Figure 2. Hyper-methylation of mitochondrial 12S rRNA leads to activation of E2F1 and E2F1-dependent apoptosis.

(A) Western blot analysis of whole cell extracts for E2F1, Rb and phosphorylated-Rb, (Rb[T268]-P) in HeLa cells that over-express h-mtTFB1 (h-mtTFB1-OE) compared to cell containing an empty-vector negative control (pcDNA), and in human 143B cybrids containing wild-type (wt) or A1555G mtDNA. Tubulin was probed as a loading control. Quantification is shown in Figure S1B.

(B) Effect of h-mtTFB1 knock-down (to reduce 12S hyper-methylation) on E2F1 levels in A1555G cybrids by Western blot using tubulin as a loading control. Two independent knock-down constructs were used (+), and a scrambled shRNA was used as a negative control (−). 143B cybrids with wild-type (wt) mtDNA treated with the negative control shRNA are shown in lane 1. Quantification is shown in Figure S1C.

(C) Quantitation of apoptosis (caspase 3/7 activity) in 143B cybrids containing wild-type (wt) or A1555G mtDNA without (−) or with (+) h-mtTFB1 knocked down by shRNA (see Figure S2A). Basal and induced apoptosis refer to the presence and absence of staurosporine. The values plotted represent mean ± standard deviation (n=12), with t-test p-values indicated.

(D) Quantitation of apoptosis (caspase 3/7 activity) in 143B cybrids containing wild-type (wt) or A1555G mtDNA without (−) or with (+) E2F1 knocked down by shRNA. Basal and induced apoptosis refer to the presence and absence of staurosporine. The values plotted represent mean ± standard deviation (n=12), with t-test p-values indicated.

(E) Western blot analysis of full-length and cleaved PARP and of E2F1 in wild-type (wt) and A1555G treated with scrambled (−) or E2F1 (+) shRNA without (−) or with (+) induction of apoptosis by starosporine. E2F1, full-length (PARP) and caspase-3-cleaved forms of PARP are shown, with tubulin probed as a loading control. Quantification is shown in Figure S1D.