Calcium-mediated NFAT activation is deregulated in DLBCL cell lines. (A) Intracellular calcium levels of splenic primary mouse B cells or of the indicated cell lines measured by flow cytometry. Frequencies of positive cells were normalized to the control cell line Ramos. (B) To measure the permeability of cation channels at the plasma membrane, MnCl₂ was added to the medium and Fura-2 quenching was assessed by flow cytometry over time. Fura-2 excitation was normalized to the fluorescence of each individual cell line before MnCl₂ was added. Immunoblot analysis of (C) ABC DLBCL, (D) GCB DLBCL, or the (E) control cell lines Ramos and Jurkat, which were either treated with solvent, 600 nM CsA, or 600 nM FK506 for 4 hours to assess the phosphorylation status of NFATc1 and NFATc2. Tubulin served as loading control. (F) To investigate the importance of extracellular calcium, HBL-1 cells were kept for 4 hours in calcium-free and/or in fetal calf serum-free medium as indicated and analyzed for NFATc1/2 phosphorylation status by immunoblotting. Subcellular localization of NFATc1/2 in cytoplasmic and nuclear fractions of (G) solvent- or CsA-treated ABC DLBCL, (H) GCB DLBCL, or (I) control cell lines. Lamin A/C and GAPDH served as markers for N or C fractions, respectively. Data in panels A-E and G-I are representative of at least 3 independent experiments, in panel F of 2 independent experiments. (C-E) Black arrowheads mark hypophosphorylated and hence activated NFAT isoforms, whereas gray arrowheads indicate the hyperphosphorylated forms of the transcription factors. C, cytoplasmic; N, nuclear.