Pioglitazone (PGZ) disassembled complex I of the mitochondrial respiratory chain and altered supercomplex formation. (A) Mitochondria isolated from a mouse liver were treated with PGZ and analyzed as described in the Methods section. Proteins were separated using blue native polyacrylamide gel electrophoresis (BN-PAGE). Western blot analysis was performed using antibody against complex I (CI) subunit NDUFA9, complex II (CII) subunit SDHA, complex III (CIII) subunit core 2, and complex IV (CIV) subunit COX4. SC, subcomplexes. (B) Mouse mitochondria were incubated with PGZ and proteins were separated by BN-PAGE. Complex I ingel activity was displayed as described in the Methods section. Lanes: Ctr, control activity; PGZ, isolated mitochondria treated with 10 μM PGZ for 15 or 30 minutes. (C) Mitochondrial complexes isolated from an untreated mouse and three mice treated with 10 mg/kg/day PGZ for 12 weeks were run under native conditions on a BN-PAGE system and analyzed by western blot. Lanes: Ctr, control mouse; lane PGZ-1, PGZ-2, PGZ-3, PGZ-treated mice. (D) Mitochondrial complexes isolated as indicated in (C) were subjected to a complex I in-gel enzyme activity assay as described in Figure 2B. (E) Mouse liver mitochondria were solubilized in 4 g/g protein digitonin and complexes and supercomplexes were separated by BN-PAGE. Complexes and supercomplexes were identified by western blot using specific antibodies against subunits NDUFA9, SDHA, core 2, and COX1. CI+CIII2+CIVn, supercomplex formed by complex I, complex III dimer, and complex IV. CI+CIII2, supercomplex formed by complex I and complex III dimer. C, untreated mitochondria; P, PGZ-treated mitochondria. (F) Liver mitochondria isolated from PGZ-treated (P) and untreated (C) mice were solubilized in digitonin and complexes and supercomplexes were separated on a BN-PAGE gel. Complexes and supercomplexes were identified by western blot using antibodies described in (E). These results are representative of three controls and three PGZ-treated mice.