Ocarcinoma Cell ProliferationWe also checked the Microcystin-LR web expression of SULT2B1 isoforms in human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Standard PCR analysis showed that only the SULT2B1b isoform was expressed, although with varying levels (Figure S2). As such, a quantitative 
PCR analysis was also performed (Fig. 6A) along with an analysis of protein levels (Fig. 6B). Moreover, we assessed different growth ability of those hepatocarcinoma cells by CCK-8 assay (Fig. 6C). Slopes were calculated from the cell growth curve of these four cell lines to reveal their INCB039110 web proliferation ability. A regression analysis was performed comparing the relative cell proliferation to the relative SULT2B1 mRNA expression levels. As shown in Fig. 6D, the relative SULT2B1 mRNA expression in human hepatocellular carcinoma cell lines showed a significant and direct correlation with the rate of cell proliferation (r = 0.931, R2 = 0.867). As qPCR results indicated that, SULT2B1 mRNA levels in the human hepatocarcinoma tumor tissues showed much higher levels than those of the para-tumor tissues in the clinical samples. Besides, the PCR products were visualized on a 2 agarose gel, which was consistent with the results above (Figure 6E).Effect of SULT2B1b Interference or Overexpression on the Expression of FAS, BCL2 and MYC in Hepa1-6 CellsThe expression of proliferation and apoptosis related genes was evaluated. To identify important signaling pathways affected by SULT2B1b, the mRNA expression of the pro-apoptotic factor FAS, and the anti-apoptotic factor, BCL2, was measured. Overexpression SULT2B1b, signifcantly decreased the mRNA level of the pro-apoptotic factor, FAS (Fig. 3A), while the expression of the anti-apoptotic factor, BCL2, was upregulated (Fig. 3C). Similarly, knock-down of SULT2B1b significantly increased FAS mRNA level (Fig. 3B), but inhibited BCL2 mRNA expression (Fig. 3D). The protein expression of FAS, BCL2 and MYC was evaluated in NC-GFP-LV and SULT2B1-RNAi-LV Hepa1-6 cells that were serum-deprived or were treated with a combination of tumor necrosis factor-a (TNF-a, 10 ng/mL) and cycloheximide (CHX, 10 mg/mL). As seen in Fig. 3, both serum deprivation (Fig. 3E, F) and TNF-a/CHX treatment (Fig. 3G, H) reduced BCL2 and MYC protein levels, while FAS protein levels were increased.Analysis of Cell Cycle Arrest with SULT2B1 Knock-down in Hepa1-6 CellsTo elucidate the mechanisms of cell-cycle arrest induced by SULT2B1b interference that presented 15755315 in Fig. 4, we analyzed the expression of cyclinD1 and cyclinB1 by qPCR and Western-blot assays. Both the mRNA and protein levels of cyclinD1 presented no significant difference between siSULT2B1 and control Hepa16 cells (data not shown). However, as shown in Fig. 4A, cyclinBThe Suppressive Effects of SULT2B1b Inhibition on Cell Growth, CyclinB1 Expression and Tumorigenicity in Human Hepatocarcinoma CellsImmunocytochemical localization of SULT2B1b in BEL-7402 and SMMC-7721 cells using anti-SULT2B1 antibody revealed a widespread cytoplasmic distribution (Figure S3A,B). Normal rabbit IgG served as negative controls exhibited no green staining. To investigate the effects of SULT2B1b inhibition in human hepatocarcinoma cells, we designed siRNA targeted against SULT2B1 (both SULT2B1a and SULT2B1b isoforms) andSULT2B1b Promotes Hepatocarcinoma Proliferationspecifically against SULT2B1b. qPCR analysis revealed that both SULT2B1 siRNA and SULT2B1b-specific siRNA treatment decreased SULT2B1b mRNA.Ocarcinoma Cell ProliferationWe also checked the expression of SULT2B1 isoforms in human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Standard PCR analysis showed that only the SULT2B1b isoform was expressed, although with varying levels (Figure S2). As such, a quantitative PCR analysis was also performed (Fig. 6A) along with an analysis of protein levels (Fig. 6B). Moreover, we assessed different growth ability of those hepatocarcinoma cells by CCK-8 assay (Fig. 6C). Slopes were calculated from the cell growth curve of these four cell lines to reveal their proliferation ability. A regression analysis was performed comparing the relative cell proliferation to the relative SULT2B1 mRNA expression levels. As shown in Fig. 6D, the relative SULT2B1 mRNA expression in human hepatocellular carcinoma cell lines showed a significant and direct correlation with the rate of cell proliferation (r = 0.931, R2 = 0.867). As qPCR results indicated that, SULT2B1 mRNA levels in the human hepatocarcinoma tumor tissues showed much higher levels than those of the para-tumor tissues in the clinical samples. Besides, the PCR products were visualized on a 2 agarose gel, which was consistent with the results above (Figure 6E).Effect of SULT2B1b Interference or Overexpression on the Expression of FAS, BCL2 and MYC in Hepa1-6 CellsThe expression of proliferation and apoptosis related genes was evaluated. To identify important signaling pathways affected by SULT2B1b, the mRNA expression of the pro-apoptotic factor FAS, and the anti-apoptotic factor, BCL2, was measured. Overexpression SULT2B1b, signifcantly decreased the mRNA level of the pro-apoptotic factor, FAS (Fig. 3A), while the expression of the anti-apoptotic factor, BCL2, was upregulated (Fig. 3C). Similarly, knock-down of SULT2B1b significantly increased FAS mRNA level (Fig. 3B), but inhibited BCL2 mRNA expression (Fig. 3D). The protein expression of FAS, BCL2 and MYC was evaluated in NC-GFP-LV and SULT2B1-RNAi-LV Hepa1-6 cells that were serum-deprived or were treated with a combination of tumor necrosis factor-a (TNF-a, 10 ng/mL) and cycloheximide (CHX, 10 mg/mL). As seen in Fig. 3, both serum deprivation (Fig. 3E, F) and TNF-a/CHX treatment (Fig. 3G, H) reduced BCL2 and MYC protein levels, while FAS protein levels were increased.Analysis of Cell Cycle Arrest with SULT2B1 Knock-down in Hepa1-6 CellsTo elucidate the 
mechanisms of cell-cycle arrest induced by SULT2B1b interference that presented 15755315 in Fig. 4, we analyzed the expression of cyclinD1 and cyclinB1 by qPCR and Western-blot assays. Both the mRNA and protein levels of cyclinD1 presented no significant difference between siSULT2B1 and control Hepa16 cells (data not shown). However, as shown in Fig. 4A, cyclinBThe Suppressive Effects of SULT2B1b Inhibition on Cell Growth, CyclinB1 Expression and Tumorigenicity in Human Hepatocarcinoma CellsImmunocytochemical localization of SULT2B1b in BEL-7402 and SMMC-7721 cells using anti-SULT2B1 antibody revealed a widespread cytoplasmic distribution (Figure S3A,B). Normal rabbit IgG served as negative controls exhibited no green staining. To investigate the effects of SULT2B1b inhibition in human hepatocarcinoma cells, we designed siRNA targeted against SULT2B1 (both SULT2B1a and SULT2B1b isoforms) andSULT2B1b Promotes Hepatocarcinoma Proliferationspecifically against SULT2B1b. qPCR analysis revealed that both SULT2B1 siRNA and SULT2B1b-specific siRNA treatment decreased SULT2B1b mRNA.
