Fig 1: DGKa silencing or pharmacological inhibition rescues IL-2 defects in CDC42- and NCK-1-deficient lymphocytes (A) Activated lymphocytes from healthy donors were transfected using siRNAs ctrl, CDC42, and DGKa as indicated. After 4 days, transfected cells were restimulated using anti-CD3 (OKT3, 1 µg/ml) and anti-CD28 (CD28.2, 1 µg/ml) for 4 hours followed by quantitative rt-PCR gene expression analysis to verify IL-2 expression. Data are the mean ±SEM of six experiments from three individual healthy donors. (B) Activated lymphocytes from healthy donors were transfected using siRNAs ctrl or CDC42. After 4 days, transfected cells were restimulated using anti-CD3 (OKT3, 1 µg/ml) and anti-CD28 (CD28.2, 1 µg/ml) for 4 hours in the presence or absence of the DGKa specific inhibitor (AMB639752 - 10 µM), followed by quantitative rt-PCR gene expression analysis to verify IL-2 expression. Data are the mean ±SEM of eight experiments performed in triplicate from six individual healthy donors. (C) Activated lymphocytes from healthy donors were transfected using siRNAs ctrl, NCK-1, and DGKa as indicated. After 4 days, transfected cells were restimulated with anti-CD3 (OKT3, 1 µg/ml) and anti-CD28 (CD28.2, 1 µg/ml) for 4 hours followed by quantitative rt-PCR gene expression analysis to verify IL-2 expression. Data are the mean ±SEM of six experiments from four individual healthy donors. (D) Activated lymphocytes from healthy donors were transfected using siRNAs ctrl or NCK-1. After 4 days, transfected cells were restimulated using both anti-CD3 (OKT3, 1 µg/ml) and anti-CD28 (CD28.2, 1 µg/ml) for 4 hours in the presence or absence of the DGKa specific inhibitor (AMB639752 - 10 µM), followed by quantitative rt-PCR gene expression analysis to verify IL-2 expression. Data are the mean ±SEM of six experiments from six individual healthy donors for gene expression analysis and all experiments were performed in triplicate. ns, non significant. A single, double, triple, and four asterisks denote their significance of a p-value = 0.05, = 0.01, = 0.001 and = 0.0001 respectively in all experiments.