Supplemental Figure 1. Cezanne 2 downregulation and Snail1 upregulation on a larger cohort of HCC samples. Immunoblot analysis showed Cezanne1,2 and Snail1 expression in seven more freshly isolated hepatocellular carcinoma tissues with or without metastasis and adjacent non-tumorous tissues. HCC-ST stands for HCC surrounding tissue. Tubulin was treated as a reference control.
Supplemental Figure 2. Snail1 affects transcriptional repression of Cezanne2 in SK-HEP-1 cells. (A). Lysates from SK-HEP-1 cells were examined by ChIP assay using an anti-Snail1 and anti-Snail2 antibody, respectively, as well as a PCR primer pair corresponding to the promoter of the Cezanne2 gene. Recruitment to the Cezanne2 promoter was only shown for Snail1. IP using negative control rabbit immunoglobulin; Input, 10% of the cell lysate used for the IP is shown. (B). Reporter gene assays revealing inducible Cezanne2 promoter activity in SK-HEP-1 cells after transient transfection with siRNAs against Snail1 andpGL3 inserted with Cezanne2 promoter. Mutation of the consensus Snail1 binding site (Cezanne2 mut-19) led to strong promoter activity that was not affected by transfection with siRNAs against Snail1 or pcDNA Snail1. Data are given as mean ± SEM. P < 0.05 was considered for significantly changed when compared with Cezanne2 control.
Supplemental Figure 3. Cezanne2 regulates proliferation, migration, and invasion in SK-HEP-1 cells. (A). SK-HEP-1 cells were transfected with pcDNA3.1(+), Cezanne2 WT, and Cezanne2 Mut (C210S) vectors. Thirty-two hours later, SK-HEP-1 cells were seeded at 2×105 cells per ml and analyzed at 16-hour intervals by trypan blue staining. (B). Migration of SK-HEP-1 cells in spheroid migration assays. Comparison of SK-HEP-1 cells stably transduced with Cezanne2 WT, Cezanne2 C210S, or GFP vectors. Data are given as mean ± SEM. *, P < 0.05. (C) Migration of SK-HEP-1 cells in Boyden chamber assays. Data are given as mean ± SEM. *, P < 0.05 compared with Control. Experiments in A – C were repeated at least three times.
Supplemental Figure 4. Cezanne2 could inhibit proliferation and metastasis of hepatocellular carcinoma cells in vivo. To test the effect of Cezanne2 on tumor growth in vivo, SK-HEP-1 cells stably expressing Cezanne2 were injected subcutaneously into nude mice compared with cells stably expressing GFP or noninfected cells (A). 14 days after injection, none of the mice from the Cezanne2 group, but 6 out of 10 animals from the GFP and noninfected control groups, developed tumors. After 4 wk, 4 mice from the Cezanne2 group developed tumors; however, these tumors were significantly smaller (4.8±1.5 vs. 23.5±8.3 mm3 [GFP] and 25.1±12.5 mm3 [noninfected]);. (B). Hepatocyte growth factor/scatter factor (HGF/SF) serum levels were testedby ELISA as an established marker to monitor metastasis of liver cancer in the xenograft model. 4 weeks after injection, animals receiving Cezanne2 expressing hepatocellular carcinoma cells had significantly lower HGF/SF serum levels (5.7±1.4 ng/ml) than animals injected with GFPtransduced (10.4±3.8 ng/ml) or nontransduced (9.8±2.6 ng/ml) cells.Experiments in A – B were repeated at least three times.
Supplemental Figure 5. Overexpression of Cezanne2 could not regulate EMT in HepG2 cells. Both epithelia markers (E-cadherin, ZO-1 and -catenin) and mesenchymal makers (N-cadherin, Vimentin, and Fibronectin) were compared by western blot analysis between Cezanne2 overexpressed cells and untreated cells.
Supplemental Figure 6. Cezanne2 could not interact with TRAF2 (A) or TRAF3 (B) in 293 cells.
Supplemental Figure 7.The effect of Snail1 silencing on HCC migration and NF-κB activation. (A). Suppression of Snail1 inSK-HEP-1 cells resulted in significantly impaired migration rates compared with control cells. SiRNAmediated depletion of Cezanne2 could rescue migratory potential of Snail1-depleted hepatocellular carcinoma cells. (B). Western blot analysis was used to detect nuclear p-P65 expression in SK-HEP-1 cells transfected with siRNA vectors against Cezanne2 or Snail1 for 24 hours and then treatment with TNFα stimulation at 2 hours.The level of the active form of NF-κB (p-P65) in nuclei was not dramatically changed in SK-HEP-1 cells after Snail1 knockdown.