Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factor-β†
Article first published online: 28 JAN 2010
Copyright © 2010 American Association for the Study of Liver Diseases
Volume 51, Issue 5, pages 1635–1644, May 2010
How to Cite
You, H., Ding, W. and Rountree, C. B. (2010), Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factor-β. Hepatology, 51: 1635–1644. doi: 10.1002/hep.23544
Potential conflict of interest: Nothing to report.
- Issue published online: 22 APR 2010
- Article first published online: 28 JAN 2010
- Accepted manuscript online: 28 JAN 2010 12:00AM EST
- Manuscript Accepted: 20 DEC 2009
- Manuscript Received: 21 JUL 2009
- National Institutes of Health. Grant Numbers: NIDDK K08, K08DK080928
- American Cancer Society, Research Scholar Award. Grant Number: RSG-10-073-01-TBG
- Office for the Advancement of Telehealth (OAT)
- Health Resources and Services Administration, DHHS. Grant Number: D1BTH06321
- Children's Miracle Network
Additional Supporting Information may be found in the online version of this article.
|HEP_21544_sm_suppfig1.tif||826K||Supporting 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.tif||288K||Supporting 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.tif||103K||Supporting 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.tif||1174K||Supporting 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.|
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