Fig. 2

Lunasin induces PTEN expression and nuclear localization. a Transcript levels of PTEN in MCF-7 cells treated with GEN (20 nM and 2 μM) or Lunasin (50 nM and 2 μM) were quantified by qRT-PCR, normalized to that of 18S, and renormalized to those of the vehicle-treated group. Each bar represents mean ± SEM, expressed as fold-change. Results are representative of three independent experiments, with each experiment performed in triplicates. Different superscripts are different at P < 0.05 by one-way ANOVA. b MCF-7 cells were co-transfected with PTEN promoter–reporter construct (PTEN-Luc) or empty vector (pGL3-basic) and pRL-SV40 and then treated with vehicle or Lunasin (LUN) for 24 h. Luciferase activity is expressed as relative luminescence units (RLU) normalized to pRL-SV40. Means (±SEM) with different superscripts are significantly different (P < 0.05 by two-way ANOVA). c Total cell lysates from vehicle or LUN (2 μM)-treated cells were immunoprecipitated (IP) with PTEN anti-mouse antibody or control IgG followed by western blot analysis (IB) using PTEN anti-rabbit antibody. A representative blot is shown, with each lane representing individual samples. d The percent of nuclear PTEN-positive staining MCF-7 cells after treatments with GEN or LUN for 24 h was determined by counting five random areas per slide, with at least three slides per treatment group. Means (±SEM) with different superscripts are significantly different (P < 0.05, one-way ANOVA)