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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: J Neuroimmune Pharmacol. 2017 May 2;12(4):624–660. doi: 10.1007/s11481-017-9746-5

Figure 4. Mitochondrial ROS drives the NLRP3 inflammasome activation, c-Abl and PKCδ phosphorylation and inflammatory mediators release in LPS-primed rotenone stimulated BV2 cells.

Figure 4

(A) BV2 microglial cells were pretreated with MitoTEMPO (200μM or 500μM) for 1 hour, followed by priming with LPS (1μg/ml) for 3 hours. The primed cells were subsequently stimulated with ROT (0.5μM) for 6h. The reactive oxygen species production post treatment was quantified fluorometrically using CM-H2DCFDA dye. The data is expressed as percent of control and is represented as mean ± S.E.M.; n=8. *p<0.05, **p<0.01 and ***p<0.001 versus control; a ap<0.01 vs ROT; while # #p<0.01 denotes significance between LPS-primed ROT treated groups with and without MitoTEMPO (B, C) The whole cell lysate were immunoblotted for NLRP3, caspase-1 (cleaved) (B), p-c-Abl (Y412), p-c-Abl (Y245), c-Abl, p-PKCδ (Y311) and PKCδ (C). β actin was used as loading control. The densitometric analysis of normalized band intensity for each treatment group was plotted on histogram and is represented as mean ± S.E.M. for each experiment performed with n=4. *p<0.05, **p<0.01 and ***p<0.001 versus control; ap<0.05 and a ap<0.01 represents significant difference between ROT vs LPS-primed ROT treatment groups; while #p<0.05, # #p<0.01 and # # #p<0.001 denotes significance between LPS-primed ROT treated groups with and without MitoTEMPO; n.s.: not significant. (D) The cell supernatant was assessed for secreted IL-1β and IL-18 by ELISA. The data is represented as mean ± S.E.M.. n=6. ***p<0.001 and ****p<0.0001 compared with control; a a a ap<0.0001 represents significant difference between ROT vs LPS-primed ROT treatment groups; while # #p<0.01 and # # #p<0.001 denotes significance between LPS-primed ROT treated groups with and without MitoTEMPO.