Figure 1.
Deficiency in HDAC4 arrestingly facilitates the production of IFN-β. (A) Quantitative RT-PCR analysis of HDAC4 mRNA (top) and immunoblot (IB) analysis of HDAC4 and GAPDH as loading control throughout (bottom). (B and C) HEK293T cells (2 × 105) were transfected for 36 h with an IFN-β firefly luciferase reporter (IFN-β-Luc), along with non-targeting control siRNA (siNC) or siRNA targeting HDAC4 (siHDAC4-1, -2, -3, -4), and then left uninfected (UI) or infected for another 8 h with SeV (50 hemagglutination units per ml). (B) Luciferase assay analysis of IFNB1 promoter activity. Luciferase reporter activity is normalized to that of renilla luciferase. (C) ELISA of IFN-β. (D) Quantitative RT-PCR analysis of ISG15, ISG54, and ISG56 mRNA in HEK293T cells transfected for 36 h with siNC or siHDAC4 (siHDAC4-4) and then infected for another 8 h with SeV. The reference gene actin serves as a loading control. (E and F) Luciferase assay of IFNB1 promoter activity (E) and quantitative RT-PCR analysis of IFNB1 mRNA (F) in HEK293T cells transfected for 36 h with siNC or siHDAC4-4 and then treated for 8 h with SeV, VSV, HSV-1, and poly(I:C). (G) ELISA of IFN-β in culture medium of siNC or siHDAC4-treated BMDMs (4 × 105) incubated for 5 days with the cytokine M-CSF and then left unstimulated (US), infected with SeV (12 h), VSV (12 h), or HSV-1 (18 h), or stimulated with LPS (10 h) or poly(I:C) (10 h). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired t-test). Data are from three independent experiments (mean and SD of three independent biological replicates per group) or are representative of three independent experiments (A, bottom).