Figure 3. BMPER is required and sufficient to activate NFATc1.

A, BMPER knockdown blocks NFAT activation in response to LPS (10 μg/ml). MLECs were transfected with a mixture of NFAT-responsive firefly luciferase and renilla constructs, and BMPER or control siRNA. One day later, cells were treated with LPS and the luciferase activity was measured after another day’s incubation. B, BMPER increases transcriptional activity of NFAT in MLECs. MLECs were transfected with a mixture of NFAT-responsive firefly luciferase and renilla constructs. After 24 hours, cells were treated with BMPER or control for another 24 hours and the luciferase activity was measured. C–D, BMPER increases nuclear translocation of NFATc1. MLECs were treated with 10 nM BMPER for indicated time periods. Nuclear and cytoplasmic enriched fractions of cell lysates were used to determine the translocation of NFATc1. Lamin B1 (nuclear marker) and HSP90 (cytosol marker) immunoblotting were used to verify the purity of the fractions. NFATc1 protein levels in nuclear and cytosol fractions were quantified and shown in D. E, NFATc1 is required for IFNγ induction upon BMPER treatment. MLECs were transfected with NFATc1 or control siRNA. After 24 hours, cells were treated with BMPER at 10 nM or control and incubated for another 8 hours. Cells were harvested for real time PCR with IFNγ-specific probe and primers. * P<0.05, compared to cells without LPS (A) or BMPER (B, D, E) treatment; # P<0.05; n=3~6. Analysis was two-way ANOVA followed by Fisher’s LSD multiple comparison test (for A, E) and one-way ANOVA (for B, D).