FIG. 3.

Oxidative stress in GPx4 knockdown cells. NIH 3T3 cells stably transfected with pU6-m3, siGPx1, or siGPx4-3 were grown and intracellular ROS were measured following a 30 min incubation with 10 μg/ml of H₂DCFDA, as described in Materials and Methods. In (A), cells were seeded in a chamber coverglass with growth media (without phenol red), grown for 24 h, incubated with H₂DCFDA, and the cells imaged with a confocal microscope. The left panel shows the fluorescent patterns and the right panel the phase contrast in the two cell lines. In (B), cells stably transfected with pU6-m3, siGPx1, or siGPx4-3 were grown and treated with H₂DCFDA as in (A), harvested with a cell lifter and analyzed immediately by flow cytometry. H₂O₂-treated pU6-m3 was shown as a positive control. In (C), NIH/pU6-m3 and NIH/siGPx4-3 cells were treated with NAC or α-tocopherol as described in Materials and Methods and growth rate of the cells was assessed after 48 h. *p < 0.01; α-tocopherol-treated NIH/siGPx4-3 was compared with buffer-treated NIH/siGPx4-3.