Figure 7
Calcineurin/NFAT inhibitor cyclosporin A antagonized lithium-induced cell number increase in RG3.6 cell cultures. (A) Lithium did not significantly change β-catenin expression in RG3.6 cells. RG3.6 cells were grown for 3 days in culture medium containing 3 mM LiCl or 3 mM control NaCl. The cells were then lysed in RIPA for western blotting analysis on β-catenin and β-actin expression. (B) Lithium increased nuclear expression of NF-AT. Aliquot of cells from A were used for cytoplamic and nuclear fractionation, followed by western blotting assays for the subcellular expressions of NF-AT. (C) Lithium stimulated transcriptional activation of NF-AT. RG3.6 cells treated with LiCl or NaCl were used for gene reporter assays. The luciferase activity is presented as fold induction relative to that of NaCl-treated cells. *: paired t test, P < 0.01. (D) Effect of different doses of cyclosporin A (CsA) on RG3.6 cell growth. Cell count analysis was performed on RG3.6 cells grown for 5 days in culture medium containing various doses of CsA (0 (Control), 0.01 μM, 0.1 μM, 1 μM, 10 μM, 50 μM, n = 6 for each dose). *: paired t test, P < 0.005 compared to control, 0.01 μM, 0.1 μM, or 1 μM CsA condition. (E) Cyclosporin A antagonized lithium-induced RG3.6 cell growth. Cell count analysis was performed on RG3.6 cells grown for 5 days in culture medium with or without 3 mM LiCl, and with or without 1 μM CsA (n = 6 for each condition). The data represent mean ± standard error. *: paired t test, P < 0.02 compared to No CsA + No LiCl, or CsA + No LiCl condition.