Journal: PLOS Pathogens

Article Title: HCMV encoded UL84 hijacks FHL2 to suppress type I interferon production and enhance viral replication

doi: 10.1371/journal.ppat.1013895

Figure Lengend Snippet: (A) Schematic diagram of the enhancer region within the IFNB1 promoter. (B) The involvement of FHL2 in the regulation of IFN-β transcription was identified through the ChIP assay. HFF cells were infected with HCMV (MOI = 1) for 6 h, followed by ChIP assays using FHL2-specific or H3 histone control monoclonal antibodies, and IFN-β enhancer-specific P1, P2, and P3 primers. (C) FHL2 associated with IFN-β enhanceosome complex. HFFs were infected with HCMV (MOI = 1) for 6h, and cross-linked with 1% formaldehyde for 15min before CHIP. NC as non-specific control, normal rabbit IgG as a negative control, anti-IRF3 positive control, anti-FHL2 as an inductive antibody. (D) Co-IP assays were performed to investigate the interaction between FHL2 and IRF3, c-Jun, or p300. HEK293T cells were transiently co-transfected with pcDNA3.1-FHL2, pcDNA3.1-IRF3, pcDNA3.1-p300, and pcDNA3.1-c-Jun plasmids for 48 h. Total cell lysates were then prepared and immunoprecipitated with FHL2-specific antibody before being analyzed by Western blot using FHL2, IRF3, p300, and c-Jun antibodies. (E) Co-IP assay of FHL2 (ΔLIM2) with IRF3, c-Jun, or p300. Total cell lysates were prepared from HEK293T cells co-transfected with pRK-Flag-FHL2 (ΔLIM2), pcDNA3.1-IRF3, pcDNA3.1-p300, and pcDNA3.1-c-Jun plasmids for 48 h and then immunoprecipitated with FHL2-specific antibody before Western blot analysis. (F) Effect of different mutations on the interaction between FHL2 and c-Jun, IRF3, or p300. HEK293T cells were transiently co-transfected with pRK-Flag-FHL2 (full length or four single point mutants) and pcDNA3.1-c-Jun, pcDNA3.1-IRF3 or pcDNA3.1-p300 respectively for 48 h before Co-IP analysis. The displayed images were representative ones from three independent experiments. (G) Schematic diagram of the overall structural conformation of FHL2. (H) Figure of the radius of gyration results, a core indicator for characterizing molecular spatial conformation in molecular dynamics simulation. (I) Figure of the root mean square deviation results, a core indicator for characterizing molecular spatial conformation in molecular dynamics simulation. (J) Figure of the root-mean-square fluctuation results, a core indicator for characterizing molecular spatial conformation in molecular dynamics simulation. (K) Figure of the Solvent accessible surface area results, a core indicator for characterizing molecular spatial conformation in molecular dynamics simulation. The displayed images were representative ones from three independent experiments. For all figures, statistical analyses were performed using two-tailed t-test. Differences were considered statistically significant when * denoted p < 0.05, ** denoted p < 0.01, *** denoted p < 0.001, and **** denoted p < 0.0001.

Article Snippet: The antibodies against human IRF3 rabbit monoclonal antibody (Cat. No. 11904; diluted 1:1,000), human pIRF3 rabbit monoclonal antibody (Cat. No. E6F7Q; diluted 1:1,000), c-Jun mouse monoclonal antibody (Cat. No. 2315; diluted 1:1,000), STAT1 rabbit monoclonal antibody (Cat. No. 9172S; diluted 1:1,000) and pSTAT1 rabbit monoclonal antibody (Cat. No. 9167S; diluted 1:1,000) were obtained from CST (Boston, USA).

Techniques: Infection, Control, Bioprocessing, Negative Control, Positive Control, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Solvent, Two Tailed Test

Journal: PLOS Pathogens

Article Title: HCMV encoded UL84 hijacks FHL2 to suppress type I interferon production and enhance viral replication

doi: 10.1371/journal.ppat.1013895

Figure Lengend Snippet: (A) FHL2 associated with IFN-β PIC complex. HFFs were infected with HCMV (MOI = 1) for 6h, and cross-linked with 1% formaldehyde for 15min before CHIP. NC as non-specific control, normal rabbit IgG as a negative control, anti-IRF3 positive control, anti-FHL2 as an inductive antibody. (B) FHL2 associated with IFN-β PIC complex. HFFs and FHL2-knockot cell line were infected with HCMV (MOI = 1) for 6h, and cross-linked with 1% formaldehyde for 15min before CHIP. Normal rabbit IgG as a negative control, anti-IRF3 positive control. (C) Molecular docking simulation of the complex of IRF3-FHL2-TBP. The structure of the IRF3, FHL2 and TBP was downloaded from the Protein Data Bank, green refers to FHL2, grey refers to TBP, and pink refers to IRF3. (D) Co-IP analysis of FHL2 interactions with TBP, IRF3, c-Jun, or p300. Expression plasmids of Flag-FHL2, TBP, IRF3, p300 and c-Jun were transiently transfected into HEK293T cells for 48 h, and then the total cell lysates were prepared and immunoprecipitated with anti-Flag, anti-TBP, anti-IRF3, anti-c-Jun or anti-p300 antibodies, followed by immunoblot analysis. (E) Co-IP analysis of FHL2 (ΔLIM2) interactions with TBP, IRF3, c-Jun, or p300. Expression plasmids of Flag-FHL2 (ΔLIM2), TBP, IRF3, p300 and c-Jun were transiently transfected into HEK293T cells for 48 h, and then the total cell lysates were prepared and immunoprecipitated with anti-Flag, anti-TBP, anti-IRF3, anti-c-Jun or anti-p300 antibodies, followed by immunoblot analysis. (F) Effect of different mutations on the interaction of FHL2 with TBP. HEK293T cells were transiently transfected with Flag-tagged FHL2 (full-length or the four single-point mutants) and pcDNA3.1-TBP for 48 h before Co-IP analysis was performed. The displayed images were representative ones from three independent experiments.

Article Snippet: The antibodies against human IRF3 rabbit monoclonal antibody (Cat. No. 11904; diluted 1:1,000), human pIRF3 rabbit monoclonal antibody (Cat. No. E6F7Q; diluted 1:1,000), c-Jun mouse monoclonal antibody (Cat. No. 2315; diluted 1:1,000), STAT1 rabbit monoclonal antibody (Cat. No. 9172S; diluted 1:1,000) and pSTAT1 rabbit monoclonal antibody (Cat. No. 9167S; diluted 1:1,000) were obtained from CST (Boston, USA).

Techniques: Infection, Control, Negative Control, Positive Control, Co-Immunoprecipitation Assay, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: PLOS Pathogens

Article Title: HCMV encoded UL84 hijacks FHL2 to suppress type I interferon production and enhance viral replication

doi: 10.1371/journal.ppat.1013895

Figure Lengend Snippet: (A) UL84 interacts with FHL2 to repress IFN-β transcription. HFFs were transiently transfected with expression plasmids for pGL3-luci-IFN-beta, FHL2, UL84, UL84 (Δ400-460) for 24 h, HCMV (MOI = 1) reinfection of cells for 6h before luciferase assay. (B) The UL84-overexpressing cell line was infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, IRF3, pIRF3, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (C) HFF cells were transfected with the Con-RNAi, UL84-RNAi-#1, and then infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, IRF3, pIRF3, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (D) HFF cells were transfected with the Con-RNAi, UL84-RNAi-#2, and then infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, IRF3, pIRF3, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (E) Co-IP analysis of UL84 interaction with FHL2, IRF3, c-Jun, or p300. Expression plasmids of UL84, FHL2, TBP, IRF3, p300 and c-Jun were transiently transfected into HEK293T cells for 48 h, and then the total cell lysates were prepared and immunoprecipitated with anti-UL84, anti-FHL2, anti-IRF3, anti-c-Jun or anti-p300 antibodies, followed by immunoblot analysis. (F) Co-IP analysis of UL84 (Δ400-460) interaction with FHL2, IRF3, c-Jun, or p300. Expression plasmids of UL84(Δ400-460), FHL2, TBP, IRF3, p300 and c-Jun were transiently transfected into HEK293T cells for 48 h, and then the total cell lysates were prepared and immunoprecipitated with anti-Myc, anti-FHL2, anti-IRF3, anti-c-Jun or anti-p300 antibodies, followed by immunoblot analysis. (G) HEK293T cells were transfected with pCDNA3.1-FHL2, pCDNA3.1-IRF3, pCDNA3.1-p300, and pCDNA3.1-c-Jun plasmids, along with increasing amounts of the pCDNA3.1-UL84 expression vector for 48 h. Co-IP analysis of UL84 interaction with FHL2, IRF3, c-Jun, or p300. (H) Effect of different mutations on the interaction of FHL2 with UL84. HEK293T cells were transiently transfected with Flag-tagged FHL2 (four single point mutants) and pcDNA3.1-UL84 for 48 h before Co-IP analysis. (I) Co-IP analysis of UL84 interactions with TBP. HEK293T cells were transiently transfected with the indicated plasmids for 48 h, using as the indicated anti-UL84, anti-Flag or anti-TBP antibody before Co-IP analysis was performed. (J) Molecular docking simulation of the complex of UL84-FHL2-TBP. The structure of the IRF3, FHL2 and TBP was downloaded from the Protein Data Bank, green refers to FHL2, grey refers to TBP, and blue refers to UL84. The displayed images were representative ones from three independent experiments. For all figures, statistical analyses were performed using two-tailed t-test. Differences were considered statistically significant when * denoted p < 0.05, ** denoted p < 0.01, *** denoted p < 0.001, and **** denoted p < 0.0001.

Article Snippet: The antibodies against human IRF3 rabbit monoclonal antibody (Cat. No. 11904; diluted 1:1,000), human pIRF3 rabbit monoclonal antibody (Cat. No. E6F7Q; diluted 1:1,000), c-Jun mouse monoclonal antibody (Cat. No. 2315; diluted 1:1,000), STAT1 rabbit monoclonal antibody (Cat. No. 9172S; diluted 1:1,000) and pSTAT1 rabbit monoclonal antibody (Cat. No. 9167S; diluted 1:1,000) were obtained from CST (Boston, USA).

Techniques: Transfection, Expressing, Luciferase, Infection, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Plasmid Preparation, Two Tailed Test

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Stearic acid cooperative effect with TNF-α for MIP-1α/CCL3 production requires IRF3. ( A ) THP-1 monocytic cells were transfected with either control or IRF3 siRNA and incubated for 36 h. Real time PCR was done to measure IRF3 expression. ( B , C ) IRF3 deficient THP-1 cells were stimulated with stearic acid and TNF-α. MIP-1α/CCL3 expression was determined. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. ( D ) IRF3 activity reporter monocytic cells were treated with stearic acid (200 µM) or 0.1% BSA (control) or TNF-α (10 ng/mL) or in combination. Culture media were collected after 24 h. Cell culture media were assayed for luciferase activity representing the degree of IRF3/ISRE activation using Quanti-Luc medium. ( E ) Western blot analysis showed that stearic acid induced IRF3 phosphorylation in a time dependent manner in THP-1 monocytes, verifies the role of IRF3 in the cooperative effect of stearic acid in the TNF-α mediated production of MIP-1α/CCL3. ( F ) Expression of phosphorylated IRF3 is shown as determined by densitometry of western blot bands. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Transfection, Control, Incubation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Cell Culture, Luciferase, Activation Assay, Western Blot, Phospho-proteomics

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Polyinosinic-polycytidylic acid (poly I:C) act as a substitute of stearic acid cooperative effect on MIP-1α/CCL3 production. ( A , B ) THP-1 cells were treated (via transfection) with poly I:C (5 µg) for 2 h and then incubated with BSA (control) or stearic acid or TNF-α for 24 h. MIP-1α/CCL3 mRNA and protein were determined. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. All data are expressed as mean ± SEM ( n ≥ 3); * P < 0.05; ** P < 0.01, when compared with stearic acid or TNF-α alone. ( C , D ) Western blot analysis showed that individuals with obesity have significant higher levels of phospho-IRF3 in monocytes compared to lean individuals ( P = 0.0179).

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Transfection, Incubation, Control, Western Blot

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Schematic illustration of signaling pathways underlying the cooperative relationship between stearic acid and TNF-α for MIP-1α/CCL3 production. Blocking TLR4/IRF3 signaling pathways significantly suppress the cooperative production of MIP-1α/CCL3 by stearic acid/TNF-α. TLR: Toll like receptor; TNF-α: Tumor necrosis factor α; MyD88: Myeloid differentiation factor 88; IRAK: Interleukin-1 receptor-associated kinase; TRIF: TIR domain–containing adapter-inducing IFN-β; IRF3: Interferon regulatory factor-3; TBK: TANK-binding kinase 1; TRAM: Toll-receptor-associated molecule; TRAF6: tumor-necrosis factor receptor-associated factor 6: IKK-Iκ-B kinase; NF-kB: Nuclear factor-κB; TRADD: Tumor necrosis factor receptor 1-associated death domain protein; RIP: receptor-interacting protein; NEMO: NF-kappa-B essential modulator. BioRender.com was used for .

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Protein-Protein interactions, Blocking Assay, Binding Assay