Uation V (mm3) = a ?b2/2, where a is the largest diameter and b is the smallest diameter. No significant body weight loss was observed. To perform gene expression profiling, tumors (4 tumors per group) were harvested and snap frozen immediately on dry ice when the tumor size reached 500?00 mm3. Tumor tissue was dissected, homogenized in Trizol, and total RNA was isolated and subjected to gene expression analysis as described above.Additional Files Additional information about the data reported in this manuscript, including formatted gene expression data in these studies are available at BMC Genomics. Establishment of A549 0 cell cultures Candidate A549 0 lung cancer cell cultures were prepared using previously described ethidium bromide-based protocols [12]. Consistent with prior reports of 0 cell lines (e.g. [7,9,33]), the resulting cells displayed auxotrophic growth dependence on pyruvate and uridine and elongated shape (data not shown). The growth rate of the candidate 0 cells (27 hour generation time) was slower than the parental line (20 hour generation time) in pyruvate and uridine supplemented medium (Fig. 1A).from background (Fig. 1B). To demonstrate the involvement of respiration, we also treated parental A549 and A549 0 cells with antimycin A, an inhibitor of mitochondrial complex III of the electron transport chain (ETC) [34], prior to measuring oxygen consumption. As expected, antimycin A-treated A549 cells showed reduced oxygen consumption, while A549 0 cells were not significantly affected by drug-treatment (Fig. 1B). These results strongly suggest that residual mitochondria-dependent aerobic respiration in A549 0 cells, if present, is below the threshold that can be measured in this assay.A549 0 cells grow in tumor xenografts at a slower rate than their parental cells Parental A549 cells and their 0 derivatives both grew as xenografts when implanted shallowly (subcutaneously/ intramuscularly) in the flank muscle of nude mice. mtDNA-depleted cell lines have been reported to form xenograft tumors [13,18,19]. Here, we found that A549 0 cells grow readily in xenograft, although at a slower rate and after a longer induction period (approximately 30 days) relative to the parental line (Fig. 2). This is consistent with the growth characteristics of tumor xenografts derived from N2B 0 osteosarcoma [18], E3 0 serous ovarian carcinoma [18], HSA 0 cervical cancer [18], and T47D 0 breast cancer [19] cell lines relative to their parental counterparts. A549 0 cells do not express mtDNA-derived transcripts in culture or in xenografts We performed quantitative PCR analysis of mtDNAencoded MT-ATP6 and MT-CYB transcripts in A549 and A549 0 cells grown in culture and in xenografts, in the absence of ethidium bromide. In contrast to the parental A549 cells, the expression of these two transcripts could not be detected in 0 cells (Additional File 1). This strongly suggests that, in bulk, the A549 0 cells retained their mtDNA-depleted genotype over the course of their growth in culture and in xenograft. This was important to ascertain since it was possible that during their growth in xenograft, rare mtDNA-positive cells present in the injected A549 0 cell population could have PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25962748 substantially increased in abundance and affected our downstream analyses. PX-478 site Similarly, it is possible that A549 or A549 0 cells with certain genetic or epigenetic changes (e.g. those that confer a growth advantage) were selected for in culture or in xenografts and affecte.