Figure 1: Reduction of mitochondrial respiratory activity in MEFs derived from Cox7rp KO mice. | Nature Communications

Figure 1: Reduction of mitochondrial respiratory activity in MEFs derived from Cox7rp KO mice.

From: A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle

Figure 1

(a) Generation of Cox7rpKO mice. Schematic representation of the gene trap vector insertion site in the mutant gene. BTK, bruton tyrosine kinase cassette; LTR, long terminal repeat; NEO, neomycin-resistant gene cassette; pA, polyadenylation sequence; PGK, phosphoglycerate kinase-1 promoter; SA, splicing acceptor site; SD, splicing donor site. (b) Genotyping of Cox7rpKO mice. Representative litters containing WT, Cox7rp+/− (+/−) and Cox7rpKO (KO) mice were shown. Sense primer-F and -L within intron 1 and the vector, and common antisense primer-R within intron 1 (a) were used to amplify WT (489 bp) and mutant (Mut: 289 bp) alleles, respectively. (c) Western blot analysis of Cox7rpKO MEFs. Cell extracts from MEFs were resolved on SDS–polyacrylamide gel and immunoblotted with anti-COX7RP. (d) Decrease in COX activity in Cox7rpKO MEFs. COX activity was assessed using MEFs from WT and Cox7rpKO mice infected with recombinant adenovirus expressing control DsRed (Ad-DsRed) or COX7RP (Ad-COX7RP). Data are presented as means±s.d. (n=3). *P<0.05 for WT versus KO; Student’s t-test. (e) Decrease in mitochondrial ATP production in Cox7rpKO MEFs. ATP production was assessed using MEFs from WT and Cox7rpKO mice infected with Ad-DsRed or Ad-COX7RP. Data are presented as means±s.d. (n=3). *P<0.05 for WT versus KO; Student’s t-test. (f) Retardation of growth in Cox7rpKO MEFs. Data are presented as means±s.d. (n=3). *P<0.05, **P<0.01 for WT versus KO at each indicated time point; Student’s t-test. (g) Decline of cellular ATP content in Cox7rpKO MEFs. Data are presented as means±s.d. (n=3). **P<0.01 for WT versus KO; Student’s t-test. (h) Decreased oxygen consumption rates of Cox7rpKO MEFs. MEFs were permeabilized with digitonin, and subsequently substrates and inhibitors were added. Rates of oxygen consumption are expressed as nanomoles of oxygen per minute per milligram of protein. Data are presented as means±s.d. (n=3) *P<0.05, **P<0.01 for WT versus KO under each condition; Student’s t-test.

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