H₂O₂ control Bim expression through regulation of Foxo3a levels in T-cell blasts. (A) Day-6 T-cell blasts were cultured in the presence of 30 IU/mL of IL-2 for 24 hours in the absence of any additional treatment (Control) or in the presence of 5 mM GSH, 400 μM BSO, 1 μM DPI, or 1 μg/ml of NOK-1, as indicated. After the treatments, the intracellular levels of H₂O₂ were determined by DCF-DA staining and flow cytometry, with similar percentages of increase or decrease as shown in Figure 6A (not shown), or extracts were obtained and anti-Foxo3a, anti–phospho-Foxo3a (P-Foxo3a), or anti–β-actin immunoblots performed. Results are representative of experiments performed with T-cell blasts from 4 different healthy donors. (B) Anti-Foxo3a or anti–β-actin immunoblots were performed on extracts from fresh PBMCs (day 0) or from day-6 T-cell blasts (day 6), obtained from healthy donors (Control) or from ALPS-Ia patient 2 (left panels), or from day-6 T cell blasts obtained from healthy donors (Control) or from ALPS-Ic patient 1 (right panels), as indicated. The extracts used correspond to 1 × 10⁶ cells. Results are representative of experiments performed with T-cell blasts from 3 different healthy donors and of 2 different experiments performed with T cells from the ALPS patients. The vertical line in the left subpanel of panel B is due to smear of the sample, revealed in the immunoblot. The samples shown in each immunoblot panel were run in the same gel.