Overexpression of SERPINB4 in HeLa cells inhibits GrM-induced cell death.

<p>(<b>A</b>) RT-PCR analysis of stably transfected HeLa cells for SERPINB4 and GAPDH mRNA expression (upper and lower panel, respectively). (<b>B</b>) Immunoblot analysis of SERPINB4 protein expression by HeLa cells stably transfected with pcDNA3 SERPINB4, pcDNA3 SERPINB4 RCL-mutant or pcDNA3 empty vector. (<b>C</b>) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (0.5 µM), and/or recombinant GrM-SA (0.5 µM) for 16 h at 37°C. Viable cells were quantified using the methylene blue assay. Data represent the percentages of viable cells as compared to HeLa cells that overexpressed SERPINB4 RCL-mutant and were treated with buffer only, which was set as 100%. Figure represents the mean ± SD of 4 independent experiments; * <i>p</i><0.05. (<b>D</b>) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (1 µM), and/or recombinant GrM-SA (1 µM) for 20 h at 37°C. Cell viability was determined using flow cytometry, with AnnexinV and PI negative cells considered viable (mean ± S.D., <i>n</i> = 3, ** <i>p</i><0.005). (<b>E</b>) HeLa cells stably transfected with SERPINB4 or SERPINB4 RCL-mutant were treated with the indicated combinations of a sublytic dose of SLO (500 ng/ml), recombinant GrM (1 µM), and/or recombinant GrM-SA (1 µM) for 4 h at 37°C. Total cell lysates were immunoblotted using antibodies against nucleophosmin and nm23H-1 (which served as a loading control).