Fig. 6.

Overexpression of GCLC and GCLM in Ikkβ(−/−) cells partially restores GSH levels. Wild-type and Ikkβ(−/−) cells were transfected with empty or GCLC and GCLM expression vectors for 24 h. A, GSH and GSSG levels were measured and redox potentials calculated as in Fig. 1. B, cells were treated with sodium arsenite (50 μM) for 6 h, followed by CM-H₂DCFDA labeling. The nuclei were identified after staining with DAPI (blue) and the ROS-activated dichlorofluorescein fluorescence (green) was observed with fluorescence microscopy. Wild-type and/or the Ikkβ(−/−) cells transfected with empty or GCLC and GCLM expression vectors were treated with 50 μM sodium arsenite for 6 h. C, the cells were stained with DAPI, and cells with condensed nuclei (i.e., apoptotic cells) were identified under fluorescence microscopy. Numbers represent three fields of triplicate samples. Results were the mean values ± S.E. from at least three independent experiments. Statistical analyses were done by comparing the mean values in untransfected Ikkβ(−/−) cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 are considered significant. D, the cells were subjected to Annexin V staining and TUNEL analyses. The number of Annexin V and TUNEL-positive cells was counted, and the percentage of apoptosis was calculated based on total cell number.