Research Paper Volume 12, Issue 13 pp 13023—13037

Figure 2. The phosphorylation of Akt and mTORC1 positively correlates with cell invasion ability and is regulated by the Gαh-PLC-δ1 pathway in TNBC cells. (A) Results from the western blot analysis for the Gαh, phosphorylated Akt (p-Akt), Akt, p-mTOR, mTOR and GAPDH proteins derived from the indicated TNBC cell lines. (B) Giemsa staining of the invaded cells of the tested TNBC cell lines after a 16-hour invasion assay. (C) Correlation of mRNA expression levels between Gαh and the mTORC1 gene set in a panel of breast cancer cell lines derived from the Cancer Cell Line Encyclopedia (CCLE) database. Spearman’s correlation test was used to estimate the statistical significance. (D–E) Results from the western blot analysis for the Gαh, p-Akt, Akt, p-mTOR, mTOR and GAPDH proteins derived from the parental (PT) HCC1806 cells without (vector control, VC) or with Gαh overexpression (D) and the parental MDA-MD231 cells without (nonsilenced, NS) or with Gαh knocked down using two independent shRNA clones (E). (F–H) MDA-MD231 cells treated without or with 10 μM Gαh/PLC-δ1 protein-protein interaction (PPI) inhibitor for 2 hours were subjected to a reciprocal immunoprecipitation for detecting the PPI of Gαh/PLC-δ1 (F), Western blot analysis for measuring the protein levels of p-Akt, Akt, p-mTOR, mTOR and GAPDH (G), and immunofluorescent staining for visualizing the intracellular protein levels of p-Akt and p-mTOR (H). In A, D, E, G, GAPDH was used as an internal control of protein loading. The protein intensities of representative blots from three independent experiments were normalized by GAPDH levels and presented as a ratio to the control group.