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 7

SIRT1 selectively attenuates transcription activity of NF-κB and suppresses IL-12 expression in LPS-stimulated RAW 264.7 cells. (a) Cells were treated with 1 μM STO-609 alone or combined with 2 μM SRT1720 or 1 mM AICAR for 30 min before 100 ng/mL LPS stimulation (left). Cells were treated with 5 μM Compound C alone or with 2 μM SRT1720. Cells were also treated with 1 mM AICAR alone or with 2 μM EX527 (right). Supernatants were collected at 24 h and IL-12 p40/IL-12 p70 (c) and IL-6 (d) levels were detected by ELISA (). (b) Cells were transfected with plasmid pGL-luc2P/NF-κBRE for 24 h and further treated with 100 ng/mL LPS in normal DMEM, calcium-free DMEM, DMEM with 2 mM CaCl₂, DMEM with 1 mM AICAR, or DMEM with 2 μM SRT1720 for 6 h. Relative luciferase activity of NF-κB against medium was presented. versus medium; versus LPS (). Cells were also transfected with plasmids pAP1-luc and pIRF3-luc for 24 h and further treated with 100 ng/mL LPS and LPS plus calcium-free DMEM for 6 h. Relative luciferase activity of AP-1 and IRF3 was detected (). (c) Cells were treated with LPS alone with 5 μM SR11302 or 10 μM wedelolactone (Weldo) for 24 h. Supernatant IL-12 p40/p70 and IL-6 levels were detected by ELISA. versus LPS (). (d) Cells were treated with LPS, LPS with calcium-free medium, or LPS with calcium-free medium alone with 5 μM SR11302 or 10 μM wedelolactone (Weldo) for 24 h. Supernatant IL-12 p40/p70 and IL-6 levels were detected by ELISA. versus LPS; versus LPS plus calcium-free medium ().

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