(A) Mitochondrial ROS level in scrambled shRNA-transfected (shControl) and RecQL4-suppressed U2OS cells (shRecQL4) as well as in human fibroblasts derived from unaffected heterozygous mother (AG18459) and affected homozygous son (AG18375). Cells were stained with MitoSOX™ Red for 30 min and then analyzed by flow cytometry; (B) Alteration of mitochondrial morphology in shControl and RecQL4-suppressed U2OS cells 48 h post menadione treatment. Cells were stained with 25 nM DiOC6 for 15 min, fixed and analyzed by confocal microscopy analysis; (C) Long range QPCR result of full length mitochondrial DNA (mt-FL) in shControl and shRecQL4 U2OS cells at 0, 5, 24, 48 h post menadione treatment, and in AG18459 and AG18375-RTS cells at 0, 24, 48 h post menadione treatment. Equal amount of mtDNA template DNA in each sample normalized by mtND1 gene was used for long range QPCR reaction.