Figure 8.

Effect of transfected vector on survival after oxidative stress. (A) MTT assay. Cells transfected with empty vector (sham transfectants), wild-type PIF or the N32Q, N44Q or N32Q N44Q mutant forms of PIF were seeded at 15 × 10⁴ cells per well in 96-well plates, incubated overnight, then treated with 75 mU ml⁻¹ glucose oxidase for 2 h prior to MTT assay. Wild-type PIF transfection resulted in a 26% increase in viable cells following glucose oxidase treatment in comparison with sham transfection. Transfection with the N32Q and N44Q mutants similarly protected HuH7 cells from oxidative stress. In contrast, transfection with the double mutant N32Q N44Q did not bestow a survival benefit. (B) Flow cytometry.Transfected cells were seeded at 1 × 10⁶ cells per well in six-well plates, incubated overnight, treated for 2 h with 75 mU ml⁻¹ glucose oxidase, then harvested. Flow-cytometric gating of cells by forward and side scatter revealed 37% survival of wild-type PIF transfected cells in comparison with 26% survival of sham transfectants, a survival benefit of 42%. Transfection with the N44Q mutant bestowed a similar benefit to transfection with wild-type PIF. Neither the N32Q nor the N32Q N44Q mutants conferred any survival benefit.