts of the indicated expression plasmids for 20 h before immunoblot analysis with the indicated antibodies. (B) VISA is serine/threonine phosphorylated by PKACs. HEK293 cells were transfected with HA-VISA and the indicated expression plasmids for 20 h. Cell lysates were immunoprecipitated with anti-HA, and the immunoprecipitates were treated with buffer or lambda protein phosphatase (λ-PPase) and then analyzed by immunoblots with the indicated antibodies. (C) Effects of PKACα on VISA and its mutants. HEK293 cells were transfected with the indicated expression plasmids for 20 h and then analyzed by immunoblots with the indicated antibodies. (D) Effects of VISA and its mutants on IFN-β promoter activation. HEK293 cells were transfected with the IFN-β promoter luciferase and Flag-VISA or its mutants plasmids for 20 h before luciferase assays were performed. The blot shows the expression levels of the transfected VISA and its mutants. (E) Effects of VISA and its mutants on SeV-induced transcription of IFNB1 gene. VISA-deficient HEK293 cells reconstituted with VISA or its mutants were infected with SeV (MOI = 1) for the indicated times before qPCR analysis. (F) Effects of VISA and its mutants on SeV-induced TBK1 and IRF3 phosphorylation. VISA-deficient HEK293 cells reconstituted with VISA or its mutants were infected with SeV (MOI = 1) for the indicated times before immunoblot analysis with the indicated antibodies. (G) PKACs phosphorylate VISA at T54. HEK293 cells were transfected with HA-VISA, Flag-tagged PKACα and Flag-PKACβ and their mutants for 20 h. Cell lysates were analyzed by immunoblots with the indicated antibodies. (H) Endogenous VISA is phosphorylated at T54 by PKACs after SeV infection. HEK293 cells were transfected with the indicated plasmids and selected with puromycin, then infected with SeV (MOI = 1) for the indicated times. Cell lysates were subjected to immunoprecipitation and immunoblot analysis with the indicated antibodies. (I) Effects of PKACs knockdown on activation of the IFN-β promoter by VISA and its mutants. HEK293 cells were transfected with the IFN-β promoter luciferase and PKACs RNAi plasmid for 36 h, then transfected with Flag-VISA or its mutants for 20 h before luciferase assays were performed.

Article Snippet: Lipofectamine 2000 (Invitrogen); RNase inhibitor (Thermo); SYBR (Bio-Rad); mouse monoclonal antibodies against Flag, HA, and β-actin (Sigma), TBK1, phospho-TBK1 (Ser172) and phospho-Ser/Thr (Abcam), phospho-PKA substrate and phospho-IRF3 (Ser396) (Cell Signaling Technology), rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology) and rabbit polyclonal antibodies against VISA (Bethyl) were purchased from the indicated manufacturers; mouse anti- PKACα/β antisera were raised against recombinant human full-length PKACβ; SeV, EMCV and HSV-1 were previously described [ , ]; HEK293 and THP1 cells (ATCC) were purchased from the indicated manufactures; HEK293T cells were originally provided by Dr. Gary Johnson (National Jewish Health).

Techniques: Modification, Transfection, Expressing, Western Blot, Immunoprecipitation, Activation Assay, Luciferase, Infection, Phospho-proteomics, Knockdown, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways

doi: 10.3390/ijms20184658

Figure Lengend Snippet: CCL4 co-induction by TNF-α/palmitate is TLR4 dependent. To investigate the involvement of TLR4 in the cooperative induction of CCL4 by TNF-α and palmitate, TLR4 signaling activity was blunted in THP-1 cells by TLR4 siRNA, the TLR4 neutralizing antibody or the TLR4 chemical inhibitor OxPAPC as described in the materials and methods section. The data (mean ± SEM) show that the cooperative effect of TNF-α/palmitate co-treatment was predominantly lost following siRNA-mediated ablation of TLR4 in THP-1 cells, resulting in significantly reduced expression of ( A ) CCL4 mRNA ( p = 0.019) and ( B ) protein ( p = 0.026) compared to control (scramble siRNA). Similarly, the cooperative induction of CCL4 by TNF-α/palmitate was abrogated after the TLR4 receptor was intercepted by treatment with an anti-TLR4 neutralizing antibody, displaying the diminished expression of ( C ) CCL4 mRNA ( p = 0.002) and ( D ) CCL4 protein ( p = 0.003) compared to the control (isotype-matched antibody). In addition, THP-1 cells treated with the the TLR4 inhibitor OxPAPC before TNF-α/palmitate co-stimulation also exhibited reduced expression of ( E ) CCL4 mRNA ( p = 0.004) and ( F ) secreted CCL4 protein ( p = 0.006) compared to the control (mock treatment). * Significant ( p < 0.03), ** Very significant ( p < 0.007).

Article Snippet: For siRNA-mediated TLR4 or IRF3 genetic suppression, THP-1 cells were washed, resuspended in 100 μL nucleofector solution (Amaxa NucleofectorTM Kit V, Lonza Bioscience, Cologne, Germany) and transfected separately with TLR4 siRNA or IRF3 siRNA (30 nM each; OriGene Technologies Inc. MD, USA), scrambled (control) siRNA (30 nM; OriGene Technologies Inc. MD, USA) and pmaxGFP (0.5 μg; Amaxa NucleofectorTM Kit V, Lonza Bioscience, Cologne, Germany).

Techniques: Activity Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways

doi: 10.3390/ijms20184658

Figure Lengend Snippet: TNF-α/palmitate cooperativity for CCL4 induction is MyD88 dependent. To determine whether the MyD88 adaptor protein was required for the cooperative CCL4 expression, MyD88-deficient THP1-XBlue-defMyD cells were co-stimulated with TNF-α/palmitate. The data (mean ± SEM) show that the co-induction effect was lost in MyD88 −/− cells as TNF-α/palmitate co-stimulation induced similar expression of ( A ) CCL4 mRNA ( p = 0.735) and ( B ) CCL4 secreted protein ( p = 0.859) as compared to treatment with TNF-α alone. In parallel, IRF3 silencing was carried out as described in the materials and methods section and the data (mean ± SEM) show that IRF3 ablation did not downmodulate the expression of ( C ) CCL4 mRNA and ( D ) CCL4 secreted protein in IRF3-ablated cells compared to the control (scrambled siRNA-transfected cells). Together, these results support that the co-induction of CCL4 by TNF-α/palmitate in human monocytic cells is MyD88 dependent or IRF3 independent.

Article Snippet: For siRNA-mediated TLR4 or IRF3 genetic suppression, THP-1 cells were washed, resuspended in 100 μL nucleofector solution (Amaxa NucleofectorTM Kit V, Lonza Bioscience, Cologne, Germany) and transfected separately with TLR4 siRNA or IRF3 siRNA (30 nM each; OriGene Technologies Inc. MD, USA), scrambled (control) siRNA (30 nM; OriGene Technologies Inc. MD, USA) and pmaxGFP (0.5 μg; Amaxa NucleofectorTM Kit V, Lonza Bioscience, Cologne, Germany).

Techniques: Expressing, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways

doi: 10.3390/ijms20184658

Figure Lengend Snippet: TNF-α/palmitate cooperativity for CCL4 induction is MyD88 dependent. To determine whether the MyD88 adaptor protein was required for the cooperative CCL4 expression, MyD88-deficient THP1-XBlue-defMyD cells were co-stimulated with TNF-α/palmitate. The data (mean ± SEM) show that the co-induction effect was lost in MyD88 −/− cells as TNF-α/palmitate co-stimulation induced similar expression of ( A ) CCL4 mRNA ( p = 0.735) and ( B ) CCL4 secreted protein ( p = 0.859) as compared to treatment with TNF-α alone. In parallel, IRF3 silencing was carried out as described in the materials and methods section and the data (mean ± SEM) show that IRF3 ablation did not downmodulate the expression of ( C ) CCL4 mRNA and ( D ) CCL4 secreted protein in IRF3-ablated cells compared to the control (scrambled siRNA-transfected cells). Together, these results support that the co-induction of CCL4 by TNF-α/palmitate in human monocytic cells is MyD88 dependent or IRF3 independent.

Article Snippet: For siRNA-mediated TLR4 or IRF3 genetic suppression, THP-1 cells were washed, resuspended in 100 μL nucleofector solution (Amaxa Nucleofector™ Kit V, Lonza Bioscience, Cologne, Germany) and transfected separately with TLR4 siRNA or IRF3 siRNA (30 nM each; OriGene Technologies Inc. MD, USA), scrambled (control) siRNA (30 nM; OriGene Technologies Inc. MD, USA) and pmaxGFP (0.5 μg; Amaxa Nucleofector™ Kit V, Lonza Bioscience, Cologne, Germany).

Techniques: Expressing, Transfection