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 4

Activation of CaMKKβ mediates the inhibition of calcium-dependent IL-2 production in RAW 264.7 cells. (a) Cells were treated with LPS in normal DMEM, calcium-free DMEM, calcium-free DMEM with 2 mM CaCl₂, or DMEM with 5 mM EGTA for 30 min. Protein levels of CaMKKβ and pCaMKKβ were detected by western blot. Comparison was made for pCaMKKβ/CaMKKβ. versus medium; and versus LPS (). (b) Cells were treated with LPS or LPS plus 1 μM STO-609 for 24 h. Supernatants IL-12 p40 and IL-12 p70 were detected. versus LPS for each cytokine (). (c) Cells were treated with negative control siRNA (NC siRNA) or CaMKKβ siRNA for 24 h. mRNA and protein levels of CaMKKβ 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 were detected. versus LPS for each cytokine ().

(a)

(b)

(c)

(d)