Fig 2: Increased Par-4 expression enhances the sensitivity of MCF-7 breast cancer cells to docetaxel. MCF-7-pcPar-4 and MCF-7-pcNeo cells were cultured in 8-well chamber slides, treated with low (5 nM) and high (100 nM) doses of docetaxel for 24 h, fixed in 4% paraformaldehyde and co-stained with acridine orange (AO) and Hoechst 33342. (A) Nuclear morphology was examined by fluorescence microscopy. (B) For quantification, attached cells stained with AO displaying abnormal nuclear morphology from 10 different areas of the well were counted using ImageJ software. Data are expressed as mean ± SD of three experiments. *Statistically significant differences, P=0.05.

Fig 3: Increased Par-4 protein expression inhibited cell proliferation and ERK phosphorylation in MCF-7 breast cancer cells. (A) Cell proliferation and viability were measured by MTT assay in MCF-7-pcPar-4 and MCF-7-pcNeo cells (1×104 cells) seeded in 96-well plates and maintained in normal serum (NS) for 24, 48 and 72 h. Experiments were performed in triplicate and the values are given as mean absorbance (OD 570 nm) with SD. (B and C) Cellular pERK protein levels were determined by western blot and densitometric analysis, respectively. (D) Relative PAR-4, BAX, BID, BCL-2 and BECLIN mRNA expression were determined by qRT-PCR, normalized to GAPDH. Bar height represents gene expression in cells maintained in NS relative expression in control cells (MCF-7-pcNeo). All data are expressed as mean ± SD of three experiments. *Statistically significant differences, P=0.05. (E) Flow cytometric analysis of MCF-7-pcPar-4 or control MCF-7-pcNeo using propidium iodide (PI) staining. (F) Proportion of cell death, evaluated by the cell population in sub-G1.

Fig 4: Increased Par-4 expression results in induction of early apoptosis and increased sensitivity to docetaxel. MCF-7-pcPar4 and MCF-7-pcNeo cells were treated with docetaxel for 24 h, harvested, stained with Annexin V-FITC (AnV) and/or propidium iodide (PI) and analyzed by flow cytometry. The proportion of cells positive for AnV and negative for PI (early apoptosis) and positive for both are shown in each panel.