Fig. 5
Effect of valproic acid (VPA) on somatostatin (SST) production and secretion of ADMSC. a SST mRNA expression was increased 3.6 ± 0.3 fold by 1 mM VPA after 5 days of culture compared to the absence of VPA (n = 5, p < 0.001). b (1) Pro-SST protein was released to the culture medium. An aliquot of the supernatant was sampled 24 h after medium change for each time point. SST 25–116 peptide concentrations were measured by Elisa as described in the ‘Materials and methods’ section normalised to the protein content (mg) of the supernatant. Secretion was calculated from three independent experiments at five time points (0, 24, 48, 72 and 120 h) of ADMSC cultivation. Cells were cultured in 5-ml media per well, and secretion rates were given in pmol SST 25–116 per mg protein an hour. b (2) Cellular SST 25–116 production was upregulated by VPA added at 0, 0.25, 0.5, 1, 1.5 and 2.0 mM final concentrations. Analysis of variance resulted in significant difference (p = 0.008). b (3) SST 25–116 production in response to VPA concentration was plotted. Data were fit to a non-linear function with the assumption that intracellular SST 25–116 was increased by the presence of VPA in the medium of cultivated ADSC. Half-maximal effective concentration of VPA to stimulate SST synthesis was 0.4 mM (R2 = 0.79). c Staining of ADMSC with rabbit anti-somatostatin antibody. c (1) ADMSC cultivated in normal medium showed aggregates of SST within the cytoplasm. c (2) Cultivation in the presence of 1 mM VPA for 120 h resulted in higher density of aggregates adjacent to cell membrane. For PCR analysis, HPRT was considered as a housekeeping gene and fold change was achieved with 2−∆∆Ct method. To compare the groups, one-way ANOVA with Turkey’s post hoc test was utilised. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus control condition without VPA