Research Paper Volume 14, Issue 11 pp 4673—4698

Figure 5. Characterizing the role of the PI3K/Akt/mTOR pathway in promoting CXCR4 overexpression-mediated ovarian cancer invasion, EMT, and CSC stemness. PI3K/Akt/mTOR pathway-related protein phosphorylated states indicated were analysed in both CXCR4 shRNA knockdowned-OVCA420 and CXCR4 overexpressed SKOV3 cells by WB with the indicated antibody against each protein, respectively (A, C). Band density ratios of phosphorylated-PI3K (p-PI3K), -Akt (p-Akt) and -mTOR to total-PI3K (PI3K), -Akt (Akt) and -mTOR (mTOR) were determined by densitometry analysis, respectively (B, D). Effects of MK-2206 on inhibiting SKOV3 cell invasion induced by CXCR4 overexpressing were analysed by a transwell tumour cell invasion assay (E, upper panel). Effects of MK-2206 on inhibiting SKOV3 cell spheroid formation capacity induced by CXCR4 overexpressing were analysed by a spheroid culture in hanging drops assay (E, lower panel), which were quantified by counting the total number of cells (invasion rate) from 10 random fields (magnification, 200X) (F), and the total spheroid hanging drop area (percentage of control) from the CXCR4-overexpressed SKOV3 culture cell experiments, respectively (G). Furthermore, effects of MK-2206 on inhibiting the expression of p-Akt, Akt, EMT-related proteins (E-cadherin, N-cadherin, vimentin and snail) in the CXCR4 overexpressed SKOV3 cells were analysed by WB with the indicated antibody against each protein examined (H). Band density ratios of p-Akt to Akt, E-cadherin, N-cadherin and vimentin to β-actin were determined by densitometry analysis, respectively (I). Data are presented as the mean ± SD of three independent experiments. Asterisk indicates P<0.05 compared with the controls as determined by t test. Please note that CSC related protein expression profiles in the CXCR4 overexpressed SKOV3 cell line in the presence or absence of MK-2206 treatment were described in the supplementary materials (Supplementary Figure 5).