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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Stroke. 2015 Apr 28;46(6):1681–1689. doi: 10.1161/STROKEAHA.115.009099

Fig. 3. LPS reduces mitochondrial function in cultured and primary cerebrovascular endothelial cells.

Fig. 3

(A) Immunofluorescence staining of mitochondrial (MitoTracker, purple) co-stained with MyD88 (green), suggests that the MyD88 expresses on mitochondria of cultured cerebral vascular endothelial cells (cCVECs). Nuclei were stained with DAPI (blue). Results are representative from four independent experiments. Images were taken under 63× objective. Scale bars = 10 μm. (B) Bioenergetics functional assay in cCVECs exposed to various concentration of LPS compared to vehicle control for 24 hours. Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the assay presented in supplementary Figure III A. Results are representative from four independent experiments. N = 4 per group; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. vehicle group. One way ANOVA followed by post-hoc Tukey's test was used for data analysis. (C) Bioenergetics functional assay in pCVECs exposed to various concentration of LPS compared to vehicle control for 24 hours. Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the assay presented in supplementary Figure III B. Results are representative from four independent experiments. N = 4 per group; *, P < 0.05 vs. vehicle group. One way ANOVA followed by post-hoc Tukey's test was used for data analysis. (D) Analysis of mitochondrial specific proteins for complex I proteins (NDUFAF1, NDUFS2, NDUFA2), complex II protein (succinate dehydrogenase, SDH), complex III protein (cytochrome c, Cyc), and complex IV protein (cytochrome c oxidase, cox IV) in cCVECs after a 1 μg/ml LPS challenge for 48 hours. Flow cytometry confirmed that LPS decreases the expression of complex I (NDUFS2 and NDUFA2), complex III (cytochrome c) and complex IV (cox IV) proteins. Results are representative from three independent experiments. N = 3 per group; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 vs. vehicle group. Student's t test was used for data analysis.