Figure 6

Wnt5B initiated WNT/ β-catenin to govern mitochondrial biogenesis through MCL1. (A) Dual luciferase assay of TCF promoter activity. Twenty thousand cells were seeded into 12-well plates, and the cells were transduced with shCtl or shWNT5B lenti-particles and treated with vehicle control or 50 nM mWNT5B. The plasmid Topflash and Renilla luciferase were co-transfected into MDA-MB-231 cells at 24 h using lipofectamine 2000. The cells were harvested for the dual luciferase assay at 48 h. Bars represent the average luciferase activities normalized to internal control (n = 3). **P < 0.01; ***p < 0.001. (B) IHC staining of Myc and MCL1 in tissue array samples. FFPE tissue array slides were stained with Myc or MCL1 antibody, and the staining was graded into three different levels. Nuclear staining of Myc and cytosolic staining of MCL1 were observed. (C) Western blot of proteins related to mitochondrial structure and function. MDA-MB-231/shWNT5B and control cells were collected for cell lysates. The protein expression of MCL-1, AIF and PGC-1α was evaluated. (D) Western blot of Myc-regulated genes. MCL1 protein was inhibited with Myc knockdown by Myc siRNA; mitochondrial structural protein TOM20 also decreased. (E) MCL1 induced proteins in MDA-MB-231/shWNT5B cells. TOM20 was upregulated by MCL1 independent of WNT5B expression status.