Figure 5

RSV reduced mtROS generation through the promotion of Sirt3-mediated mtDNA transcription. Cells were pretreated by RSV (10 μM) for 2 h, then washed, and followed by treatment with or without t-BHP (80 μM) for an additional 4 h. (a) Representative images show Sirt3 expression within mitochondria in HUVECs by immuofluorescence assay. Red fluorescence indicates mitochondria labeled with Mito-tracker Red. Green fluorescence originates from anti-Sirt3 labeling. And blue fluorescence indicates nuclei stained with DAPI. (b) The bar graph shows quantification of Sirt3 expression in the mitochondria. (c) The expressions of mtRNAPol, FoxO3A and Sirt3 in the mitochondria were determined by western blot. (d) The bar graph shows quantification of the indicated proteins. Sirt3 was knocked down by Sirt3 siRNA transfection as described in the Materials and Methods section. The cells were pretreated with RSV of 10 μM for 2 h, then washed, and followed by incubation with fresh medium in the presence or absence of t-BHP of 80 μM for an additional 4 h. (e) Cells were harvested for ChIP analysis and the recruitment of FoxO3A was measured by real-time PCR assay using primers targeted to Sirt3. The results were plotted relative to those of the total input controls (untreated chromatin). (f) The expressions of mtDNA-encoded genes of ATP6, CO1, Cytb, ND2 and ND5 along with the housekeeping genes of β-actin, GAPDH and 18s rRNA were determined by real-time PCR assay. In order to generate a relative expression ratio, the expressions of the genes of interest were normalized to the geometric average of the housekeeping genes. (g) The enzyme activity of complex I was determined using the corresponding assay kit, according to the manufacturer’s instructions. (h) ATP content was determined using an ATP determination kit. All results are presented as means±S.E.M. of at least three independent experiments. ^aP<0.05, ^bP<0.01 versus the control group; ^cP<0.05, ^dP<0.01 versus the t-BHP-treated group; ^eP<0.05, ^fP<0.01 versus RSV (10 μM) and t-BHP (80 μM) co-treated group with control siRNA transfection