Figure 4

tBHP induces formation of BAX clusters close to the mitochondrial network. (Top panel) Control and tBHP treated cells were incubated with 125 nM Mitotracker red (red) for 30 min previous to fixation and subsequently labeled with an antibody to BAX (green) and counterstained with Hoechst (blue). (A-C) Control cells showing a homogeneous BAX distribution throughout the cell and filament-shaped mitochondria. (D-F) and (G-I) H9c2 cells treated with 50 μM and 100 μM tBHP, respectively, during 1 hour, showing BAX aggregates located close to the mitochondrial network and a change in mitochondria morphology from a filament-like form to small round mitochondria. (J) and (K) Magnification of the cell indicated by an arrow in Panel E (J-BAX immunolabeling; K-Mitotracker red labeling). Images are representative from 3 different cell preparations. Scale bar corresponds to 20 μM. (Bottom panel) Quantification of the cell fluorescence intensity of Hoechst, Bax immunolabeling and Mitotracker red. Data represent the mean ± SEM of 3 different cell preparations. *p < 0.05 vs 0 μM tBHP, ^# p < 0.05 vs 50 μM tBHP.