Figure 4.

JUN activation was crucial for autophagy induction and anchorage-independent growth inhibition by ISO treatment. (A and D) The stable transfectants, UMUC3 (TAM67) vs. UMUC3 (Vector) or UMUC3 (shJUN) vs. UMUC3 (Nonsense) cells, were treated with 10 μM ISO for 24 h. The cells were then extracted and the cell lyses were subjected to western blotting for determination of JUN phosphorylation, SESN2 induction and LC3-II generation as indicated. (B and E) Representative images of anchorage-independent growth of UMUC3 (TAM67) vs. UMUC3 (Vector) or UMUC3 (shJUN) vs. UMUC3 (Nonsense) in the absence or presence of various concentrations of ISO were visualized and captured using a microscope. (C and F) The colony formation was counted under a microscope with more than 32 cells of each colony, and the results presented as colonies/10⁴ cells. The bars indicate mean ± SD from 3 independent experiments. The symbol (*) shows a significant decrease from the vehicle control (p < 0.05).