Figure 3 | Scientific Reports

Figure 3

From: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo

Figure 3

Effects of peretinoin on the mRNA level, protein expression, and enzymatic activity of SPHK1. (a) effects of peretinoin on the mRNA level of sphingolipid-related genes in Huh-7 cells. Huh-7 cells were treated with peretinoin at 10, 25, or 50 μM or with 0.5% DMSO for 72 h and total cellular RNA was extracted. The mRNA levels of SPHK1, SPHK2, S1P lyase, S1P receptor 1–3 (S1P1, S1P2, and S1P3), and β-actin were quantitated by qRT-PCR (SYBR green assay), and the mRNA levels of SPHK1, SPHK2, S1P lyase, S1P1, S1P2, and S1P3 were normalized to that of β-actin. The relative mRNA level of each gene under each condition was normalized to that of DMSO control. (b) Effects of peretinoin on the protein expression of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin 10, 20, or 40 μM. Total cell lysates were collected 12, 24, 48, and 72 h later and then probed by western blotting with anti-SPHK1 and β-actin antibodies. Full-length gels and blots before cropping are shown in supplemental Fig. S8. (c) Effects of peretinoin on the enzymatic activity of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin at 10, 20, and 40 μM and a plasmid encoding SPHK1 cDNA or an empty vector were transfected into Huh-7 cells. Total cell lysates were collected 72 h after initiation of peretinoin treatment and transfection and used in an in vivo SPHK activity assay. Synthesized S1P was visualized by using radioisotopes. The radioactive spots corresponding to S1P were scraped into vials and the radioactivity was measured in a scintillation counter and normalized to that of DMSO-treated cells or empty vector-transfected cells. 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. (d), effects of peretinoin on the enzymatic activity of SPHK1 in vitro. Human recombinant SPHK1 was incubated with DMSO or peretinoin at 5, 10, 25, or 50 μM or with SKI II at 5, 10, 25, or 50 μM as described in the Experimental procedures. Synthesized S1P was quantitated by a fluorescence-based method. The SPHK activity in each condition was normalized to that of DMSO control. *p < 0.05, **p < 0.01, ***p < 0.005.

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