Figure 4: The critical role of Sam68 in the expression of the CD25 gene in T cells.

(a) Jurkat cells were transfected with nonspecific (NS) or three different Sam68-specific siRNAs. After 72 h, the knockdown of Sam68 was examined by immunoblot (IB) for Sam68 with β-actin as a loading control. (b) NF-κB luciferase assay (mean±s.d., n=3) of Jurkat cells transfected with nonspecific (NS) or Sam68 siRNAs, together with indicated amount of p65 plasmids and a luciferase reporter gene driven by 5 × CD25 κB sites. (c) Real-time PCR quantization (mean±s.d., n=3) of mRNA level of CD25 normalized to GAPDH in Jurkat cells silenced with NS or pooled Sam68 siRNAs and stimulated by 50 ng ml⁻¹ PMA plus 1.5 μM ionomycin (PMA/I) for indicated time. (d) Human peripheral blood T lymphocytes were transfected with nonspecific (NS), p65, or Sam68 siRNAs and then stimulated with PMA/I or left unstimulated (Unstim). Flow cytometry histograms of T cells with CD25 induction and carboxyfluorescein succinimidyl ester (CFSE) dilution were assessed 12 h and 72 h later, respectively. The percentage of CD25⁺ cells and of dividing cells is shown. (e) Flow-sorted in vitro activated human CD4⁺CD25⁺ cells were transfected with nonspecific (NS), p65 or Sam68 siRNAs. The surface CD25 expression was analysed at 72 h, and the percentage of CD25⁺ cells is shown.