Figure 4. (A, B) Western blots of proteins related to and AMPK, LKB1, and Sirt1 signaling and mitochondrial dysfunction at PID 15 in shNegative, shSirt1, and shSirt1+LKB1 K48R cells. p-T172 AMPK and AMPK alpha2 were increased despite decreased p-MARK (LKB1 substrate) in Sirt1 knockdown cells. LKB1 K48R increased p-MARK and decreased p-T172 AMPK. It also significantly upregulated mitochondrial complex I proteins. The ratio of phospho-to-total MARK: A decrease in this ratio indicates a decrease in LKB1 activity caused by Sirt1 knockdown and upregulated by LKB1 K48R expression. Complex 1 expression normalized by HSP90: As seen in (A), expression of LKB1 K48R not only ameliorates LKB1 activity, it also increases mitochondrial complex I expression. (n=4, *p<0.05 vs shNegative). AMPK activity was increased by shSirt1 cells: AMPK activity was assessed by using 32P-ATP in vitro in the SAMS peptide assay. (*p<0.05 vs shNegative, n=6).