Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2012 Aug 13;40(19):9717–9737. doi: 10.1093/nar/gks774

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

Figure 2. — Reduced mitochondrial respiratory activities in p32^−/− MEFs. (A) The activity of each complex was measured using cell lysates as described in ‘Materials and Methods’ section. Reduced enzymatic activities of mitochondrial complexes I, III and IV in p32-knockout cells were observed. In contrast, complex II activity was not decreased. Re-expression of p32 in p32^−/− cells restored the enzymatic activities of complexes I, III and IV. The results represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 versus controls (p32^+/+ versus p32^−/− and p32^−/−versus p32^−/−::p32). (B) Oxygen consumption by digitonin-permeabilized wild-type (p32^+/+) and knockout (p32^−/−) MEFs. Glutamate and malate (glu+mal) respiration depends on the activities of complexes I, III and IV; glycerol-3-phosphate and succinate (G3P+suc) respiration depends on the activities of complexes III and IV. Rotenone (a complex I inhibitor) and Antimycin A (a complex III inhibitor) completely inhibit O₂ consumption at each step. Data show the mean ± SD of triplicate experiments and **P < 0.01 versus controls (p32^+/+ versus p32^−/−). (C) Decreased mitochondrial membrane potential in p32-knockout MEFs. MEFs were stained with the fluorescent dye JC-1 and then analyzed by flow cytometry at 527 and 590 nm. The fluorescence ratio of JC1 dimer (Red)/JC-1 monomer (Green) is shown. Dissipation of the membrane potential with CCCP was used as a control. (D) Effects of 2-deoxy-d-glucose (2DG) (20 mM) and oligomycin (10 μM) on intracellular ATP content in MEFs. The ATP content in untreated MEFs is presented in lane 1. The ATP concentration of untreated MEFs (lane 1) was subtracted from that of 2DG-treated MEFs (lane 2) for assessment of glycolytic ATP production (lanes 1 and 2). The ATP concentration in 2DG-treated cells (lane 2) was subtracted from that in cells treated with 2DG + oligomycin (lane 3) for mitochondrial ATP production (lanes 2–3). Wild-type (p32^+/+), p32-knockout (p32^−/−), re-expressed p32 (p32^−/−::p32) and vector only-transfected (p32^−/−::vector) MEFs were used. The ATP response was measured using a Luminescence ATP assay kit in a 96-well plate. Data show the mean ± SD of triplicate experiments. (E) Cell proliferation monitored in MEFs. Upper panel: the diamonds, squares, triangles and circles represent wild-type MEFs (p32^+/+), p32-knockout MEFs (p32^−/−), knockout MEFs with reintroduced p32 cDNA (p32^−/−::p32) and knockout MEFs with introduced vector-only cDNA (p32^−/−::vector), respectively. Lower panel: pyruvate (1 mM) was added to wild-type and p32^−/− MEFs. The cell number was counted at 24, 48, 72 and 96 h after seeding. Here, we used DMEM (1000 mg/l glucose) supplemented with 10% dialyzed FBS without pyruvate.

Figure 2. — Reduced mitochondrial respiratory activities in p32^−/− MEFs. (A) The activity of each complex was measured using cell lysates as described in ‘Materials and Methods’ section. Reduced enzymatic activities of mitochondrial complexes I, III and IV in p32-knockout cells were observed. In contrast, complex II activity was not decreased. Re-expression of p32 in p32^−/− cells restored the enzymatic activities of complexes I, III and IV. The results represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 versus controls (p32^+/+ versus p32^−/− and p32^−/−versus p32^−/−::p32). (B) Oxygen consumption by digitonin-permeabilized wild-type (p32^+/+) and knockout (p32^−/−) MEFs. Glutamate and malate (glu+mal) respiration depends on the activities of complexes I, III and IV; glycerol-3-phosphate and succinate (G3P+suc) respiration depends on the activities of complexes III and IV. Rotenone (a complex I inhibitor) and Antimycin A (a complex III inhibitor) completely inhibit O₂ consumption at each step. Data show the mean ± SD of triplicate experiments and **P < 0.01 versus controls (p32^+/+ versus p32^−/−). (C) Decreased mitochondrial membrane potential in p32-knockout MEFs. MEFs were stained with the fluorescent dye JC-1 and then analyzed by flow cytometry at 527 and 590 nm. The fluorescence ratio of JC1 dimer (Red)/JC-1 monomer (Green) is shown. Dissipation of the membrane potential with CCCP was used as a control. (D) Effects of 2-deoxy-d-glucose (2DG) (20 mM) and oligomycin (10 μM) on intracellular ATP content in MEFs. The ATP content in untreated MEFs is presented in lane 1. The ATP concentration of untreated MEFs (lane 1) was subtracted from that of 2DG-treated MEFs (lane 2) for assessment of glycolytic ATP production (lanes 1 and 2). The ATP concentration in 2DG-treated cells (lane 2) was subtracted from that in cells treated with 2DG + oligomycin (lane 3) for mitochondrial ATP production (lanes 2–3). Wild-type (p32^+/+), p32-knockout (p32^−/−), re-expressed p32 (p32^−/−::p32) and vector only-transfected (p32^−/−::vector) MEFs were used. The ATP response was measured using a Luminescence ATP assay kit in a 96-well plate. Data show the mean ± SD of triplicate experiments. (E) Cell proliferation monitored in MEFs. Upper panel: the diamonds, squares, triangles and circles represent wild-type MEFs (p32^+/+), p32-knockout MEFs (p32^−/−), knockout MEFs with reintroduced p32 cDNA (p32^−/−::p32) and knockout MEFs with introduced vector-only cDNA (p32^−/−::vector), respectively. Lower panel: pyruvate (1 mM) was added to wild-type and p32^−/− MEFs. The cell number was counted at 24, 48, 72 and 96 h after seeding. Here, we used DMEM (1000 mg/l glucose) supplemented with 10% dialyzed FBS without pyruvate.