Fig. 4
Tyrphostin A9 acts as ROS-reducing mitochondrial uncoupler in mDA neurons. (A) mDA neurons were treated with 0.1–10 µM Tyrphostin A9 for 72 h and the mitochondrial membrane potential was measured using the JC1 dye. JC1 fluoresces red at high membrane potential and green at lower membrane potentials. Three independent experiments with at least three technical replicates each were performed. (B) mDA neurons were treated with 0.5, 1, and 2.5 µM Tyrphostin A9 for 72 h and the ROS level was determined using the CellROX dye. The obtained signal was normalized to total protein content. Three independent experiments with at least three technical replicates each were performed. (C) Left panel: Scheme illustrating the protonophore mode of action. Right panel: mDA neurons were treated with 0.5, 1, and 2.5 µM Tyrphostin A9 for 72 h and the intracellular Calcium level was measured using the Rhod-FF dye for 250 s before and after the addition of Ionomycin. The obtained signal was normalized to total protein content. Two independent experiments with at least three technical replicates each were performed. (D) Neuronal activity was measured in ca. 60-day old mDA neurons using a microelectrode array (MEA). The neuronal firing rate was assessed before and up to 72 h after treatment with 2.5 µM Tyrphostin A9. Neuronal activity was measured in two independent experiments with at least three technical replicates each. For all data, the mean and 95% confidence interval is shown.
