Figure 3

MTS- and phosphorylation-dependent mitochondria localization of CTMP in U2OS cells. U2OS cells were transfected with (A) An N-terminal deletion mutant of CTMP (GFP-CT-CTMP ΔN31) or (C) The S37D/S38D negatively charged side group-mimic mutant form of CTMP (GFP-CT-CTMP-S37D/S38D) for 24 h. Differential localization of CTMP (Cyt: cytoplasm, PM: plasma membrane, Mit: mitochondria, Nuc: Nucleus) was examined using confocal microscopy. At least 200 cells were counted from three distinct fields for each transfected group. U2OS cells were co-transfected with (B) pEGFP-N3-Δ31-CTMP and pDsRed-mito for 24 h and analyzed. (D) HeLa cells were transduced with the indicated virus for 24 h, treated with 1 uM staurosporine for 3 and 6 h, harvested, and analyzed. Results are representative of three independent experiments. (E, F) Bands of cleaved PARP and caspase 9 were further analyzed by densitometry. Statistical differences of cleaved protein were determined by comparing values for actin at each lane. The results are mean ± SD of three independent experiments. Asterisk, p < 0.05; double asterisk, p < 0.01.