Nix induces mitochondrial autophagy in platelets in response to FCCP. Platelets were isolated from the blood of Nix^−/− mice and WT mice. (A-B) Platelets (3 × 10⁸/mL) were treated with FCCP at 5 μM in panel A or at the indicated concentrations in panel B at room temperature for 2 hours, and the samples were subjected to western blotting analysis using the indicated antibodies. (A) Each lane in the same treatment of mouse represents a different mouse and 3 independent experiments with a total of 9 mice were used. (B) Three mice of WT and 3 mice of Nix^−/− were used and the platelets were pooled in 1 experiment, and 3 independent experiments with a total of 9 mice were used. The grayscale values of all the bands were measured with ImageJ software and the statistical data are illustrated. ANOVA was used. (C) Platelets were treated as described in panel A, and platelet mitophagy was detected by electron microscopy. Mitophagy phenomenon of mitochondria enclosed in autophagosomes was quantitated, and the data demonstrated that 4 of 318 mitochondria were observed within the autophagosomes in 60 WT platelets treated with FCCP and 0 of 387 mitochondria were observed within the autophagosomes in 60 Nix^−/− platelets treated with FCCP. (D-E) Analysis of the interaction between Nix and LC3 by coimmunoprecipitation. The IgG-treated sample served as a control; n = 9 for each group. Red asterisk, mitochondrion. Red arrow, double-membrane autophagic structures (mitophagosomes).