Figure 5.

Cells carrying the deafness-associated A1555G mtDNA mutation, like those that over-express h-mtTFB1, exhibit mitochondrial 12S hypermethylation, aberrant mitochondrial biogenesis, and increased susceptibility to cell death. (A) Shown are the results of methylation-sensitive 12S rRNA primer extension reaction from total RNA of wild-type and A1555G mutant cybrids, exactly as described in the legend to Figure 1, except in this case read-through products (*) were terminating due the presence of ddGTP (to yield a product of known length for quantification purposes, see Materials and Methods). The right panel shows quantification of results with means and standard deviations calculated from six separate measurements. (B) Immunoblots of whole cell extracts from wild-type and A1555G mtDNA mutant cybrids were serially probed for mitochondrial h-mtTFB1, h-mtTFB2, COX1, COXII and VDAC and tubulin (as a loading control) as indicated. (C) Mitotracker Green fluorescence values from three separate samples from wild-type and A1555G cybrids. (D) Results of sorbitol-induced cell death assays of wild-type and A1555G cybrids (upper graph) and h-mtTFB1-overexpression and empty-vector (vector) control cell lines. Graphed are the mean fold changes in percent dying cells due to sorbitol treatment of the indicated cell lines.