Figure 7.

The effect of dynasore is mediated by ROS. (A) Western immunoblot of NF-κB-p-p65 and IκBα: cells were pre-incubated with NAC (100 mM) or vehicle for 30 min in HBSS, followed by dynasore (80 μM) or DMSO for additional 15 min. Cells were then treated in 2% FBS with the inhibitors for the times indicated. NF-κB-p-p65 and IκBα representative blots were from two independent separate experiments. Therefore, loading was ascertained by reprobing with p65 or GAPDH respectively. Note the inhibition of dynasore induction of NF-κB-p-p65 and IκBα degradation with NAC, suggesting that dynasore induction is mediated by ROS. (B) NF-κB-luciferase: cells were transfected and maintained as described in the Methods section (n = 3). (C) H₂O₂ assay: cells were plated and treated as described earlier. Aliquots of conditioned media were assayed for H₂O₂ using Amplex red kit. Note the enhanced levels of H₂O₂ in dynasore-treated cells (n = 3). (D) Western immunoblot showing that H₂O₂ increase p-DRP1 and reduces MAVS monomer levels. Cells were treated with 300 μM H₂O₂ for 15 min and processed for Western blotting for p-DRP1, MAVS and GAPDH. (E, F) Densitometry of blots shown in panel D: bands were scanned, corrected for GAPDH levels and normalized to the corresponding controls. Increase in p-DRP1 (E) and decrease in MAVS (F) are statistically different (P < 0.03; n = 3) from DMSO-treated control. (G) NF-κB-luciferase: cells were pretreated for 20 min with Mito-Tempo (25 μM; inhibitor of mitochondrial ROS), apocynin (600 μM; inhibitor of NADPH oxidase) or DMSO in HBSS, followed by treatment with dynasore (80 μM) for another 15 min. Cells were then treated with the inhibitors in 2% FBS overnight and processed for luciferase measurement. Both ROS inhibitors reduced dynasore-induced activation of NF-κB-luciferase (anova: P < 3 × 10⁻⁶; d.f. within groups = 12).