Figure 2.

Effects of treatments with Doxo alone and in combination with radicicol on (A) mitochondrial Cx43 expression, (B) mitochondrial ROS production, and (C) mitochondrial membrane potential of H9c2 cells. Doxo (1 µM) was administered for 3 and 6 h, and where indicated, radicicol (1 µM) was administered 30 min prior to Doxo. (A) Mitochondrial Cx43 expression was detected by western blot analysis. TOM20 protein expression was used as the loading control. Inset, top panel, representative western blot for Na⁺/K⁺ATPase and Ox-phos complex II is shown as markers of mitochondria and sarcolemma, respectively, to demonstrate the purity of the mitochondrial extracts. (B) Mitochondrial superoxide production evaluated using MitoSOX red in the H9c2 cells by flow cytometry, as the percentages of MitoSOX Red-positive cells. (C) Mitochondrial membrane potential was evaluated by flow cytometry analysis with tetramethylrhodamine ethyl ester (TMRE), a cell permeant, positively charged, red-orange dye that penetrates cells and accumulates in the mitochondria in inverse proportion to the membrane potential. A low percentage of TMRE-positive cells indicates that the TMRE dye was not trapped within the mitochondrial membranes, due to its depolarization. Data are the means ± SEM of fluorescence intensity, from at least 3 independent experiments, each performed in duplicate. ^*P<0.05, ^**P<0.005, ^***P<0.001 vs. control; °P<0.05, °°P<0.005 vs. cells treated with Doxo under the same conditions (one-way ANOVA and multiple comparisons by Bonferroni's test). Doxo, doxorubicin; Rad, radicicol.