Combined treatment with CsA plus NGF inhibits cell growth inducing p21 promoter activity.

<p>(A) MTT growth assays in HK-2 cells untreated (c) or treated with CsA (1–10nM) (A) or NGF (100–250–500ng/ml) (B). Cell proliferation is expressed as fold change respect to control. *p<0.05 vs c ; NS: not significant. (C) MTT growth assay in HK-2 cells untreated (c) or treated with CsA 10nM, NGF 100 ng/ml, alone or in combination for 48h. Results are expressed as fold change respect to c. *p<0.05 compared with c. **p<0.05 compared with CsA-treated cells. (D) CD1 and p21 mRNA content, evaluated by real time RT-PCR after 24 hours of exposure to CsA 10nM and NGF 100ng/ml alone or in combination. Each sample was normalized to its GAPDH mRNA content. *p<0.05 compared with c; **p<0.05 compared with CsA-treated cells. (E) HK-2 cells were untreated (c) or treated for 24h with CsA 10nM and NGF 100ng/ml alone or in combination. CD1 (Antibody dilution 1:500) and p21 (1:500) protein levels were determined by WB. β-Actin (1:10000) was used as loading control. Immunoblots show a single representative of 3 separate experiments. The upper panels represent the means ± SD of 3 separate experiments between CD1/β-Actin and p21/β-Actin levels in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05; **p<0.05 compared with CsA-treated cells. (F) HK-2 cells, transiently transfected with p21 promoter, were untreated (c) or treated for 15h with CsA 10nM, NGF 100ng/ml, Mithramycin (M) 100nM alone or in combination and then luciferase activity was measured. Luciferase activity of untreated cells was set as 1-fold induction, upon which treatments were calculated. *p<0.05 compared with c; **p<0.05 compared with cells CsA+NGF-treated. Bars represent the means ± SD of 3 different experiments each performed in triplicate. Secondary antibodies dilutions used are: goat anti-mouse (1:2000) or anti-rabbit (1:7000).