Figure 1.
Sorting and validating replicative senescent yeast cells.
a. Biotin-Streptavidin method for sorting senescent cells was performed as depicted. b. A comparison of bud scars on adsorbed and not adsorbed yeast cells stained by Solophenyl Flavine 7GFE 500%. c. Yeast cells were cultured for 48 hours and then divided into three groups: no-sorting, adsorbed by magnets and those not adsorbed by magnets. The bud scars for a hundred cells were counted. The average number of bud scars on the adsorbed cells was approximately three times the figures obtained for not-adsorbed cells.
Figure 2.
Mitochondrial network morphology and fusion/fission gene expression levels in replicative senescent cells.
a. The mitochondria in yeast cells were labeled by green fluorescence protein (pVT100-MtGFP) for visualization. The morphology of mitochondrial network were classified into three categories: fragmented, tubular, and hyperfused/elongated. b. Senescent wild type cells were sorted at various time points after biotin labeling. Mitochondria network morphology of log phase cells and adsorbed senescent cells were classified. The ratio of senescent cells with fragmented mitochondria gradually increased with the duration of the culturing period. c. Senescent dnm1 and fzo1 cells at different time points were sorted and subjected for classifying mitochondrial network morphology. Almost none of the senescent cells from fission genes DNM1 deleted strain presented a fragmented network. The same was true for senescent cells from the FZO1 deleted strain, which presented all fragmented networks. d. Relative mRNA levels of DNM1 and FZO1 in wild type cells at the log phase of growth and senescence were measured by quantitative RT-PCR. DNM1 levels in 48 h senescent cells were 2.5 fold higher than those in log phase and significantly higher than in young, not adsorbed cells (*: p<0.05).
Figure 3.
Mitochondrial DNA copy number, membrane potential and superoxide level in wild type cells.
a. Relative copy numbers of the mtDNA in log phase and 48 h senescent cells were presented as fold changes. The relative mtDNA was normalized to genomic DNA by quantitative PCR. Our results indicate that the number of mtDNA copies in senescent cells was significantly higher than that obtained from log phase cells (p<0.05). b. Mitochondrial membrane potential was measured by Rhodamine 123 staining. Rho 123+ cells were determined by flow cytometry. The ratio of Rho 123+ cells in log phase cell was higher than 48 h senescent populations (p<0.05). c. Mitochondrial superoxide level was assayed by MitoSOX Red staining. MitoSOX Red+ cells were determined by comparing stained and unstained control cells. Log phase cells presented lower ratio of MitoSOX Red+ cells in the population compared to 48 h senescent cells. d. Intracellular superoxide level was examined by DHE staining. The ratio of DHE+ cells was significant higher in senescent yeast cells.
Figure 4.
The effects of resveratrol on mitochondrial network morphology and fusion/fission gene expressions in replicative senescent cells.
a. Mitochondria network morphology of control senescent cells and resveratrol-treated sample was classified. Resveratrol was added to the medium 14 hours after biotin labeling. The ratio of cells with fragmented mitochondria was 45% in resveratrol-treated sample, compared to 70% in the control group (p<0.05). b. Relative mRNA levels of DNM1 and FZO1 in senescent cells from the control sample and 30 µM resveratrol-treated senescent cells were measured by quantitative RT-PCR. Individual gene expression levels were normalized to that of the control cells (*: p<0.05). The FZO1 gene level in resveratrol-treated senescent cells was approximately 2 times higher than that of the control cells. c. The Western blot and quantitation of Dnm1-HA in in log phase cells, senescent cells and senescent cells treated with 30 µM resveratrol. The relative Dnm1-HA protein levels were determined by Odyssey Imaging Systems. By normalizing to log phase cells, we found Dnm1 level in resveratrol treatment senescent cells was lower than untreated cells. The results demonstrated that Dnm1 level is correlated to fragmented status of mitochondrial network.
Figure 5.
The effects of resveratrol on mtDNA, membrane potential and superoxide level in senescent cells.
a. Relative mtDNA copy number of the control cells and senescent cells treated with resveratrol were measured by quantitative PCR. Cells treated with resveratrol had 1.73 folds higher of relative mtDNA contents than that of the control groups (*: p<0.05). b. Mitochondrial membrane potential was measured by DiOC6 (3) staining and flow cytometry. No significant differences were observed in the ratio of DiOC6 (3)+ cells between the control and resveratrol-treated cells. c. Mitochondrial superoxide level was assayed using MitoSOX Red staining. No significant difference was observed in the ratio of cells with MitoSOX Red+ between the control and resveratrol-treated senescent cells. d. Intracellular superoxide levels of control and resveratrol-treated cells were assayed by DHE staining. The ratio of DHE+ cells was lower in resveratrol-treated cells (*: p<0.05).