Positive regulation of transcription and enzymatic function of <i>SOAT1</i>.

<p>(A-D) Effects of forskolin and dibutyryl-cyclic AMP (db-cAMP) on <i>SOAT1</i> mRNA accumulations in EECs and hepatocytes. EECs and hepatocytes were treated with different concentrations of forskolin or db-cAMP for 24 hours. <i>SOAT1</i> mRNA accumulation was measured and normalized to <i>β-actin</i> mRNA. (n = 4) (E-F) H89 inhibited IBMX-induced <i>SOAT1</i> mRNA accumulations in EECs and hepatocytes. <i>SOAT1</i> mRNA accumulation was increased by 0.5 mM IBMX treatment for 24 hours, whereas the PKA inhibitor, H89 (10 μM) abolished the IBMX-mediated increase of <i>SOAT1</i>. (n = 6) (G-H) The IBMX treatment increased PKA activity in EECs and hepatocytes. The cells were treated with 0.5 mM IBMX with or without 10 μM H89 for 24 hours. The cell lysates were assayed for PKA activity using the PepTag assay. Typical gel patterns are represented (three independent experiments) for EECs and hepatocytes with statistical analysis of the densitometric analysis represented by the graphs (quantified by ImageQuant software). (I) Fluorescence examination of <i>SOAT1</i> activity by applying NBD-cholesterol as the substrate. EECs were incubated with 10 μg/mL of NBD-cholesterol at 37°C for 30 mins or 1 hour. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. (J-K) The fluorescent intensity was quantified by ImageJ software. EECs were treated with or without db-cAMP for 24 hours. EECs were then incubated with 10 μg/mL of NBD-cholesterol for 30 min or 1 hour and examined by confocal microscopy. (J) Fluorescent intensity of total area. K: Fluorescent intensity per area of lipid droplets in EECs. (n = 4) Statistical significance was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05). (L) The <i>SOAT1</i> protein levels after db-cAMP or IBMX treatments. EECs were treated with 0.5 or 1 mM db-cAMP or 0.5 mM IBMX for 48 hours and protein levels were examined by Western blotting. The <i>β-actin</i> was used as internal control. Statistical significance was determined by one-way analysis of variance. Dunnett's multiple comparisons test was used to evaluate differences between means. Data were expressed as means ± S.E.M. (n = 8) and different letters indicate the significant difference (P≤0.05). (M-N) The effects of H89 with or without db-cAMP and IBMX treatments on NBD-cholesterol uptake and storage. (M) EECs were pre-treated with or without H89 (10 μM) for 2 hours, and added db-cAMP (1 mM) or IBMX (0.5 mM) for 24 hours. Cells were then incubated with 10 μg/mL NBD-cholesterol for one hour, fixed with 4% paraformaldehyde and finally examined by confocal microscopy. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. Scale bar = 50 μm. (N) The quantitation of fluorescent intensity on NBD-cholesterol uptake and storage by ImageJ software in cells treated with H89 with or without IBMX or db-cAMP. Data were expressed as means ± S.E.M. (n = 4) Statistical significance among more than three different experimental groups was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Different letters indicate a significant difference (P≤0.05).