Figure 4

JY-1-106 activates apoptosis via intrinsic pathway. (A) 5 × 10⁶ A549 cells were transfected with Mcl-1 siRNAs or control siRNAs for 24 hours and then exposed to 5 μM JY-1-106 or 5 μM ABT-737 for 12 hours. Cleaved PARP and PARP proteins were then evaluated by western blotting using anti-PARP antibodies. Immunoblotting for β-actin was performed to normalize the loading. (B) 5 × 10⁶ I45 cells were exposed to 5 μM JY-1-106 or DMSO control for 12 hours. Bax and dimerized Bax were then assayed by western blotting using anti-Bax antibodies. (C) Determination of mitochondrial membrane potential through JC-1 staining and detection using fluorescent microscopy. I45 cells were exposed to 5 μM JY-1-106 or DMSO control for 12 hours. JC-1 dye was then loaded into the medium for the final 20 minutes of culturing. After JY-1-106 treatment, the mitochondrial membrane potential was found to be interrupted, as evidenced by the migration of JC-1 dye from the mitochondria into the cytoplasm of treated cells, and the subsequent reduction in the mitochondrial red fluorescence signals. (D) 2 × 10⁶ A549 cells were exposed to 5 μM JY-1-106 or DMSO control for 12 hours. These cells were then collected, fixed and subjected to a TUNEL reaction. Apoptosis signals were measured using flow-cytometry. The data shown are the average of triplicate assessments for each condition. The differences between these two groups were measured using the Student’s t-test (p < 0.01). These experiments were repeated twice.