Figure 1.
Identification of HSPD1 as an interacting protein of IRF3.
A. FLAG-tagged IRF3 was exogenously expressed in the HEK293T cell line, and then the cells were activated by overexpression of RIG-IN or mock-transfected with the respective vector. The proteins were extracted and purified with anti-FLAG agarose gel. Next, the purified proteins were analyzed by LC-MS/MS. In comparison with the control sample, 53 peptides and 18 corresponding unique peptides of HSPD1 (coverage: 39.8%) indicated in red were identified in the induced samples. In the blue frame is mitochondrial transit peptide and ΔHSPD1 is lack of the mitochondrial transit peptide. B. Myc-tagged HSPD1 was co-expressed with FLAG-tag, FLAG-tagged IRF3, or FLAG-tagged IRF3/5D in HEK293T cells. The proteins were co-precipitated with anti-FLAG agarose gel and then probed with antibodies against Myc-tag or FLAG-tag. C. FLAG-tagged IRF3/5D was co-expressed with Myc-tagged HSPD1, Myc-tagged HSPD1 without the mitochondrial transit peptide or control vector in HEK293T cells. The proteins were co-precipitated with anti-FLAG agarose gel and then probed with antibodies against Myc-tag or FLAG-tag.
Figure 2.
Co-localization of IRF3 and HSPD1.
A. HeLa cells were transfected with the MAVS or control plasmid. At 8 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 and mouse antibody against HSPD1 and further incubated with goat anti-mouse IgG H&L (Cy3) and goat anti-rabbit IgG H&L (FITC). Nuclei were stained with DAPI. B. HeLa cells were transfected with the MAVS or control plasmid. At 16 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 (phospho S386) and mouse antibody against HSPD1 and further developed with goat anti-mouse IgG H&L (Cy3) and goat anti-rabbit IgG H&L (FITC). Nuclei were stained with DAPI.
Figure 3.
Overexpression of HSPD1 facilitated IFN-β induction.
A. The expression of Myc-tagged HSPD1 was detected using an antibody against the Myc tag. B and E. The HEK293T cells were co-transfected with the luciferase reporter plasmid pIFN-β-Luc or pIRF3-Luc, the Renilla luciferase plasmid phRL-TK and plasmid encoding Myc-tagged HSPD1 or control vector. After incubation for 24 h, the cells were infected with SeV or mock-treated with the same buffer. After infection for 8 h, all of the cells were collected and the luciferase activity was measured using a dual-luciferase assay system. Data represent the relative firefly luciferase activity normalized to the Renilla luciferase activity. C and F. The HEK293T cells were co-transfected with 200 ng of the luciferase reporter plasmid pIFN-β-Luc or pIRF3-Luc, 20 ng of the Renilla luciferase plasmid phRL-TK (Promega), 200 ng of plasmid encoding Myc-tagged HSPD1 or control vector, and 200 ng of plasmid encoding RIG-IN or control vector for 36 h. The cells were then collected, and the luciferase activity was measured using a dual-luciferase assay system (Promega) and a luminometer (Turner BioSystems, CA). D. The HEK293T cells were co-transfected with 200 ng of the luciferase reporter plasmid p NF-κB -Luc or pIRF3-Luc, 20 ng of the Renilla luciferase plasmid phRL-TK (Promega), 400 ng of plasmid encoding Myc-tagged HSPD1 or control vector. After incubation for 24 h, the cells were infected with SeV or mock-treated with the same buffer. After infection for 8 h, all of the cells were collected and the luciferase activity was measured using a dual-luciferase assay system.
Figure 4.
Knockdown of endogenous HSPD1 impaired IFN-β induction.
A. Hela cells transfected with HSPD1 shRNA or control shRNA showed a significant reduction of HSPD1 expression in cells treated with HSPD1 shRNA compared with control shRNA in a Western blot assay. B. SeV infection activated the IFN-β luciferase reporter with control shRNA, and the induction was significantly inhibited with HSPD1 shRNA. C. Hela cells transfected with HSPD1 shRNA displayed significant inhibition of the expression of HSPD1 in comparison with control shRNA in a quantitative PCR assay. D. Knockdown of endogenous HSPD1 significantly inhibited the induction of IFN-β mRNA induced by overexpression of MAVS for 8 h in a quantitative PCR assay. E. HEK293T cells transfected with HSPD1 shRNA displayed significant inhibition of the expression of HSPD1 in comparison with control shRNA in a quantitative PCR assay. F. Knockdown of endogenous HSPD1 significantly inhibited the induction of IFN-β mRNA induced by SeV infection for 8 h in a quantitative PCR assay. G. Knockdown of endogenous HSPD1 significantly inhibited the expression of IP-10 induced by SeV infection for 8 h in a quantitative PCR assay.
Figure 5.
HSPD1 regulated IFN-β induced by various components of the signaling.
HEK293T cells were co-transfected with the luciferase reporter plasmid pIFN-β-Luc, the Renilla luciferase plasmid phRL-TK, HSPD1 plasmid or control plasmid, and RIG-IN, MDA5-IN, MAVS, TBK1, IKKε, IRF3-5D or control plasmid. At 32 h post-infection, all of the samples were collected and the luciferase activity was measured using a dual-luciferase assay system. Data represent the relative firefly luciferase activity normalized to the Renilla luciferase activity.
Figure 6.
Overexpression of HSPD1 facilitated the activation of IRF3.
A and C. HEK293T cells were transfected with plasmid encoding Myc-tagged HSPD1 or control vector, shRNA or control vector. After incubation for 24 h, the cells were infected with SeV. At 8 h post-infection, the proteins were extracted and analyzed by SDS-PAGE and further blotted with antibody against IRF3, phospho S386-IRF3 or HSPD1. GAPDH served as an internal control. B and D. The proteins were analyzed by native PAGE and then blotted with antibody against IRF3.