Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK-AMPK-SIRT1 Signaling Pathway

Figure 5

AMPK is activated downstream of CaMKKβ for the negative control of the LPS induced IL-12 production in RAW 264.7 cells. (a) Cells were treated with LPS alone or with calcium-free DMEM, 2 mM CaCl₂, 5 mM EGTA, or 1 μM STO-609 for 30 min. Protein levels of AMPKα and pAMPKα were detected by western blot. versus medium; or versus LPS (). (b) Cells were treated with LPS or LPS plus 1 mM AICAR in calcium-free DMEM (left) or with LPS, LPS plus 5 μM Compound C, and LPS plus 1 mM AICAR in normal DMEM (right). Supernatants IL-12 p40 and IL-12 p70 were detected by ELISA. and versus LPS (). (c) Cells were treated with NC siRNA or AMPKα siRNA for 24 h. mRNA and protein levels of AMPKα were detected by real-time PCR or western blot. versus NC siRNA () for mRNA. (d) siRNA-treated cells were further stimulated by 100 ng/mL LPS for 24 h. Supernatants IL-12 p40 and IL-12 p70 (24 h) were detected. and versus LPS for each cytokine ().

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