Fig. 1.

Knockdown of DNA ligase III delays the recovery of mtDNA copy number. (A) Experimental design. The presence and absence of 2′,3′-dideoxycytidine (ddC) in the medium is indicated by dark and pale grey stripes respectively. (a) Time course of the ddC treatment and dsRNA knockdown. R2 and R3 represent the cell harvest points 2 and 3 days after ddC removal, whilst an arrowhead indicates the timing of dsRNA transfection. (b and c) Incubation of cells in medium in the absence or presence of ddC for 3 days as the non-treated or ddC-treated controls. NT and D3 represent the point of their harvest. (B) Western blot analysis of DNA ligase III levels in cell lysates prepared from cells treated with scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA. The top panel shows the DNA ligase III band and the middle panel is a digitally enhanced image of the top panel. Tubulin was used as a loading control (bottom panel). (C) The relative copy number of mtDNA analysed with real-time quantitative PCR. The mtDNA content was normalised against nuclear gene content in each sample. The relative mtDNA copy number in NT sample was expressed as 100 in each experiment and those of the other samples were displayed relative to this. Data represent the mean of 3 independent transfection experiments ± SEM. * (p < 0.05).