Additional Supporting Information may be found in the online version of this article.

HEP_21544_sm_suppfig1.tif826KSupporting Figure 1: Epigenetic regulation of CD133 expression. A: CD133- Huh7 cells were treated with 5μM DAC and/or 300nM TSA for 72 hrs, and DMSO was used as a vehicle control. Bars represent mean±SD, (n=3). B: CD133- Huh7 cells were treated with 5 μM DAC for indicated time, and DMSO was used as a vehicle control. C: CD133- Huh7 cells were treated with DAC at defined dosage for 72 hrs, and DMSO was used as a vehicle control. For each series, immuno-blot was performed to determine CD133 expression, and densitometry analysis was utilized to calculate relative CD133 expression levels after normalization to loading control β-Actin.
HEP_21544_sm_suppfig2.tif288KSupporting Figure 2: Verification of in-vitro DNA methylation using HpaII. Methods: In-vitro DNA methylation was performed using a recombinant CpG methyltransferase (M.SssI, New England Biolabs, Beverly, MA). 5μg DNA plasmids were incubated with 5 units M.SssI supplemented with 160μM S-adenosylmethionine overnight at 37°C. The methylated DNA was incubated with HpaII at 37°C for 1 hr to confirm the completion of in vitro methylation at CpG sites. Results: Methylated and un-methylated constructs were subsequently digested using methylation-sensitive restriction enzyme HpaII, which cleaves un-methylated but not methylated DNA. Products were electrophoresed in a 2% agarose gel with DNA molecular standard was shown in the left. This figure clearly shows that in-vitro methylation was successful at preventing restriction by methylation sensitive HpaII.
HEP_21544_sm_suppfig3.tif103KSupporting Figure 3: In-vitro methylation significantly reduces CD133 promoter-1 activity. Huh7 cells were plated into 24-well plates, cultured to 60% confluence, transfected with 50 ng CD133 promoter-1 luciferase reporter vector, with and without in-vitro DNA methylation (as described in Supporting Figure 2) and co-transfected with 25ng renilla luciferase vectors as internal control. The cells were washed twice with ice-cold 1×PBS, lysed with 100μL Passive Lysis Buffer after 24 hrs of transfection. Firefly and renilla luciferase activity were measured using a dual luciferase reporter assay system (Promega) according to the manufacturer's instructions. Promoter activity was presented as the ratio of Firefly to control Renilla activity (p<0.01). Bars represent mean±SD, (n=4).
HEP_21544_sm_suppfig4.tif1174KSupporting Figure 4: TGFβ1 induces de-methylation in CD133-promoter-1. Raw pyrosequencing data demonstrates that TGFβ1 stimulation is capable of reducing methylation in the corresponding CpG sites in CD133 promoter-1.
HEP_21544_sm_supptable.doc28KSupporting Information.

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