Figure 7.

Vascular endothelial growth factor receptor (VEGFR) 2 blockade by Ki8751 interferes with VEGF intracellular signaling in breast cancer cells. The breast cancer cells, MCF-7 and MDA-MB-231, were seeded in 6-well plates (1.5 × 10⁵ cells/well), and cultured with vehicle (0.01% dimethyl sulfoxide), 2.5, or 5 μM Ki8751 for 24 or 48 h. The whole cell lysates were prepared using EBC buffer (50 mM Tris at pH8.0, 120 mM NaCl, 0.5% NP-40) containing protease inhibitors and phosphatase inhibitors. Samples containing the same protein amounts were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Representative blotting images of p-Akt, Akt, p-PGC1α, PGC1α, and TFAM of MCF-7 cell (panel A) and MDA-MB-231 cell lysates (B) are shown from not less than 3 independent experiments. To demonstrate PGC1α intracellular mobilization, MCF-7 cells and MDA-MB-231 cells were treated with 5 μM Ki8751 for 24 h and fixed with 4% paraformaldehyde. Representative fluorescent images of PGC1α and 4′6′-diamidino-2-phenylindole are shown in (C and E) and representative immunoblotting analysis of cytoplasmic and nuclear fractions from MCF-7 and MDA-MB-231 cells are shown in (D and F); mean ± SEM are plotted in the bar charts, n = 4, **P < 0.01. Panel G: MCF-7 cells were treated with vehicle or 2.5 μM Ki8751 in the presence or absence of siRNA SMARTPools specific PGC1α or mitochondrial transcription factor A for 48 h. Afterwards, cancer cells were harvested for flow cytometric analysis of mitochondrial mass. Mean ± SEM, n = 4, **P < 0.01 compared to the control, †P < 0.05, compared MCF-7 cells treated by Ki8751 alone.