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Figure 5 | Molecular Cancer

Figure 5

From: Hepatitis B virus X protein specially regulates the sialyl lewis a synthesis among glycosylation events for metastasis

Figure 5

The inhibition of cell adhesion to endothelial cells and metastasis through the suppression of SLA synthesis in HBx-transfected cells by β1,3-galactosyltransferase shRNA. (A) Total RNA from each cell was isolated using the Trizol reagent. The mRNA expressions of β1-3 and 4 galactosyltransferase genes were detected by RT-PCR using the primers indicated in Table 1. β-Actin was included as an internal control. (B) After transfection with β1-3Gal-T 5 promoter, luciferase activity from the cells was analyzed as described in Materials and Methods. Relative luciferase activity was normalized with the activity of pCMVβ-gal plasmid. (C) Each cell was transfected with the shRNA-1 and 2 against β1-3Gal-T 5 gene. Total RNA from these cells was isolated. The mRNA expression of β1-3Gal-T 5 gene was detected by RT-PCR using the primers indicated in Table 1. β-Actin was included as an internal control. (D) To check whether silalyl lewis A expression induced in HBx-transfected cells was suppressed by the shRNAs against β1-3Gal-T 5 gene, we examined this using flow cytometric analysis. Fluorescence histograms of a representative experiment indicate the baseline expression of SLA. The y-axis gives the percentage of positive cells (counts); the x-axis gives fluorescence intensity. (E) Each cell was transfected with the shRNA against β1-3Gal-T 5 gene. Each cell was added on TNF-α-stimulated HUVECs, and was incubated for 20 min at 37°C with rotation at 60 rpm in a shaking incubator. The attached cells were then visualized using a microscope. (F) Hematoxylin and eosin staining of the lungs. The stained sections of the lungs were photographed at 40× magnification. M, metastatic nodule; N, normal.

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