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. 2022 Oct 17;31(6):1688–1704. doi: 10.1016/j.ymthe.2022.10.005

Figure 7.

Figure 7

Interaction of circSlc8a1 and the mitochondrial proteins

(A–C) Mitochondrial proteins ATPB, COX5B, NDUFS2, and TOMM22 were pulled down by circSlc8a1 probe in the heart tissues of circSlc8a1 (A) and cA-circSlc8a1 (B) transgenic mice, and in HL-1 cells transfected with circSlc8a1 or cA-circSlc8a1 plasmids (C) followed by western blotting. n = 3–4. (D and E) Antibodies against ATPB, COX5B, NDUFS2, and TOMM22 were used to precipitate circSlc8a1 from the heart tissues of circSlc8a1 (D) and cA-circSlc8a1 (E) transgenic mice. The immune-precipitated circSlc8a1 was measured by qPCR. n = 6–9; ∗∗p < 0.01 versus negative mice. (F) circSlc8a1 was immune-precipitated by ATPB, COX5B, NDUFS2, and TOMM22 antibodies in HL-1 cells transfected with circSlc8a1 or cA-circSlc8a1 plasmids followed by qPCR to detect the circSlc8a1. n = 6–9; ∗∗p < 0.01 versus vector. (G and H) Mitochondrial proteins were isolated from the heart tissue of circSlc8a1 (G) and cA-circSlc8a1 (H) transgenic mice. The mitochondrial translocation of ATPB, COX5B, and NDUFS2 proteins increased in the circSlc8a1-transgenic mice (G) but decreased in the cA-circSlc8a1-transgenic mice (H) compared with their counterpart negative litters. The outer mitochondrial membrane protein TOMM22 was not affected by circSlc8a1 expression. n = 3–4. (I) The mitochondrial and the cytosolic proteins were separated from HL-1 cells transfected with circSlc8a1 or cA-circSlc8a1 plasmids. The mitochondrial translocation of ATPB, COX5B, and NDUFS2 proteins increased in the circSlc8a1-transfected cells but decreased in the cA-circSlc8a1-transfected cells compared with the vector controls. The purity of mitochondria and cytosol was confirmed by TOMM22 and β-actin antibodies. n = 3–4. (J) ATP levels were significantly increased by circSlc8a1 overexpression and decreased by cA-circSlc8a1 in the heart tissues of the transgenic mice (left) and HL-1 cells (right). n = 4; ∗∗p < 0.01. (K) A model depicting the proposed mechanism of circSlc8a1 facilitating the translocation of pre-proteins from the cytosol into the mitochondria.