Fig. 7: CBD directly interacts with mitochondria.
a Isolation of mitochondria from Jurkat cells was corroborated by flow cytometry analysis. Representative experiment is shown. The purity of mitochondria population was determined as a percentage of double positive particles after staining (20 min) with MtGreen (200 nM) and Rhod-2 (2 μM). First, the mitochondria population was visualized in FSC/SSC dot blot (upper left panel). Non-stained mitochondria were used to determine the autofluorescence interval (gray histograms and corresponding intervals in upper right and lower left panels). Accordingly, positive intervals for MtGreen (green histogram and corresponding interval in upper right panel) and Rhod-2 (red histogram and corresponding interval in lower left panel) were determined. More than 90% of freshly isolated mitochondria were double-positive (lower right panel). Freshly isolated mitochondria were assayed immediately. b [Ca2+]m uptake by freshly isolated mitochondria in a response to vehicle or CBD (30 μM) treatments. Traces are mean ± SD for three independent experiments. c CBD effect on membrane potential (Δψm) in isolated mitochondria. Freshly isolated mitochondria were stained with TMRE (400 nM) and exposed to different CBD concentrations for 10 min. FCCP was used as a positive control. Data are mean ± SD of at least three independent experiments (****p < 0.0001, one-way ANOVA test). d In silico analysis predicts that CBD interacts with VDAC1 at N-terminus and β9–11 residues. e Residues, interacting with CBD are depicted; hydrogen bonding interactions are colored in red, whereas steric interactions are depicted in blue
