NFAT dephosphorylation and nuclear translocation are not driven by BCR signaling. (A) Measurement of intracellular calcium levels in the presence of either agonistic anti-IgM antibodies, the ionophore ionomycin, the Ca²⁺ chelator EGTA, the Src kinase inhibitor saracatinib, the SYK inhibitors GS-9973 and R406, the BTK inhibitor ibrutinib, or the PI3K inhibitor AZD8835. The intensity of the calcium sensor Calbryte 520 was measured over time by flow cytometry. (B) Cytoplasmic and nuclear fractions of DLBCL cell lines treated with solvent or the SYK inhibitor GS-9973 for 4 hours. NFATc1 localization was visualized by immunoblotting. P-AKT was used to control the efficacy of SYK inhibition in the ABC DLBCL cell lines TMD8 and HBL-1. Lamin A/C and GAPDH served as nuclear and cytoplasmic markers, respectively. (C) HBL-1 cells were treated with either solvent or the indicated inhibitors for 4 hours before quantification of NFATc1 binding to its consensus nucleotide sequence by TransAM assay. (D) Localization of NFATc1 in control or CD79B-silenced HBL-1 cells visualized by immunoblot analysis. (E) HBL-1 cells were transfected with scrambled or with increasing amounts of small interfering RNA targeting IgM. NFATc1/2 expression was analyzed by immunoblotting. (F) The ABC DLBCL cell lines were treated with solvent or the indicated inhibitors for 24 hours, before NFATc1/2 levels were measured by immunoblotting. (G) HBL-1 cells were treated with inhibitors targeting PI3K, SYK, or BTK and NFATc1/2 mRNA levels were quantified by qPCR. All samples were normalized to the solvent control. (H) The NFATc1 P1 luciferase-based reporter construct was transfected into HBL-1 cells, which were subsequently treated with inhibitors targeting PI3K, BTK, or IKK for 24 hours. Luciferase signals were normalized to the DMSO-treated sample. (I) The ABC DLBCL cell lines HBL-1 and OCI-Ly3 as well as the GCB cell line HT were lentivirally transduced with a DN-IκBα expression construct and NFATc1 levels were visualized by immunoblotting. (A-I) Data are representative of at least 3 independent experiments. Error bars correspond to the mean ± SD. Statistical significance was calculated using Student t test (*P < .05, **P < .01, ***P < .001). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction.