Fig. 5

Arf6–AMAP1 pathway via ILK negatively regulates RhoT1–TRAK2 interaction for efficient distribution of mitochondria. a–c MDA-MB-231 cells were transfected with siRNA targeting TRAK1 or TRAK2. Protein expression of TRAK1 or TRAK2 was analyzed by western blotting (a). Mitochondria (green), microtubules (red), and nuclei (blue) were fluorescently visualized by specific antibodies or dyes. Bar, 10 μm (b). Mitochondrial distribution was quantified (c). d–f MDA-MB-231 cells were transfected with siRNA targeting AMAP1, Arf6, ILK, TRAK1, or TRAK2, in the combinations indicated. Expression of each protein was analyzed by western blotting (d). Mitochondria (green), microtubules (red), and nuclei (blue) were fluorescently visualized by specific antibodies or dyes. Bar, 10 μm (e). Mitochondrial distribution was quantified (f). g, h MDA-MB-231 cells stably expressing V5-RhoT1 were serially transfected with the indicated siRNAs and then plasmid DNA encoding Xpress-tagged TRAK1 or TRAK2. Parental cells were also used as a control. Immunoprecipitation by the anti-V5 antibody was performed using lysates of the cells expressing Xpress-TRAK1 (g) or TRAK2 (h). Asterisks indicate non-specific bands. (i) Immunoprecipitation using lysates of MDA-MB-231 cells transfected with the indicated siRNAs, and the anti-ILK antibody. j–l MDA-MB-231 cells were transfected with siRNA targeting TRAK1 or TRAK2. ROS production was visualized by DHE (j), and quantified (k). Bar, 50 μm. Cumulative cell death after IR treatment was also measured (l). All graphs indicate the mean ± SEM of three independent experiments. * and ^#P < 0.05; ** and ^##P < 0.005 (two-tailed t-test, adjusted by the Holm–Sidak method). * and **, comparison to si-NC samples; ^# and ^##, comparison to the corresponding samples (i.e., si-AMAP1 + si-NC vs. si-AMAP1 + si-TRAK2)