Simultaneous knockdown of PKACα and PKACβ potentiates RNA virus-triggered signaling.

(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.