Figure 4

Effects of peretinoin on SPHK1 promoter activity. A effects of peretinoin on the promoter activity of SPHK1 and SPHK2. (a) Plasmids encoding luciferase under the control of the promoter region of SPHK1, SPHK2, β-actin, and GAPDH were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 1, 5, 10, 20, or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. The luciferase activity in each condition was normalized to that of DMSO control. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by one-way ANOVA. (b) Possible binding sites of Sp1 in the SPHK1 promoter region and a representation of deletion mutants. This figure shows the possible binding sites of Sp1 in the promoter region of SPHK1 and the series of deletion mutants analyzed. (c) Enhancement of the promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was co-transfected with a plasmid encoding luciferase under the control of the SPHK1 promoter region and, 48 h later, luciferase activity was determined. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by the Student’s t test. (d) Restoration of the suppressive effects of peretinoin on the promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was transfected into Huh-7 cells. After 24 h, the cells were transfected with a plasmid encoding luciferase under the control of the promoter region of SPHK1 or a control vector without any promoter regions. After another 24 h, DMSO or peretinoin at 20 or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity from peretinoin-treated cells to that of DMSO-treated cells in cells transfected with the plasmid encoding Sp1 or the control empty vector. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by the Student’s t test. (e), attenuation of the suppressive effects of peretinoin on the promoter activity of SPHK1 by Sp1 knockdown. Three kinds of siRNA to Sp1, control siRNA (siCNT), or mock were transfected at 20 nM into Huh-7 cells and, 48 h later, the cells were transfected with the plasmid encoding luciferase under the control of the promoter region of SPHK1 or a control vector without any promoter regions. After another 24 h, DMSO or peretinoin at 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity of cells treated with 40 μM peretinoin to that of DMSO-treated cells for each siRNA or mock transfection. (f), effects of deletion of the Sp1 binding site on the promoter activity of SPHK1. The plasmids encoding luciferase under the control of various lengths of promoter regions of SPHK1 were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 20 or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity of peretinoin-treated cells to that of DMSO-treated cells for each plasmid. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.005.