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Fig. 5 | Acta Neuropathologica Communications

Fig. 5

From: Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis

Fig. 5

β-sitosterol reduces mitochondrial respiration through complex I inhibition. a-b Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured to assess rates of mitochondrial respiration and glycolysis, respectively, in H1_DL2 cells treated with 50 μM β-sitosterol or 0.05% DMSO for 24 h (both: n = 4). a Basal respiration was determined, followed by sequential additions of oligomycin (3 μM) to assess respiration due to proton leak, carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 1.5 μM) to measure respiratory capacity, rotenone (1 μM) to assess Complex I (CI) independent respiration and antimycin A (AMA; 1 μM) to determine background OCR. b Glucose (10 mM) was provided to determine basal glycolysis, followed by sequential additions of oligomycin (3 mM) to obtain glycolytic capacity, CCCP (1.5 μM) to evaluate the influence of uncoupling and 2-deoxyglucose (2-DG; 100 mM) to measure the non-glycolytic background. c High-resolution respirometry in H1_DL2 cells to detect direct effects of β-sitosterol. First, the maximal CI + CII driven respiratory capacity was measured in the presence of digitonin (8.1 μM), malate (2 mM), pyruvate (1 mM), succinate (10 mM) and carbonylcyanide-4-(trifluoromethoxy)-phenylhydraqone (FCCP, 0.18 μM). The respiratory rate was then measured after adding β-sitosterol (50 μM) or DMSO (0.05%), followed by rotenone (0.5 μM) to inhibit CI, and AMA (2.3 μM) to determine residual oxygen consumption. The experiment was repeated 3 times. a-c Student’s t-test: n.s. = not significant, P ≥ 0.05, **** P < 0.0001. Values are given as the mean ± s.d

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