Research Paper Volume 15, Issue 6 pp 2275—2292

Figure 5. VO-OHpic protects CEP against degeneration and calcification via mitophagy stimulation. CEP chondrocytes were pretreated with 3-MA for 10 hours, then culture medium was changed with VO-OHpic (1 μM) for 18 hours and 100 μM TBHP was added for 6 hours. ROS production was evaluated with DCFH-DA staining, (A) Representative fluorescence microscopy photomicrographs of intracellular ROS in chondrocytes. (B) Flow cytometric analysis of endplate chondrocytes stained with Annexin V-FITC/PI. Percentage apoptosis rates were expressed as means ± SD. (C) Western blot was conducted to examine the protein levels of LC3, SOX9, COL2, MMP3 and MMP13. The band density of SOX9, COL2, MMP3, MMP13 and the ratio of BCL-2/BAX were quantified and normalized to control. (D) Western blot was conducted to examine the protein levels of CO10 and RUNX2. The band density of CO10 and RUNX2 was quantified and normalized to control. Data are presented as mean ± SD from three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.