Effects of MA on cardiac cell death and nuclear translocation of NF-κB.

<p>(<b>A</b>) Cell viability by MTT assay. H9C2 cells were incubated with glucose (5.5, 22, and 33 mM) for 48 hrs. Different concentrations of multiple antioxidant mixture of N acetyl cysteine (5 mM), Ascorbic acid (100 µM), α-tocopherol acetate (50 µM), β-carotene (5 µM) and Selenium (100 nM) (0.01, 0.05, 0.1, 0.5 and 1X) were added for 48 hrs and cell viability was determined by MTT assay; significance of high glucose over control at <i>P</i><0.05. <b>(B)</b> Western blot analyses of nuclear and cytosolic fractions prepared from low and high glucose treated H9C2 cells with and without multiple antioxidants mixture (0.1X) for 48 hrs. Membranes were probed with anti-NF-κB p65 antibodies, stripped, and re-probed with GAPDH or anti-PARP antibodies to determine even protein loading and purity of cytosolic [C] and nuclear [N] fractions, respectively. (<b>C</b>) Immunoblot analysis of pro-oxidant genes in cells transfected with p65/RelA- siRNA or the vector alone, after transfection H9C2 cells were treat with 5.5 and 33 mM of glucose for 48 hrs. Membranes were probe anti-NF-κB p65, 5-LO, MAO-A, XO and Nrf-2 antibodies. β-actin antibodies to ensure even protein loading in each lane.