Figure 3.

The effects of sinularin on the OCRs (Oxygen consumption rates) parameter, ECARs (extracellular acidification rate), and 5 OXPHOS (Oxidative phosphorylation) enzymatic complexes in SK-HEP-1 cells. The OCRs (pMoles/min/mg protein) were measured before and after the addition of pharmacological agents to living cells. The ECARs (mpH/min/mg protein) were measured before the addition of pharmacological agents to living cells. Four measurements were taken and averaged to provide reliable data as a base value, followed by sequential and continuous injections of Seahorse XF Cell Mito Stress Test reagents, including oligomycin, FCCP, and antimycin/rotenone. (A) A graph of OCR values versus time-course (min). (B) Quantification of basal respiration OCRs. (C) Quantification of couple respiration OCRs (ATP production) (D) Quantification of maximal respiration capacity OCRs. (E) Quantification of proton leak respiration OCRs. (F) Quantification of ECARs. (G) The Western blot profile showed the effects of sinularin on the expression levels of complex I-NDUFB8, complex II-SDHB, complex III-UQCRC2, complex IV-COX II, and complex V-ATP5A. β-actin was used as the internal control. Full, uncropped Western blot gels are shown in Supplementary Figure S2D. The complex I-NDUFB8 (H), complex II-SDHB (I), complex III-UQCRC2 (J), complex IV-COX II (K), and complex V-ATP5A (L) protein levels were quantified by densitometry analysis using ImageJ software after being normalized with the β-actin level. Each bar represents the mean ± SE of three independent experiments. The results were analyzed using Student’s t-test to determine the significance, in which * p < 0.05 and ** p < 0.01 relative to the control (sinularin-untreated cells).