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HEP_25568_sm_SuppFig1.tif1369KSupporting Information Figure 1. Rnd3 mRNA is down-regulated in HCC (continued). (A) Microarray data revealed the down-regulation of Rnd3 mRNA in HCC. Data extracted from Boyault et al., 2007 transcriptomic analysis comparing non-tumorous liver (n=5, pools of 3) and HCC (n=57) human samples. Probe set #212724 corresponds to Rnd3. ***P=0.0009 in Mann Whitney test. (B) Correlations between microarray data and qRT-PCR data performed on the same samples. Statistical analysis was performed with the Pearson correlation test. (C-E) Correlations between the Rnd3 mRNA levels analyzed by qRT-PCR and clinicopathological features of HCC. The Rnd3 mRNA level was measured by quantitative RT-PCR. The gene expression results were first normalized to internal control ribosomal 18S in all samples. Then, the results for HCC samples were expressed as a ratio with respect to the mean expression level in a pool of 15 non-tumor samples. P values from the Mann Whitney test are indicated. (C) Rnd3 expression according to the HBV status (HBV-infected, n=21; non HBV-infected, n=99). (D) Rnd3 expression according to the HCV status (HCV-infected, n=13; non HCV-infected, n=107). (E) Rnd3 expression according to the alcohol consumption status (alcohol, n=18; non alcohol, n=102). (F) Rnd3 protein levels in matched HCC (T) and non-tumoral (NT) livers were assessed using immunoblotting. Fold changes in Rnd3 expression (T/NT) are shown for 27 patient samples. Of the 27 cases, 23 HCCs (about 85%) showed a decreased Rnd3 expression in the tumor tissue when compared to the peri-tumoral tissue (***P=0.00048).
HEP_25568_sm_SuppFig2.tif19804KSupporting Information Figure 2. Rnd3 knockdown induces E-cadherin down-regulation in HCC cells. (A) Huh7 cells were transfected with two different Rnd3 siRNAs (S1 or S3) or with control siRNA (siCtrl). After 72 hours, protein extracts were analyzed by immunoblotting using anti-E-Cadherin, Rnd3 and GAPDH antibodies. Quantification of E-cadherin protein levels normalized using GAPDH is represented in the bar graph. Each bar represents the mean±SE (n=3 for S1, n=5 for S3). *, *** P<0.05 by ANOVA when compared with siCtrl. (B) Rnd3 knockdown induces E-cadherin loss at cell-cell junctions. S1-transfected Hep3B cells (72 hours post-transfection) were stained with anti-E-cadherin antibodies (red) and DAPI (blue). Bar, 25 μm.
HEP_25568_sm_SuppFig3.tif6619KSupporting Information Figure 3. E-cadherin and Rnd3 are down-regulated in the same HCC samples. Rnd3 and E-cadherin protein levels in matched HCC (T) and non-tumoral (NT) livers were assessed using immunoblotting. Quantitation on 27 matched human samples reveals that in 78% (18/23) of samples where Rnd3 is downregulated, E-cadherin is also reduced in the tumor tissue.
HEP_25568_sm_SuppFig4.tif959KSupporting Information Figure 4. Silencing of Rnd3 affects the ZEB1/2-miR200 pathway (continued). (A) Hep3B-KRAB-shRnd3 and Hep3B-KRAB-shGL2 cells were treated (+Dox) or not (-Dox) with doxycycline (30 ng/ml) for 5 days. Protein extracts were analyzed by immunoblotting using Rnd3 and GAPDH antibodies. In contrast to controls, treatment with doxycycline in Hep3B-KRAB-shRnd3 cells led to Rnd3 knockdown. (B-C) Cells were treated as described in (A). After 5 days of Dox treatment, Rnd3, E-cadherin, vimentin, Snail1 and Slug mRNA expressions were analyzed by qRT-PCR. Each bar represents the mean±SE of three independent experiments. *P=0.0126, **P=0.0025 by ANOVA when compared with control.
HEP_25568_sm_SuppFig5.tif2252KSupporting Information Figure 5. The miR-200/ZEB/E-Cadherin pathway is intact in Huh7 hepatoma cells. (A) Huh7 cells were transfected with miR-200b and/or miR-200c mimics or a miRNA control (miR-Scr). After 72 hours, cells were photographed. Forced over-expression of miR-200b or/and miR200c increased cell-cell contacts. (B) Cells were transfected as described in (A). After 72 hours, E-cadherin, ZEB1 and ZEB2 mRNA expressions were analyzed by qRT-PCR. Forced over-expression of miR-200b or/and miR200c in Huh7 cells downregulated ZEB1 and ZEB2 expression leading to E-Cadherin upregulation.
HEP_25568_sm_SuppFig6AC.tif6622KSupporting Information Figure 6AC. Rnd3 depletion favors HCC cell invasion through an amoeboid (filopodal)-like mechanism (continued). (A) Invasion induced by Rnd3 loss is MMP-independent. Hep3B cells doubled-transfected with siRNA Rnd3 S3 were assayed in a Matrigel-coated chamber to monitor invasion in the presence of GM6001 or DMSO (as a control). The graph shows the quantification of cell invasion. (B) Rnd3-KD cells develop filopodia-like protrusions (arrows) during invasion into type I collagen gel. Hep3B cells transfected with siRNA S1, with or without rescue-Rnd3 cDNA, were embedded in thick type I collagen gel and stained for actin. Bar, 50 μm (top panel) and 25 μm (bottom panel). (C) Optical sections (0.33 μm) were taken from the top to the bottom of the cell by confocal microscopy and the Z-projection of confocal sections shows filopodial-like protrusions over the cell (arrows). Bar, 10 μm. (D) Rnd3 depletion-induced cell invasion occurs in a Rac1-dependent manner. Hep3B cells transfected with siRNAs targeting (S3) or not (siCtrl) Rnd3 were assessed in invasion assays in the presence of Rac1 inhibitor (EHT1864) or control compound (EHT4063).
HEP_25568_sm_SuppFig6D.tif6617KSupporting Information Figure 6D. Rnd3 depletion favors HCC cell invasion through an amoeboid (filopodal)-like mechanism (continued). (A) Invasion induced by Rnd3 loss is MMP-independent. Hep3B cells doubled-transfected with siRNA Rnd3 S3 were assayed in a Matrigel-coated chamber to monitor invasion in the presence of GM6001 or DMSO (as a control). The graph shows the quantification of cell invasion. (B) Rnd3-KD cells develop filopodia-like protrusions (arrows) during invasion into type I collagen gel. Hep3B cells transfected with siRNA S1, with or without rescue-Rnd3 cDNA, were embedded in thick type I collagen gel and stained for actin. Bar, 50 μm (top panel) and 25 μm (bottom panel). (C) Optical sections (0.33 μm) were taken from the top to the bottom of the cell by confocal microscopy and the Z-projection of confocal sections shows filopodial-like protrusions over the cell (arrows). Bar, 10 μm. (D) Rnd3 depletion-induced cell invasion occurs in a Rac1-dependent manner. Hep3B cells transfected with siRNAs targeting (S3) or not (siCtrl) Rnd3 were assessed in invasion assays in the presence of Rac1 inhibitor (EHT1864) or control compound (EHT4063).
HEP_25568_sm_SuppInfo.doc101KSupporting Information

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