Figure 1

Analysis of NFATc expression and calcium/Calcineurin/NFAT signalling in glioma cells. (A) NFATc1, NFATc2, NFATc3, and NFATc4 mRNAs from U251 and different human Glioblastoma lines (hGB) from xenografts were amplified by TaqMan RT-PCR. NFATc mRNA was normalized to the expression of TBP as endogenous gene. Results are shown as dCt (Ct NFATc − Ct TBP). (B) Representative immunoblot showing endogenous expression of NFATc3 and NFATc1 in U251 total protein lysates. Cells were 1 hour pre-treated with 200 ng/mL CsA (lanes 2, 5 and 6) and then, non-stimulated (ns) as control or stimulated for 30 minutes with 1 μM ionophore alone (Io) or in combination with 20 ng/mL PMA (PIo). (C) Representative immunoblot showing endogenous RCAN1-4 protein expression. β-actin expression was used as loading control. Glioma total protein lysates from U251 or hGB were pre-treated without (lanes 1 to 3) or with CsA (200 ng/mL) (lanes 4 to 6) and then stimulated for 4 hours with Io (1 μM) or in combination with PMA (20 ng/mLPIo as indicated. (D) In the upper panel, RCAN1-4 mRNA was amplified from total RNA by TaqMan RT-PCR. U251 were exposed 4 h to vehicle, Io (1 μM) or thapsigargin A (Tp, 10 nM). RCAN1-4 mRNA was quantified in arbitrary units normalized to the expression of human TBP. Representative experiments of a minimum of three are shown; values are the mean ± SD of triplicate RT-PCR determinations for each condition. ***P < 0.001; **P < 0.01 (ANOVA) versus ns that was given a value of 1. Panel below, representative immunoblots for RCAN 1-4 protein expression with ponceau staining as loading control in U251 cells treated as above (n = 3).