Figure 3.

HOXA5 downregulates p53/p21 expression via activation of the PI3K/AKT signaling pathway in TAMR cells. (A) Western blotting for basal PI3K/AKT activity between parent MCF7 and TAMR cells. (B) Western blotting for PI3K/AKT pathway activation and the downstream p53 and p21 expression in MCF7 cells transfected with either the empty vector or HOXA5-overexpressing plasmids. (C) Western blotting for PI3K/AKT activation and downstream p53 and p21 expression in TAMR cells transfected with control or HOXA5 siRNAs. (D) Western blotting of PI3K/AKT activity and p53/p21 levels in HOXA5-overexpressing MCF7 cells after treatment with DMSO and LY294002 - a highly selective PI3K inhibitor for 24 hrs. (E) Representative scatter plots and quantification graphs showing the distribution of Annexin V and Propidium iodide (PI) staining from flow cytometry analysis. Parent cell lines and TAMR cells transfected with control or HOXA5 siRNAs were used for this analysis. Cells are classified as “viable” (Q3; bottom left), “early apoptotic” (Q4; bottom right), “late apoptotic” (Q2; top right), or “necrotic” (Q1; top left). (F) Western blotting for basal protein levels of full-length and cleaved caspases and PARP in parent MCF7 and TAMR cells. (G) Western blotting for the cleaved forms caspases and PARP after tamoxifen treatment in parent MCF7 and TAMR cells. (H) Western blotting for the cleaved forms of caspases and PARP after HOXA5 knockdown in TAMR cells. β-Actin was used as an internal control. All experiments were performed in triplicate.