Figure 2

Cell death induced by AEZS-136 correlates with JNK-dependent ROS production.

(a) Live cell imaging of ROS production in AEZS-136 (10 μM)-treated L-540 and L-428 cells. Representative image captured 120 minutes after drug treatment are shown. Objective lens, 1.0 NA oil objective; original magnification, 40x. (b) L-540 cells were pre-labeled with H2DCFDA and then treated with the ROS inhibitor YCG063 (20 μM). After 5 min, the indicated samples were stimulated with 10 μM AEZS-136 or DMSO vehicle. ROS inhibition was quantified using Cell R 5 software. Representative image captured 30 min after drug treatment are shown. Objective lens, 1.0 NA oil objective; original magnification, 40x. (c,d) L-540 cells were treated with or without the ROS inhibitor YCG063 (20 μM) or necrostatin-1 (60 μM). After 1 hour, the appropriate samples were stimulated with 10 μM AEZS-136 or DMSO vehicle for 48 hours and (c) ROS generation or mitochondrial membrane depolarization were assessed. The experiments were repeated twice with similar results. Representative dot plots are shown. (d) Cell death was measured via Annexin-V/PI double-staining. The mean (±SEM) values represent three independent experiments. *P ≤ 0.0001 compared with the cells treated with AEZS-136 and AEZS-136 plus YCG063 or necrostatin-1. Vehicle-treated controls are shown. (e) Western blot analysis of extracts from HL cells treated with 10 μM AEZS-136 or DMSO vehicle for the indicated periods. Total levels of JNK and β-actin are shown. (f) L-540 cells were treated with or without the ROS inhibitor YCG063 (20 μM), necrostatin-1 (60 μM), or the JNK inhibitor SP600125 (12.5 μM). After 1 hour, the indicated samples were stimulated with 10 μM AEZS-136 or DMSO vehicle for 15 min. JNK expression was measured via flow cytometry. (g) L-540 cells were incubated for 1 hour in the presence or absence of the JNK inhibitor SP600125 (12.5 μM). Following exposure to AEZS-136 (10 μM, 24 hours) or DMSO vehicle, mitochondrial membrane depolarization, cell death, and ROS generation were analyzed via flow cytometry. (h) Proposed model for the mechanism of action of AEZS-136. Activation is represented by continuous black lines, and inhibition is represented by dotted gray lines.