Research Paper Volume 5, Issue 12 pp 884—901

Figure 5. Increased ROS levels in eIF2αP-deficient immortalized cells. (A, B) Primary fTg/eIF2αA/A MEFs were subjected to immortalization by the expression of SV40 large T Ag followed by lentivirus-mediated expression of Cre (A) or retrovirus-mediated expression of Cre-ERT2 (B). Cre-ERT2 cells were treated with 1μM tamoxifen for 72 hours (B, lane 2). As control, cells were infected with insert-less retroviruses (A,B, lane 1) in the presence of 1 μM tamoxifen (B, lane 1). Protein extracts (50 μg) from proliferating cells were used for immunoblot analysis for the indicated proteins. (C,D) ROS levels were measured in immortalized fTg/eIF2αA/A MEFs expressing either Cre-ERT2 (C) or constitutive Cre (D) by Cell-RoxTM Deep Red staining and FACS analysis. Histograms represent the average ROS levels measured by CellRox fluorescence in three independent experiments. (E,F) Colony formation assays of untreated as well as Trolox-treated immortalized fTg/eIF2αA/A MEFs expressing a constitutive Cre (E) or a tamoxifen inducible Cre-ERT2 (F). Cells were visualized by crystal violet staining. Values represent ratios of optical density (OD) in arbitrary units.