Fig. 4
mRNA of intra- and extracellular IGFBP-3 are increased by Aβ via calcineurin in cultured cells. Human astrocytoma cells (H4 cells) were treated with 1 μM Aβ1–42 or control peptide for 24 h. mRNA was extracted and subjected to quantitative RT-PCR. The levels of IGFBP-3 mRNA were significantly increased after treatment with Aβ1–42, compared with the control peptide (a, n = 6, 170.2 ± 8.7 %, p < 0.001). Simultaneous treatment with both 1 μM Aβ1–42 and 1 μM FK506 for 24 h downregulated the levels of IGFBP-3 mRNA, compared with Aβ1–42 treatment alone (a, n = 6, p < 0.001). The data represent the mean ± SE. * indicates statistically significant differences (***p < 0.001). b-d Western blotting and ELISA were performed to verify the upregulation of both intra- and extracellular IGFBP-3 protein levels after 48 h of treatment with 1 μM Aβ1–42 or control peptide. Increased levels of IGFBP-3 were observed in the Aβ1–42-treated cell preparations in comparison to preparations treated with the control peptide treatment, as indicated by western blot (c, n = 4, 552.5 ± 14.1 %, p < 0.001) and ELISA (d, n = 7, 255.4 ± 26.0 %, p < 0.001). FK506 treatment significantly reduced the levels of intra- and extracellular IGFBP-3, as demonstrated by western blot (c, n = 4, p < 0.001) and ELISA (d, n = 7, p < 0.001), respectively. The data represent the mean ± SE. * indicates statistically significant differences (***p < 0.001)