Fig 2: Construction of the SDHD mutant. The gene sequence of SDHD with CRISPR targeting sites is shown a. The sgRNA and PAM sequences predicted by the online idtdna program are depicted in blue and red colors, respectively; the specific site for mutation (at 118-bp) is shown with a green arrow. Expression of SDHD protein is shown b. The protein extracts reacted with rabbit polyclonal antibodies to SDHD and rabbit polyclonal antibodies to ß-Actin are shown. The DNA level recombination events in the genome of the mutant are illustrated c. The bases c and a (highlighted and in purplr color) of the parent have been replaced with the base g (highlighted and in red color) in the mutant. Expression of representative subunits from all five ETC complexes are shown d-i. The expression of NDUFB8 (complex i; d and e), SDHB (comlex II; d and f), UQCRC2 (complex III; d and g), MTCO1 (complex IV; d and h) and ATP5A (complex V; d and i) were normalized to the expression of ß-Actin

Fig 3: Actin is associated with the CIV subunit MTCO1 and localizes inside brain mitochondria. (A) Silver staining of gels after SDS-PAGE of the immunoprecipitates from brain mitochondria isolated from middle-aged male mice with normal IgG and anti-MTCO1 antibody. (B) Immunoblot analysis of the immunoprecipitates with normal IgG and anti-MTCO1 antibody using antibodies against MTCO1 and actin. (C) Isolated brain mitochondria were digested with trypsin at 37 °C for 30 min, followed by immunoblotting with antibodies against the indicated proteins. (D) Confocal images of isolated brain mitochondria after immunofluorescence staining with antibodies against Tom20, actin, and MTCO1. Arrows indicate the contact sites of actin with MTCO1.

Fig 4: Inhibiting actin polymerization enhances the OCR, with no significant alterations in the association of actin with MTCO1. (A) Measurement of the OCR of isolated brain mitochondria during incubation with DMSO or CB with high-resolution respirometry (n = 14, mean ± S.D.). p < 0.001. (B) Isolated brain mitochondria were incubated with DMSO or CB for 30 min, immunoprecipitated with anti-MTCO1 antibody, and immunoblotted for MTCO1 and actin. The ratio of coprecipitated-actin to MTCO1 was determined (n = 4, mean ± S.D.). (C) After incubation of the isolated brain mitochondria with DMSO or CB for 30 min, whole mitochondrial lysates were subjected to SDS-PAGE and immunoblotted for MTCO1 and actin. The total amount of MTCO1 and actin in arbitrary units (A.U.) were compared between samples treated with DMSO and CB (n = 6, mean ± S.D.). (D) Confocal images of immunofluorescence staining with anti-MTCO1 (green) and anti-actin (red) antibodies after the treatment of isolated brain mitochondria with DMSO or CB for 30 min. Arrows indicate the contact sites of actin with MTCO1.

Fig 5: miR-181c alters MICU1 expression through Sp1 alteration. A, Primary NMVMs were isolated from WT (C57BL6/j) and miR-181c/d-/- (c/d KO) pup hearts. MICU1 expression was altered with small interfering RNA against MICU1 (siMICU1). Western blot analysis (B and C). mt-COX1, (B and D). Sp1, and (B and E). MICU1 expression. B through E, mt-COX1 (upper bands), Sp1 (second from the top bands) and MICU1 (third from the top bands) were normalized to a-tubulin (lower bands). F, Amplex red assay was performed to measure the rate of ROS production from the isolated NMVMs with/without siMICU1 transfection. *P<0.05, vs WT Scr, and † P<0.05, vs c/d KO Scr. n=3 to 5. KO indicates knockout; NMVMs indicates neonatal mouse ventricular myocytes; ROS, reactive oxygen species; Sp1, specificity protein; WT, wild-type.