shWNT5B suppressed mitochondrial biogenesis in MDA-MB-231 cells. (A) qPCR analysis of mtDNA content. mtDNA was quantified by comparing the Cox2 gene to an intron of HBB in cells transduced with shWNT5B or shCtl virus. Bars represent the mean ± SD (n = 3), P = 0.0089. (B) Cellular ATP was extracted from shWNT5B- or shCtl-infected cells right before the assay, and the average single cell ATP level was calculated and graphed. The mean values represent 3 independent experiments. P = 0.0072. (C) RT-qPCR analysis of OXPHOS-related gene expression. CYC1 and ATP5G1 mRNA was detected and normalized with GAPDH. The results represent the mean of 3 independent experiments and 3 wells for each sample at each time point. **P = 0.0091 for CYC1 and P = 0.0084 for ATP5G1. (D) The OCR was measured and normalized by the cell number after oligomycin, FCCP and Rotenone were sequentially added to the cells. The values were plotted as the mean ± SD. P = 0.036. *P < 0.05; **P < 0.01.