IL6, IL10, and JUN expression is directly regulated by NFAT. (A) c-Jun overexpressing or control OCI-Ly10 cells were treated every second day with CsA and/or IL-6/IL-10 as indicated. Treated cells were counted and normalized to the solvent controls. (B) OCI-Ly10 cells, which had been transduced with either a c-Jun or an empty expression vector, were treated for 48 hours with solvent, CsA, and/or IL-6/IL-10 as indicated. P-STAT3 and c-Jun protein levels were assessed by immunoblotting. A JUN reporter construct was cotransfected with either empty vector control, RelA, or NFATc1/αA in the indicated (C) GCB DLBCL cell lines or (D) in U2932. Luciferase activity was measured (C) 48 or (D) 36 hours after transfection and normalized to the empty vector control. (E,G) HEK293T cells were transfected with IL-6 or IL-10 reporter constructs in combination with RelA, c-Jun, and NFATc1/αA as indicated. Luciferase activity was measured 24 hours after transfection and normalized to the empty vector control. (F,H) The ABC DLBCL cell line U2932 was electroporated with either a proximal/distal IL10 or an IL6 reporter construct. Expression vectors containing RelA, NFATc1/αA, or c-Jun were cotransfected as indicated. Thirty-six hours after transfection, luciferase activity was quantified and normalized to the empty vector control. (I) ChIP analysis from CsA- or solvent-treated (4 hours) HBL-1 cells. NFATc1 binding to the JUN, IL10, IL6, and myoglobin (MB) promoters was compared with the IgG control. The data are representative of (A-H) at least 3 or (I) 2 independent experiments. Error bars correspond to the mean ± SD. Statistical significance was calculated using Student t test (*P < .05, **P < .01, ***P < .001).