Effects of Lusianthridin in intact cells.A, U2OS cells were treated with Lusianthridin, AICAR, or A-769662 for 30 min before lysing and performing an HTRF assay with the phospho-ACC (pACC) assay kit, and results are presented as the ratio of 665/620 nm (pACC HTRF). B, Western blot analysis of U2OS cells treated with Lusianthridin or AICAR with the indicated antibodies. Representative blots of n = 3 are shown along with the quantification of the pACC and pThr172 antibodies. C, U2OS cells were treated with varying concentrations of Lusianthridin for 30 min with or without prior treatment with STO-609 (10 μg/ml, 1 h). pACC was determined by performing an HTRF assay with the pACC assay kit, and results are presented as the ratio of 665/620 nm (pACC HTRF). A representative blot is displayed for the ionomycin-treated cell lysates using an automated capillary immunoblotting system (Sally Sue). AICAR, 5-aminoimidazole-4-carboxamide riboside; HTRF, Homogenous Time-Resolved Fluorescence.