Combined treatment with CSA plus NGF activates intrinsic apoptosis via p75^NTR.

<p>(A) Real time RT-PCR analysis of TrKA^NTR and p75^NTR mRNA treated for 24h with CsA 10nM and NGF 100ng/ml alone or in combination. *p<0.05 compared with c; **p<0.05 compared with cells treated with CsA alone. (B) HK-2 cells were untreated (c) or treated with CsA 10nM and NGF 100ng/ml alone or in combination. Levels of phosphorylated (p) Akt (Ser473) (Antibody dilution 1:1000), mtor (1:500), MAPK (Thr202/Tyr204) (1:1000), JNK (Thr183/Tyr185) (1:200) and total non-phosphorylated proteins (1:1000) were measured in cellular extracts by WB. β-Actin (1:10000) was used as loading control. Numbers over the blots represent the average fold change versus control. (C) p53 and BAD mRNA expression in HK-2 cells untreated (c) or treated for 24h with CsA 10nM and NGF 100ng/ml alone or in combination. *p<0.05 compared with c; **p<0.05 compared with CsA-treated cells. (D) WB of p75^NTR in HK-2 cells transfected for 72h with siRNA targeted human p75^NTR mRNA sequence or with a control siRNA. β-Actin was used as loading control. The upper panels represent the means ± SD of 3 separate experiments between NGF/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. (E) p53 and BAD mRNA levels in HK-2 cells transfected with p75^NTRsiRNA or control-siRNA for 72 hours followed by CsA 10nM and NGF 100ng/ml treatment alone or in combination for 24 hours. *p<0.05 compared to untreated cells (c), **p<0.05 compared with CsA-treated cells; § p<0.05 compared with cells CsA+NGF-treated and transfected with control-siRNA. All mRNA sample were normalized to its GAPDH mRNA content. Bars represent the means ± SD of 3 independent experiments, each performed in triplicate. All immunoblots show a single representative of 3 separate experiments. Secondary antibodies dilutions used are: goat anti-mouse (1:2000) or anti-rabbit (1:7000) IgG or donkey anti-goat (1:3000) .