Blockade of CaMKII activation with KN62 or silencing of CaMKII expression attenuates TLR4-activated proinflammatory cytokine and type I IFN production in macrophages. (A) Mouse peritoneal macrophages (4 × 105) were pretreated with 15 μM KN62 for 30 minutes followed by stimulation with 0.1 μg/mL LPS for the indicated time. The production of IL-6, TNF-α, or IFN-β was measured by ELISA, and IFN-4α mRNA expression was measured by Q-PCR. (B) (Top) RAW264.7 cells were transfected with control small RNA (Ctrl) or CaMKII-α siRNA 1. After 48 hours, CaMKII-α and β-actin expression in the cells was detected by immunoblot. (Bottom) CaMKII-α–silenced RAW264.7 cells were transfected with CaMKII-α full-length expression vector. After 36 hours, CaMKII-α and β-actin expression in the cells was detected by immunoblot. Similar results were obtained in 3 independent experiments. (C) Mouse peritoneal macrophages (4 × 105) were transfected with control small RNA (Ctrl) or CaMKII-α siRNA1. After 48 hours, the cells were stimulated with 0.1 μg/mL LPS for the indicated time. IL-6, TNF-α, or IFN-β in the supernatants was measured by ELISA, and IFN-4α mRNA expression was measured by Q-PCR. (D) RAW264.7 cells (1.5 × 105) were transfected with control small RNA (Ctrl) or CaMKII-α siRNA1. After 36 hours, the cells were transfected with full-length CaMKII-α plasmid. Thirty-six hours later, the cells were stimulated with 0.1 μg/mL LPS for the indicated time. IL-6 and IFN-β in the supernatants were measured by ELISA. Data are shown as mean plus or minus SD of 3 independent experiments. **P < .01.
