Figure 9.

MRPL52 activates the ROS-Notch1-Snail signaling pathway to mediate EMT of BC cells exposed to hypoxia. (A) MDA-MB-231 cell was transfected with GFP-tagged MRPL52 in order to analyze the subcellular localization of MRPL52. (B) Levels of mitochondrial 12 S and 16 S rRNA were evaluated by RT-qPCR (mean ± SD, n = 4). *P < 0.05. (C) Mitochondrial proteins were isolated and treated by CAP (0.15 μg/μL) for 10 min at 4 ℃ as required. WB was performed to assess protein levels of mitochondrial respiratory complexes components, including ND2 and ND5 (complex I), CYTB (complex III), COX1 and COX2 (complex IV) encoded by mt-DNA, as well as ATP5A and NDUFA9 encoded by nuclear genome. (D) DCFH-DA and (E) MitoSOX fluorescence in cells measured by fluorescence microscopy. The fluorescence mean intensity of DCFH-DA and MitoSOX represent cytosolic ROS and mitochondrial ROS, respectively (mean ± SD, n = 3). *P < 0.05. Scale bars, 100 µm. (F) Cells were preincubated with MitoTEMPO (20 μM) for 1 h. MitoSOX fluorescence in cells measured by fluorescence microscopy (mean ± SD, n = 3). *P < 0.05. Scale bars, 100 µm. (G) Cells were preincubated with tBHP (200 μM) for 4 h; or with NAC (2.5 or 5 mM) for 1 h. WB for detecting the expression levels of MRPL52, Notch1 and N1ICD in MDA-MB-231 and MCF-7 cells exposed to 1% O₂ with treatments as indicated.