FIGURE 7.

Inhibition of CaMKKβ abolishes NO-enhanced AMPK activation in LKB1-deficient HeLa S3 cells and in HUVECs. A, HeLa S3 cells were pretreated with STO-609 (0.5 μm; 30 min) prior to stimulation with SNP (50 μm, 1 h). B and C, AMPK phosphorylation induced by SNP in HeLa S3 cells was significantly inhibited by pretreatment with CaMKKβ siRNA. HeLa S3 cells were preincubated with synthetic RNA duplexes targeted to human CaMKKβ or a control RNA duplex. Following siRNA treatment, cells were stimulated with SNP (50 μm, 1 h), and AMPK activation was monitored using antibody against phospho-AMPK (Thr-172). Expression of AMPK and CaMKKβ was measured by Western blot analysis using antibodies against AMPK-α and CaMKKβ, respectively. D and E, AMPK phosphorylation induced by SNP in HUVECs was significantly inhibited by pretreatment with CaMKKβ siRNA as described above. Cell lysates were subjected to Western blot analysis using antibodies against phospho-AMPK (Thr-172), AMPK-α, or β-actin. Typical blots and densitometric analysis (means ± S.E., n >= 3) are presented. *, p < 0.05 versus samples pretreated with control siRNA.