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. 2020 Dec 8;12(12):3679. doi: 10.3390/cancers12123679

Figure 3.

Figure 3

Induction of autophagy initiation by CWS-loaded formulations via mTOR signaling inhibition and AMPK activation in bladder cancer cells. (A) Cells were treated with 1 μg/mL of CWS-loaded formulations for 24 h, and the lysates were subjected to Western blotting. (B) 5637 cells were treated with 1 μg/mL of CWS or CWS-Nano-CL for 24 h, and then, the phosphorylated AMPK to total AMPKα protein expression ratio was assessed by Western blotting. As they are autophagy-initiation-related proteins, the ratio of phosphorylated ULK1 (Ser555 and Ser757) to total ULK protein expression was detected by Western blotting. (C) Cells were treated with 1 μg/mL of CWS, CWS-Nano-CL, or 0.1 µM bafilomycin (Bafilo) for 5 min, 3 h, or 24 h, and the cell lysates were subjected to Western blot analysis using anti-LC3B-I/II antibodies. Actin was used the loading control. The blots are representative of three independent experiments. The quantification graphs represent p-mTOR/mTOR, p-4EBP1/4EBP1, p-AMPK/AMPK, pULK1(Ser555)/ULK1, pULK1(Ser757)/ULK1, and LC3B-II/Actin ratios determined by densitometric analyses. All expression ratios were normalized to the untreated group.