E cells. 56104 Hepa1-6 cells treated with GFP-LV or SULT2B1-RNAi-LV were cultured in 24-well plates with 500 mL culture medium. After attachment, cells were incubated with 20 nM [3H] cholesterol (53 Ci/mol; Perkin Elmer, San Jose, CA, USA) and 50 mM PAPS for the sulfation assay. After 16 h, the attached cells were washed with PBS, and then harvested in 50 mL of PBS. Conversion of [3H] cholesterol to [3H] methanol-water-soluble products wasdetermined by scintillation counting after extraction with 3.3 volumes of chloroform-methanol (1:1, v/v) from cells. The rates of cholesterol sulfation following GFP-LV or SULT2B1-RNAi-LV infection were calculated as the ratio of [3H] methanol watersoluble counts to the sum of chloroform/methanol water-soluble counts.Murine Xenograft Model for Tumorigenicity AssaySix-week old male BALB/c-nude mice were used for experimental tumorigenicity assays.Mouse Hepa1-6 cells (1.56106) that were transduced with NC-GFP-LV or mSULT2B1-RNAi-LV and human BEL-7402 cells (1.56106) transduced with NC-RFP-LV or hSULT2B1-RNAi-LV were injected subcutaneously into the subaxillary space of each mouse. Mice were weighed and the tumor width (W) and length (L) were measured every three days. Tumor volume was estimated according to the standard formula 1/26L6W2. Observation continued until day 18,21. TumorsSULT2B1b Promotes Hepatocarcinoma ProliferationFigure 7. SULT2B1b MedChemExpress PS 1145 knock-down suppressed the growth of human hepatocarcinoma cells in vivo and in vitro. (A) qPCR analysis of SULT2B1b mRNA levels in BEL-7402 and SMMC-7721 cells with SULT2B1 or SULT2B1b-specific siRNA treatment. Cell proliferation rates of BEL-7402 (B) and SMMC-7721 (C) cells treated with SULT2B1 or SULT2B1b-specific siRNA was assessed by CCK-8 assay. (D) cyclinB1 mRNA levels in BEL-7402 and SMMC-7721 cells with SULT2B1b-specific siRNA treatment was detected by qPCR assay. (E) cyclinB1 protein levels in BEL-7402 and SMMC-7721 cells with SULT2B1b-specific siRNA treatment detected by Western-blot analysis, b-actin as internal control. *P,0.05 vs. NC group. BEL-7402 cells (16106) infected with NC-RFP-LV or hSULT2B1-RNAi-LV were injected subcutaneously into right subaxillary region of each nude mouse. The xenograft tumor growth curve (F), representative images (G) and the weights (H) of dissected order Finafloxacin xenografts were shown. *P,0.05 vs. NC-RFP-LV control group. doi:10.1371/journal.pone.0060853.gwere excised from the animals, and then frozen in liquid nitrogen and stored at 1662274 280uC.Results SULT2B1b Promoted the Growth of Hepa1-6 Cells in vitroSULT2B1 expression was detected in C57BL/6 mouse liver, primary mouse hepatocytes purified from C57BL/6 mice liver as described in [22], and in the mouse hepatocarcinoma cell line, Hepa1-6, by Western-blot assay. As shown in Figure S1A, the SULT2B1 protein level in Hepa1-6 cells was much higher than normal mouse 
liver tissue and primary mouse hepatocytes. Figure S1B demonstrates SULT2B1 localization in Hepa1-6 cells by immunofluorescence. SULT2B1 isoforms were detected in Hepa16 cells transduced with NC-GFPLV or SULT2B1-RNAi-LV byStatistical AnalysisFor all experiments, each treatment condition was conducted in triplicate and repeated at least three times. The results of multiple observations are presented as the mean 6 standard error of the mean of at least three separate experiments. Statistical significance was determined by one-way ANOVA for multiple comparisons or by independent-sample Student’s t-test, Pearson correlation and simpl.E cells. 56104 Hepa1-6 cells treated with GFP-LV or SULT2B1-RNAi-LV were cultured in 24-well plates with 500 mL culture medium. After attachment, cells were incubated with 20 nM [3H] cholesterol (53 Ci/mol; Perkin Elmer, San Jose, CA, USA) and 50 mM PAPS for the sulfation assay. After 16 h, the attached cells were washed with PBS, and then harvested in 50 mL of PBS. Conversion of [3H] cholesterol to [3H] methanol-water-soluble products wasdetermined by scintillation counting after extraction with 3.3 volumes of chloroform-methanol (1:1, v/v) from cells. The rates of cholesterol sulfation following GFP-LV or SULT2B1-RNAi-LV infection were calculated as the ratio of [3H] methanol watersoluble counts to the sum of chloroform/methanol water-soluble counts.Murine Xenograft Model for Tumorigenicity AssaySix-week old male BALB/c-nude mice were used for experimental tumorigenicity assays.Mouse Hepa1-6 cells (1.56106) that were transduced with NC-GFP-LV or mSULT2B1-RNAi-LV and human BEL-7402 cells (1.56106) transduced with NC-RFP-LV or hSULT2B1-RNAi-LV were injected subcutaneously into the subaxillary space of each mouse. Mice were weighed and the tumor width (W) and length (L) were measured every three days. Tumor volume was estimated according to the standard formula 1/26L6W2. Observation continued until day 18,21. TumorsSULT2B1b Promotes Hepatocarcinoma ProliferationFigure 7. SULT2B1b knock-down suppressed the growth of human hepatocarcinoma cells in vivo and in vitro. (A) qPCR analysis of SULT2B1b mRNA levels in BEL-7402 and SMMC-7721 cells with SULT2B1 or SULT2B1b-specific siRNA treatment. Cell proliferation rates of BEL-7402 (B) and SMMC-7721 (C) cells treated with SULT2B1 or SULT2B1b-specific siRNA was assessed by CCK-8 assay. (D) cyclinB1 mRNA levels in BEL-7402 and SMMC-7721 cells with SULT2B1b-specific siRNA treatment was detected by qPCR assay. (E) cyclinB1 protein levels in BEL-7402 and SMMC-7721 cells with SULT2B1b-specific siRNA treatment detected by Western-blot analysis, b-actin as internal control. *P,0.05 vs. NC group. BEL-7402 cells (16106) infected with NC-RFP-LV or hSULT2B1-RNAi-LV were injected subcutaneously into right subaxillary region of each nude mouse. The xenograft tumor growth curve (F), representative images (G) and the weights (H) of dissected xenografts were shown. *P,0.05 vs. NC-RFP-LV control group. doi:10.1371/journal.pone.0060853.gwere excised from the animals, and then frozen in liquid nitrogen and stored at 1662274 280uC.Results SULT2B1b Promoted the Growth of Hepa1-6 Cells in vitroSULT2B1 expression was detected in C57BL/6 mouse liver, primary mouse hepatocytes purified from C57BL/6 mice liver as described in [22], and in the mouse hepatocarcinoma cell line, Hepa1-6, by Western-blot assay. As shown in Figure S1A, the SULT2B1 protein level in Hepa1-6 cells was much higher than normal mouse liver tissue and primary mouse hepatocytes. Figure S1B demonstrates SULT2B1 localization in Hepa1-6 cells by immunofluorescence. SULT2B1 isoforms were detected in Hepa16 cells transduced with NC-GFPLV or SULT2B1-RNAi-LV byStatistical AnalysisFor all experiments, each treatment condition was conducted in triplicate and repeated at 
least three times. The results of multiple observations are presented as the mean 6 standard error of the mean of at least three separate experiments. Statistical significance was determined by one-way ANOVA for multiple comparisons or by independent-sample Student’s t-test, Pearson correlation and simpl.
