FIGURE 5.

a, FACS analysis of the antiapoptotic protein Bcl-xL following SB216763 treatment. Our co-cultured group of cells treated with SB216763 demonstrates a higher intensity of Bcl-xL after 24 h than untreated cells. Moreover, the kinetics of Bcl-xL loss is significantly slower temporally in the treated group. Collectively, these findings suggest that the GSK-3β inhibitor SB216763 may work through Bcl-xL to protect Treg cells from apoptosis in vitro. All data are representative of at least two independently performed experiments, gating on CD4⁺CD25^+hiFoxP3⁺ cells. B, Western blot analysis of CD4⁺CD25⁺ cells treated in vitro with SB216763. Cells treated with SB216763 maintain increased Bcl-2 levels up to 66 h versus untreated cells. The Western blot was stripped and reprobed with anti-β-actin antibody as a loading control. inh., inhibitor. C, effect of caspase-3 inhibition, using DEVD-CHO, on Treg cell suppression. Performing a classical suppression assay, the use of 100 μm DEVD-CHO, a caspase-3 inhibitor (Casp. Inh), resulted in a 20% increase in suppression activity. When caspase-3 inhibitor was used with GSK-3β inhibitor (GSK-3 Inh), we observed no additive affect, suggesting that the two inhibitors may be acting on the same or parallel pathways. Data are represented as means ± S.E. of triplicate samples. All data are representative of at least two independently performed experiments.