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. 2021 May 25;11(15):7337–7359. doi: 10.7150/thno.57804

Figure 8.

Figure 8

MRPL52 reduces apoptosis of hypoxic breast BC cells by PINK1/Parkin-mediated mitophagy. (A) Protein levels of Parkin and PINK1 assessed by WB. (B) Transfection efficacy evaluated by WB. (C) Autophagy induction was investigated by examining the protein levels of p62 and LC3B by WB. The LC3BII protein level on mitochondrial fraction was assessed by WB in order to detect the mitophagy. Mito, mitochondrial protein. (D) MDA-MB-231 cells were co-transfected with GFP-LC3B and the indicated plasmid, cultured under hypoxia for 24 h, and stained with MitoTracker probe. The colocalization of LC3B puncta (green) with mitochondria puncta (red) marked by yellow fluorescent intensity was analyzed by confocal fluorescence microscopy (mean ± SD, n = 3). *P < 0.05. Scale bars, 10 µm. (E) Cell viability assessed by CCK8 assay (mean ± SD, n = 4). *P < 0.05. (F) Mitochondrial membrane potential detected by JC-1 probe (mean ± SD, n = 3). *P < 0.05. (G) Cellular apoptosis examined by TUNEL staining. (mean ± SD, n = 3). *P < 0.05. (H) Alterations in mitochondrial permeability transition pore opening were detected by calcein AM staining. The weaker the green fluorescence in cells, the higher the degree of opening of mPTP (mean ± SD, n = 3). *P < 0.05. (I) The protein expressions of cleaved PARP and cleaved Caspase-3 were analyzed using WB.