Figure 5. ZA treatment downregulates Mcl-1 expression by ROS to promote cell apoptosis.

(A) RAW264.7 cells were treated with ZA (100 μM) for the indicated time. Western blotting determined the expression of Mcl-1, Bcl-xL, and Bcl-2. (B) Mcl-1 was overexpressed in RAW264.7 cell following transfection with pcDNA₃-HA-Mcl-1, which was confirmed by a flow cytometry. The cells were then treated with ZA (100 μM) for 2 days. pcDNA₃-HA was used as the vector control. A PI-based flow cytometric analysis was used to measure apoptosis. ***p < 0.001 compared to vector; ^###p < 0.001 compared to vector with ZA treatment. RAW264.7 cells were treated with ZA (100 μM) in the presence of the ROS inhibitor NAC (20 mM). Western blotting analysis revealed the expression of Mcl-1 (C), cleaved caspase-3 (C. Caspase-3), and caspase-3 (D). β-actin was used as an internal control. One representative data set with the relative optical density from three individual experiments is shown. Full-length blots/gels are presented in Supplementary Figure 4.