Figure 2. (A) Differentiation of 3T3-L1 cells in shNegative and Sirt1 knockdown cells. Oil O red staining image (x100). Red staining was extracted with isopropanol and absorbance was measured at 400 nm. There were no apparent effects of Sirt1 knockdown on fat accumulation. (B, C) Proteins indicating adipocyte differentiation and AMPK, LKB1, and Sirt1 signaling assessed by western blot. Sirt1 knockdown was evident at 10 PID; however, pACC/tACC and pAMPK/tAMPK showed no apparent suppression of AMPK activity. pSerLKB1/LKB1 showed suppression of LKB1 at 6 and 10 PID. A similar suppression trend was observed in PGC1α/HSP90. n=4, *p<0.05 vs shNegative. (D) Effect of Sirt1 knockdown on LKB1 activity and ATP/ADP ratio. LKB1 activity was assessed by LKBtide phosphorylation in immuno-precipitated samples. Sirt1 knock-down causes LKB1 activity downregulation. (n-6, *p<0.05). ATP/ADP ratios were significantly lower with Sirt1 knock-down (n=6. P<0.05). Similar experiments were repeated three times.