Figure 2. Measurement of mtDNA levels and synthesis in PINK1-silenced SH-SY5Y cells.

A, SH-SY5Y cells were treated with PINK1 or scrambled control siRNA for 3, 6, 9 or 12 days and mtDNA levels measured by quantitative real-time PCR. MtDNA levels were expressed relative to the single copy nuclear gene TK2. Data are expressed as percentage of control siRNA mtDNA levels (n = 4). B, cells were treated with siRNA for 12 days and cell lysates probed for TFAM, GAPDH and porin by western blot. C, SH-SY5Y cells were treated with siRNA for 6 days and de novo mtDNA synthesis measured by quantifying the incorporation of [methyl-³H]thymidine into mtDNA over an 18-hour period in the presence of aphidicolin. Data are expressed as 10³ counts per minute incorporated per mg of protein and are the mean±s.e.m (n = 4). D, SH-SY5Y were treated with PINK1 siRNA for 6 days in the presence of 10 µM ddC, and then the ddC washed away, and the cells incubated for a further 3 days in the presence of siRNA before mtDNA levels were measured (n = 4). * P<0.05 vs. scrambled control siRNA; **P<0.01 vs. scrambled control siRNA.