Figure 1.Ulk1 is not required for adipogenesis in 3T3-L1 cells. (A) Knockdown of autophagy genes, except Ulk1, suppresses adipogenesis. 3T3-L1 cells were transduced by shRNAs specific to each autophagy gene. As a control, 3T3-L1 cells were transduced by scrambled (SC) shRNA. The shRNA-transduced cells were induced to be differentiated into adipocytes in medium containing methylisobutylxanthine, dexamethasone, and insulin as described previously.³⁷ At day 8, cells were stained with Oil Red O. (B) Quantitative analysis of intracellular triglyceride content at day 8. Triglycerides were extracted from Oil Red O-stained cells with isopropanol, and the contents were analyzed by measuring the optical density at 490 nm. Values were normalized by protein concentration and presented as mean ± SD *P < 0.01 relative to shRNA-SC cells. (C) Quantitative analysis of intracellular triglyceride content over the period of differentiation. Mean ± SD from 3 independent experiments. *P < 0.01 relative to scrambled control cells. (D) western blot analysis of CEBPA and the gene knockdown in shRNA-transduced cells. (E) ULK1 and ULK2 reciprocally regulate their expression in adipocytes. The expression levels of ULK1, ULK2, and ATG5 in shRNA-transduced adipocytes at day 8 were analyzed by western blotting and quantitative real time RT-PCR. (F) Knockdown of both Ulk1 and Ulk2 suppresses adipogenesis to a similar extent as knockdown of Ulk2 alone. (G) Quantitative analysis of intracellular triglyceride content over the period of differentiation. Values are mean ± SD from 5 independent experiments. *P < 0.01 relative to control cells. (H) western blot analysis of the effects of knocking down both Ulk1 and Ulk2 on the expression of PPARG and CEBPA.