Figure 1

Characterization of mtDNA-depleted HCT-8 cells. (A) Genomic DNA content. Genomic DNA was isolated from control and mtDNA-depleted HCT-8 cells, and mtDNA-encoded genes including cytochrome c oxidase subunit I (COX-I) and cytochrome c oxidase subunit II (COX-II) were amplified by PCR. GAPDH is used as a nuclear DNA-encoded control. The PCR products were electrophoresed on 2% agarose gel and then visualized by EtBr staining. A representative result of three independent experiments is shown. (B) The PCR products were quantified by densitometry, normalized to GAPDH expression, and expressed as a percentage relative to that obtained in control HCT-8 cells. The results are expressed as the means ± SD of three independent experiments. T-test: ^*P < 0.01. (C) RT-PCR analysis of mRNAs transcribed from mtDNA and nuclear DNA. Total RNA was extracted from control and mtDNA-depleted HCT-8 cells, and mtDNA-encoded transcripts such as COX-I and COX-II, and nuclear DNA-encoded transcripts such as COX-IV were analyzed by real-time PCR. The mRNA levels were expressed as a percentage relative to that obtained in control HCT-8 cells. The results are expressed as the means ± SD of three independent experiments. T-test: ^*P < 0.01. (D) Cellular ATP content. Total cellular ATP levels were measured using a somatic cell ATP assay kit as described in Materials and Methods. ATP content was expressed as a percentage relative to that observed in control HCT-8 cells. The results are expressed as the means ± SD of three independent experiments. T-test: ^*P < 0.05.