FIGURE 4.

PINK1/Parkin-mediated mitophagy regulated the osteogenic differentiation of SCCs in vitro. (A–D) Proteins associated with PINK1/Parkin-mediated mitophagy in H-SCCs and L-SCCs were tested by western blot and quantified using ImageJ (n = 5). (E) The colocalization of mitochondria and lysosomes in H-SCCs and L-SCCs was tested by fluorescence microscopy (n = 7 and 6, respectively). Red, mitochondria; green, lysosomes; blue, nucleus. (F) PINK1 mRNA expression levels in the H-SCCs or H-SCCs transfected with scrambled siRNA or PINK1 siRNA were measured by RT-qPCR (three independent experiments were performed, with similar results; data are from a single experiment performed in triplicate). (G) The expression levels of the osteogenic differentiation-related genes in the H-SCCs or H-SCCs transfected with scrambled siRNA or PINK1 siRNA were measured by RT-qPCR after 7 days of osteogenic induction (three independent experiments were performed, with similar results; data are from a single experiment performed in triplicate). (H) The formation of mineralization nodules in the H-SCCs or H-SCCs transfected with scrambled siRNA or PINK1 siRNA was tested by Alizarin red staining after 28 days of osteogenic induction (n = 3). (I) The expression levels of osteogenic differentiation-related genes in PINK1-overexpressing L-SCCs were determined by RT-qPCR (n = 3). (J) Mineralized nodule formation in PINK1-overexpressing L-SCCs was tested by Alizarin red staining (n = 3). TCs, target cells; L-Exo, exosomes secreted by L-SCCs; H-Exo, exosomes secreted by H-SCCs; H-SCCs + scramble, H-SCCs transfected with scrambled siRNA; H-SCCs + siPINK1, H-SCCs transfected with PINK1 siRNA; L-SCCs + vector, L-SCCs transfected with empty vector; L-SCCs + oePINK1, L-SCCs transfected with a PINK1 overexpression plasmid; the blue triangle represents the expected band for PINK1 or Parkin. Scale bar, 20 μm. *P < 0.05; **P < 0.01; ***P < 0.001.