Article Figures & Tables
Figures
- Fig. 1.
Sensitivity of WT and MtDNA mutator (POLGexo−) MEFs to mitochondrial toxins. Mitochondrial mutator MEFs were more sensitive than WT MEFs to inhibition of colony formation by azide (P=0.025), ethidium bromide (P=0.046), FCCP (P=0.002) and chloramphenicol (P<0.001). A very strong trend for a difference in sensitivity was also found for rotenone (P=0.053). Colony counting assays were performed to determine IC50 values for (A) azide, (B) ethidium bromide (EtBr), (C) tert-butyl hydroperoxide, (D) rotenone, (E) antimycin A, (F) hydrogen peroxide (H2O2), (G) FCCP, (H) oligomycin, and (I) chloramphenicol. Experiments were performed with three independently derived E1A immortalized WT and mtDNA mutator clone lines (n=3). Bars represent mean±s.e.m. Unpaired t-tests were performed with * indicating a P-value of <0.05 and ** indicating a P-value <0.001.
- Fig. 2.
O2 consumption rates of WT and POLGexo− MEFs treated with AICAR or rapamycin. (A) O2 consumption rates after 24 h in HGM. (B) O2 consumption rates after 48 h in HGM. (C) O2 consumption rates after 24 h in LGPM. (D) O2 consumption rates after 30 h in LGPM. (E) O2 consumption rates after 10 days in HGPUM. (F) O2 consumption rates after 20 days in HGPUM. Rapamycin transiently increases oxygen consumption in WT MEFs at the 24 h time point either in the absence or presence of pyruvate. Respiratory deficits in POLGexo− MEFs were revealed by the LGPM used in panels C and D and the HGPUM used in panels E and F. Experiments were repeated twice using three technical replicates each time. Bars represent mean±s.e.m. Two-way ANOVA with Fisher's LSD post hoc analysis was performed. *P<0.05, **P<0.001.
- Fig. 3.
Effects of AICAR or rapamycin on ROS production of WT and POLGexo− MEFs under low glucose conditions. Rapamycin treatment in LGM or LGPM had no effect on ROS production. 0.5% ethanol (EtOH) was added as a positive control to induce ROS production. (A) ROS levels after 24 h in LGM. (B) ROS levels after 48 h in LGM. (C) ROS levels after 24 h in LGPM. (D) ROS levels after 48 h in LGPM. Experiments were repeated twice using five technical replicates each time. Bars represent mean±s.e.m. Two-way ANOVA with Fisher's LSD post hoc analysis was performed. *P<0.05, **P<0.001.
- Fig. 4.
Effects of AICAR or rapamycin on ROS production of WT and POLGexo− MEFs under high glucose conditions. 0.5% ethanol (EtOH) was added as a positive control to induce ROS production. (A) ROS levels after 24 h in HGM. (B) ROS levels after 48 h in HGM. (C) ROS levels after 24 h in HGPM. (D) ROS levels after 48 h in HGPM. (E) ROS levels after 10 days in HGPUM. (F) ROS levels after 20 days in HGPUM. Experiments were repeated twice using five technical replicates each time. Bars represent mean±s.e.m. Two-way ANOVA with Fisher's LSD post hoc analysis was performed. *P<0.05, **P<0.001.
- Fig. 5.
Effects of AICAR or rapamycin on ATP levels of WT and POLGexo− MEFs under low glucose conditions. (A) ATP levels after 24 h in LGM. (B) ATP levels after 48 h in LGM. (C) ATP levels after 24 h in LGPM. (D) ATP levels after 48 h in LGPM. Experiments were repeated twice using five technical replicates each time. Bars represent mean±s.e.m. Two-way ANOVA with Fisher's LSD post hoc analysis was performed. *P<0.05, **P<0.001.
- Fig. 6.
Effects of AICAR or rapamycin on ATP levels of WT and POLGexo− MEFs under high glucose conditions. (A) ATP levels after 24 h in HGM. (B) ATP levels after 48 h in HGM. (C) ATP levels after 24 h in HGPM. (D) ATP levels after 48 h in HGPM. (E) ATP levels after 10 days in HGPUM. (F) ATP levels after 20 days in HGPUM. Experiments were repeated twice using five technical replicates each time. Bars represent mean±s.e.m. Two-way ANOVA with Fisher's LSD post hoc analysis was performed. **P<0.001.
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