Journal: International Journal of Molecular Sciences

Article Title: Cytopathological Outcomes of Knocking down Expression of Mitochondrial Complex II Subunits in Dictyostelium discoideum

doi: 10.3390/ijms23095039

Figure Lengend Snippet: SdhA reduces ATP production, and this is rescued by AMPK knockdown. SdhB and C subunits alter different parameters of mitochondrial respiration to SdhA. In each experiment, cells of the wild type and Sdh antisense transformants or SdhA/AMPK antisense cotransformants were plated into four wells per sample of a Seahorse XFe24 plate. Mitochondrial respiration was measured by the rate of oxygen consumption. The basal respiration rate ( A ) was measured followed by the addition of DCCD (dicyclohexylcarbodimide), CCCP (carbonyl cyanide m-chlorophenyl hydrazone), and rotenone. Then either Antimycin A (complex III inhibitor) or BHAM (benzohydroxamic acid, AOX inhibitor) was added to the wells. Total activity for Complex II was calculated by adding the effects of Antimycin A and BHAM. All OCR measurements were normalised to the wild type strain in each experiment. Panels B–G represent each component of mitochondrial respiration: ATP synthesis ( B ), Maximum capacity ( C ), Complex I ( D ), Complex II total which is the combination of Complex II/III/IV and Complex II/AOX ( E ), oxygen consumption by nonmitochondrial processes ( F ) and oxygen consumption attributable to mitochondrial processes that do not generate ATP termed proton “leak” ( G ). The spare capacity is the difference between the maximum, uncoupled respiratory capacity of the cells and the basal respiration rate calculated as maximum OCR minus basal OCR.

Article Snippet: Basal Oxygen Consumption Rates (OCR) were measured, followed by the injection of a series of mitochondrial inhibitors and reagents: [10 μM DCCD (N,N0-dicyclohexylcarbodimide, an ATP synthase inhibitor (Sigma-Aldrich, St. Louis, MI, USA), 10 μM CCCP (carbonyl cyanide 3-chlorophenol hydrazone, a protonophore (Sigma-Aldrich, St. Louis, MI, USA)], 20 μM rotenone [Complex I inhibitor (Sigma-Aldrich, St. Louis, MI, USA)] and either 10 μM antimycin A [Complex III inhibitor (Sigma-Aldrich, St. Louis, MI, USA)] or 1.5 mM BHAM [benzohydroxamic acid, alternative oxidase (AOX) inhibitor (Sigma-Aldrich, St. Louis, MI, USA)].

Techniques: Activity Assay