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. 2016 Jan 15;30(2):234–247. doi: 10.1210/me.2015-1281

Figure 2.

Figure 2.

Establishment of a mitochondrial reconstitution assay system. A, Electron micrograph of isolated rat adrenal mitochondria. Scale bar, 1 μm. B, Purity of recombinant SNARE proteins after Ni-NTA agarose chromatography. C, Short time course of pregnenolone production. Mitochondria were pretreated with trilostane (5 μM) and abiraterone (10 μM) for 2.5 minutes at room temperature before being added to the reconstitution assay for the indicated times. D, Effects of BSA on pregnenolone production. The incubation was conducted for 10 minutes. E, Effects of recombinant SNARE proteins and adrenal cytosol (cyto) on pregnenolone production compared with 25-hydroxycholesterol. The incubation was conducted for 10 minutes. F, Effect of recombinant SNARE proteins and cytosol on cholesterol movement to mitochondria. Mitochondria were pretreated with 100 μM aminoglutethimide (AMG) for 10 minutes at room temperature before being added to the reconstitution assay and incubated for 1 hour. G, Confocal image of the effects of SNARE proteins and BSA on cholesterol movement to mitochondria (100 times). Mitochondria (red) were labeled with MitoTracker Red FM (Invitrogen), and cholesterol (green) was labeled with bodipy-CE. Scale bar, 2 μm. In panels D, E, and G, mitochondria were pretreated with trilostane (10 μM) for 2.5 minutes at room temperature before being added to the reconstitution assay. Pregnenolone was measured by an ELISA in each group. *, P < .05; **, P < .01, ***; P < .001. All, cocktail containing all recombinant SNARE proteins; LE, lipid emulsion; M, mitochondria. Results are representative of three independent experiments, each with an n = 3..