Figure 6
Figure 6. The levels of H2O2 control Bim expression in T-cell blasts are reduced in T-cell blasts from the ALPS-Ic patient. 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 or in the presence of 400 μM BSO, 5 mM GSH, 1 μg/mL of NOK-1, or 0.5 μM DPI, as indicated. After the treatments, the intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry (A), or extracts were obtained and anti–phospho-Akt, phospho-ERK1/2, anti-Bim, or anti–β-actin immunoblots performed (C-D). In panel B, day-6 T-cell blasts were treated with increasing doses of the agonistic anti-Fas mAb CH-11 for 16 hours, and intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry. In panel A, results are expressed as the mean fluorescence intensity (MFI) values obtained in each case, and those values are also shown in panel B for each CH-11 concentration. In panel C, the levels of P-ERK1, P-ERK2, P-Akt, and BimEL were quantified in a densitometer and normalized to the same amount of β-actin, and the results obtained for the BimEL/β-actin, P-Akt/β-actin, and P-ERK1/2/β-actin ratios are shown. Results in panels A-D are representative of experiments performed with T-cell blasts from 3 different healthy donors. The samples shown in each immunoblot panel were run in the same gel. The vertical lines inside the subpanels in panel D indicate that lanes were cut from the same immunoblot membrane. (E) Day-6 T-cell blasts from a healthy control or from ALPS-Ic patient 1 were cultured for 24 hours in the presence of increasing doses of IL-2, as indicated, and the intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry. Results are expressed as the mean fluorescence intensity (MFI) values obtained in each case. (F) Day-6 T-cell blasts from a healthy control (black histogram) or from ALPS-Ic patient 1 (red histogram) were cultured for 48 hours in the presence of 300 IU/mL of IL-2, and the intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry. Numbers correspond to the MFI values in each case. Results are representative of 2 different experiments.

The levels of H2O2 control Bim expression in T-cell blasts are reduced in T-cell blasts from the ALPS-Ic patient. 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 or in the presence of 400 μM BSO, 5 mM GSH, 1 μg/mL of NOK-1, or 0.5 μM DPI, as indicated. After the treatments, the intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry (A), or extracts were obtained and anti–phospho-Akt, phospho-ERK1/2, anti-Bim, or anti–β-actin immunoblots performed (C-D). In panel B, day-6 T-cell blasts were treated with increasing doses of the agonistic anti-Fas mAb CH-11 for 16 hours, and intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry. In panel A, results are expressed as the mean fluorescence intensity (MFI) values obtained in each case, and those values are also shown in panel B for each CH-11 concentration. In panel C, the levels of P-ERK1, P-ERK2, P-Akt, and BimEL were quantified in a densitometer and normalized to the same amount of β-actin, and the results obtained for the BimEL/β-actin, P-Akt/β-actin, and P-ERK1/2/β-actin ratios are shown. Results in panels A-D are representative of experiments performed with T-cell blasts from 3 different healthy donors. The samples shown in each immunoblot panel were run in the same gel. The vertical lines inside the subpanels in panel D indicate that lanes were cut from the same immunoblot membrane. (E) Day-6 T-cell blasts from a healthy control or from ALPS-Ic patient 1 were cultured for 24 hours in the presence of increasing doses of IL-2, as indicated, and the intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry. Results are expressed as the mean fluorescence intensity (MFI) values obtained in each case. (F) Day-6 T-cell blasts from a healthy control (black histogram) or from ALPS-Ic patient 1 (red histogram) were cultured for 48 hours in the presence of 300 IU/mL of IL-2, and the intracellular levels of H2O2 were determined by DCF-DA staining and flow cytometry. Numbers correspond to the MFI values in each case. Results are representative of 2 different experiments.

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