Figure 3

Treatment with Chondramide A (ChA) leads to the dislocation of Hexokinase II (HkII) from mitochondria and activation of pro-apoptotic Bad. (a) MCF-7 cells transfected with mGFP-β-actin were treated with 300 nM ChA for 6 h, fixed and co-stained with Mitotracker red CMX-ROS and anti-Hexokinase II antibody followed by confocal microscopic analysis. Scale bar indicates 10 μm. Representative images are shown. (b) Western blot of HkII in mitochondrial fraction of MDA-MB-231 cells treated with 300 nM ChA for 6 and 24 h. Cytosolic and mitochondrial fractions were separated. Immunoblotting for β-tubulin and COX IV were used for controlling purity of mitochondrial fraction and loading control, respectively. Representative blot of the mitochondrial fraction and the respective quantification is shown. (c) Western blot analysis for Bad and phospho-(Ser112)-Bad in MCF7 and MDA-MB-231 cells treated with 100 and 300 nM ChA for 6 h and quantitative analysis of band intensities. (d) MCF7 cells transfected with mGFP-β-actin were treated with 300 nM ChA for 6 h, fixed and co-stained with Mitotracker red CMX-ROS and anti-Bad-antibody followed by confocal microscopic analysis. Scale bar indicates 10 μm. Representative images are shown. Each experiment was performed independently three times