Fig. 7: CAMK1 activation contributes to PTV-induced mitophagy.
(a–d) EPCs were pretreated with BAPTA-AM (20 mM) for 20 min followed by PTV (0.5 μM) for 24 h. Representative western blots (a) and quantitative analysis (c) indicated that BAPTA-AM significantly decreased PTV-induced CAMK1 phosphorylation at Thr177 site. Representative western blots (b) and quantitative analysis (d) indicated that BAPTA-AM significantly decreased the expression of MAP1LC3B-II induced by PTV. e Lentiviral vector carrying Camk1 shRNA was employed to knockdown Camk1 in EPCs. Representative western blots showed that Camk1 was effectively knocked down. Camk1 was knocked down by shRNA for 72 h before PTV treatment. Representative western blots (f) and quantitative analysis (g) showed that Camk1 knockdown significantly decreased the expression of MAP1LC3B-II induced by PTV. h EPCs from atherosclerotic mice were transfected by lentivirus to knock down Camk1. Camk1 knockdown group, control group, and vector control group EPCs were infected by tandem GFP-mRFP-LC3 adenovirus for 24 h before exposure to PTV (0.5 μM) 24 h. Representative LSCM images showed puncta formation in different groups. Scale bar: 10 μm. Quantitative analysis of yellow and free red puncta. PTV increased the number of yellow and free red puncta compared with control. Camk1 knockdown significantly decreased the number of yellow puncta increased by PTV. i EPCs from atherosclerotic mice were transfected by lentivirus to knock down Camk1. All the groups were infected by mtKeima plasmid for 12 h before treatment by PTV PTV (0.5 μM) 24 h. Representative images showed puncta formation in different groups. Scale bar: 25 μm. PTV treatment increased the red fluorescence intensity turnover, indicating that more mitochondria were transferred to lysosomes. This effect was blocked by silencing Camk1. Quantitative analysis of the fluorescent area showed that PTV increased mitophagy index, but knock down Camk1 blocked this effect. j Merged images revealed that Camk1 knockdown reduced the co-localization of MAP1LC3B and mitochondria in EPCs according to Pearson’s overlap coefficient analysis. Scale bar: 10 μm. (n = 10 cells per group, EPCs were isolated from 3 ApoE−/− mice fed with high fat diet for 8 weeks and 3 independent experiments were performed, mean ± SD, *P < 0.05, **P < 0.01).
