Fig. 6

Effects of SF on mitochondrial SDH activity and mitochondrial regulatory proteins in HT22 cells. (A) Effect of SF on cellular MTT formazan deposition (microscopy images, scale bar = 25 μm). HT22 cells were treated with 12.5, 25, 50, 75 and 100 mM SF for 72 h, and then incubated with MTT (0.5 mg/mL) for 2 h to allow formazan formation in the cells. (B) Concentration- and time-dependent effects of SF on cellular SDH protein levels following treatment of HT22 cells with 25, 50 and 75 mM SF for 24, 48 and 72 h. The total cellular proteins (25 μg) were separated by 10% SDS-PAGE and immunoblotted with specific antibodies for SDHA and β-actin. The corresponding unprocessed Western blot images are shown in Supplementary Fig. S6, and the quantitative intensity values are shown in Supplementary Fig. S7. (C–E) Effect of SF on the SDH activity in the cell lysates prepared from HT22 cells treated with 25, 50 and 75 mM SF for 24 h (C), 48 h (D) and 72 h (E). (n = 3). (F–H) Effect of SF on the SDH activity in the mitochondrial fraction prepared from HT22 cells treated with 25, 50 and 75 mM SF for 24 h (F), 48 h (G) and 72 h (H). (n = 3). J. Direct inhibition by SF of the SDH enzyme activity in HT22 whole cell lysates in vitro. The cell lysates were prepared from untreated HT22 cells, and were used for the in-vitro assay of the SDH activity in the presence of 25, 50, 75 and 100 mM SF (n = 5). J. Direct inhibition by SF of the mouse liver SDH activity in vitro. The mouse liver samples were homogenized for preparation of tissue lysates, which were then used for the in-vitro assay of the SDH activity in the presence of 25, 50, 75 and 100 mM SF (n = 5). (K) Levels of PGC-1α, NRF-1 and TFAM proteins following treatment of HT22 cells with SF (25, 50 and 75 mM) for 24, 48 and 72 h. The total cellular proteins (25 μg) were separated by 10% SDS-PAGE under a reducing condition and immunoblotted with specific antibodies for PGC-1α, NRF-1, TFAM and β-actin. The corresponding unprocessed Western blot images are shown in Supplementary Fig. S8, and the quantitative intensity values are shown in Supplementary Fig. S9. Each quantitative value is the mean ± S.D. * and ** denote for P < 0.05 and P < 0.01, respectively.