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HEP_23661_sm_Supptable.doc48KSupporting Table.
HEP_23661_sm_Suppinfo.doc47KSupporting Information.
HEP_23661_sm_Suppfig1.tif6068KsFigure 1 Expression of CD151 in 3 shRNA-CD151-HCCLM3 cells. Three shRNA-CD151-HCCLM3 cells were constructed to silence CD151 expression, and then validated by qRT-PCR and Immunoblotting. A showed that expression of CD151 mRNA in three shRNA-CD151-HCCLM3 cells. Of three shRNA-CD151-HCCLM3 cells, shRNA-CD151-HCCLM3 #3 cells presented with the most efficient interference of CD151 (about 85%). The results were further validated by Immunoblotting (B). Therefore, we chose the shRNA-CD151-HCCLM3 #3 cells for further study.
HEP_23661_sm_Suppfig2.tif2105KsFigure 2 Expression of FAK, p38 and phosphorylation level of counterparts in shRNA-CD151-HCCLM3. The expression of FAK and p38, and the phosphorylation level of FAKTyr397 and p38Thr180/Tyr182 in shRNA-CD151-HCCLM3 and HCCLM3 cells treated with laminin 5 were assayed by Immunoblotting (A). The result showed that the phosphorylation level of FAKTyr397 was much higher in HCCLM3-Mock cells (HCCLM3-pGCSIL-GFP) than that in shRNA-CD151-HCCLM3 cells (A).
HEP_23661_sm_Suppfig3.tif2482KFigure 3 Role of CD151 and GSK-3β in up-regulation of MMP9 in HCC cells. When the expression of CD151 in shRNA-CD151-HCCLM3 cells was up-regulated through transfection of pcDNA3-CD151 plasmids, the phosphorylation level of AktSer473 and GSK-3βSer9, and expression of Snail were strongly enhanced. Interestingly, expression of MMP9 in the modified HCC cells were rescued as well (A). Expression of GSK-3β in Hep3B cells was inhibited by siRNA, Snail and MMP9 expression, and the phosphorylation level of AktSer473 was enhanced, while CD151 and Akt expression remained unchanged (B).
HEP_23661_sm_Suppfig4.tif5156KsFigure 4 Matrigel assay. The ability of HUVECs to form capillaries was rescued, when the shRNA-CD151-HCCLM3 (A) and shRNA-MMP9-HCCLM3 groups (B) were supplemented with the supernatant from HCCLM3 cells.
HEP_23661_sm_Suppfig5.tif3309KsFigure 5 Tumor volume of xenografts. The tumor volume of HCCLM3- and HCCLM3-Mock (HCCLM3-pGCSIL-GFP)-derived xenografts was 6.4 ± 1.4 cm3 and 5.4 ± 1.2 cm3, respectively, significantly larger than that of shRNA-CD151-HCCLM3, Hep3B, and shRNA-MMP9-HCCLM3 (2.4 ± 0.3 cm3, 2.6 ± 0.6 cm3 and 2.4 ± 0.4 cm3, respectively; p<0.01). However, there was no significant difference in the tumor volume among shRNA-CD151-HCCLM3-, Hep3B-, and shRNA-MMP-9-HCCLM3-derived xenografts (p>0.05).
HEP_23661_sm_Suppfig6.tif3745KsFigure 6 Immunohistochemistry for Ki-67 in xenografts. The expression of Ki-67 in 5 HCC-derived xenografts was assayed using immunohistochemical staining. There was no significant difference in expression of the Ki-67 among the HCC-derived xenografts. Magnification: upper panel, 100×; lower panel, 400×. The scale bar represents 100μm.
HEP_23661_sm_Suppfig7.tif4191KsFigure 7 The expression of CD151 and MMP9 in xenografts and metastatic Assay. The expression of CD151 and MMP9 in HCC cell lines-derived xenografts at the mRNA level (A, upper panel) and at the protein level (A, lower panel). Metastasis assays revealed that the numbers of lung metastatic loci in HCCLM3 and HCCLM3-Mock group were much more than that in the shRNA-CD151-HCCLM3, Hep3B and shRNA-MMP9-HCCLM3 group, with statistically significant difference (B).
HEP_23661_sm_Suppfig8.tif7248KsFigure 8 The expression of VEGF-A (VEGF) in xenografts. The expression of VEGF in 5 HCC-derived xenografts was assayed using immunohistochemical staining. There was no significant difference in expression of the VEGF among the HCC-derived xenografts. Magnification: upper panel, 100×; lower panel, 400×. The scale bar represents 100μm.
HEP_23661_sm_Suppfig9.tif7581KsFigure 9 The relationship among expression of CD151, MMP9 and VEGF, and location of Snail in HCC patients and HCC cells. The expression of CD151, MMP9, VEGF and Snail were assayed by immunohistochemical staining in a tissue microarray comprised of primary tumors of 327 HCC patients. A showed that patient 5 had the characteristics of low expression of CD151 and MMP9, over-expression of Snail in cytoplasm, and over-expression of VEGF. However, patient 6 presented with over-expression of CD151 and MMP9, accumulation of Snail in the nucleus and low expression of VEGF. B showed that HCCLM3-Mock cells with CD151high had accumulation of Snail in the nucleus, while over-expression of Snail in cytoplasm of shRNA-CD151-HCCLM3, shRNA-Akt-HCCLM3, and GSK-3βS9A-HCCLM3 cells. C showed that few accumulation of Snail in the nucleus of Hep3B cells with CD151low. When pcDNA3-CD151 plasmids were transfected in Hep3B cells, accumulation of Snail in the nucleus was increased. Increased accumulation of Snail in the nucleus also was found in siRNA-GSK-3β cells. All cells were treated by Laminin 5.

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