31 The developing tumors were observed over the next 5 to 6 weeks

31 The developing tumors were observed over the next 5 to 6 weeks, and the mice were then sacrificed at the end of follow-up. All animal studies were approved by the Institutional Animal and Committee at the National Defense

Medical Center. Details regarding generation of plasmid constructs, stably or inductively expressing SOX1 clones, cell proliferation, invasion, colony formation, glutathione Nutlin-3 clinical trial S-transferase pull-down, co-immunoprecipitation, immunocytochemistry and senescence-associated β-galactosidase staining, and statistical analysis are provided in the Supporting Information. AIG, anchorage-independent growth; DOX, doxycycline; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; LEF, lymphocyte-enhanced factor; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; QMS-PCR, quantitative methylation-specific polymerase chain reaction; RT-PCR, reverse-transcription polymerase

chain reaction; SOX, SRY (sex determining Quizartinib mouse region Y)-box; TCF, T cell factor; TLCN, Taiwan Liver Cancer Network. First, we examined the messenger RNA (mRNA) and protein expression of SOX1 in eight HCC cell lines. SOX1 transcript and protein was undetectable in 100% of the HCC cell lines, but was expressed in normal liver tissue (Fig. 1A). We then checked the mRNA level of 60 primary HCCs and their corresponding adjacent nontumor tissues using quantitative RT-PCR and found that SOX1 mRNA expression was significantly downregulated

in primary HCCs compared with the adjacent nontumor tissues (P < 0.01) (Fig. 1B). There was no significant correlation between SOX1 mRNA expression and clinical characteristics (Supporting Table 2). Based on our previous data, promoter hypermethylation of SOX1 might contribute to downregulation of SOX1 in HCC. Next, we checked the methylation status of the HCC cell lines and clinical HCC tissues by QMS-PCR. Hypermethylation was confirmed in the HCC cell lines (HepG2, Hep3B, Huh7, SK-Hep-1, HA22T, Mahlauv, and Tong) and HCC tissues, which showed downregulated or silenced SOX1 expression, whereas methylation was not found in the nontumor liver tissues (P < 0.01) (Fig. 1C,D). The methylation status in the SOX1 promoter region was then validated by bisulfite sequencing. The MTMR9 bisulfite sequencing results were consistent with QMS-PCR (data not shown). To validate whether promoter methylation is involved in the regulation of SOX1, three HCC cell lines (HepG2, Hep3B, and TONG) with silenced SOX1 expression were treated with 5-AZA-2′-deoxycytidine (5-Aza-CdR) combined with or without trichostatin A. The data showed the decreased methylation status of SOX1 and re-expression of SOX1 mRNA in all cell lines examined (Fig. 1E), further implying that the transcriptional silencing of SOX1 was mediated by promoter methylation and/or histone modification.

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