1. Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer 
Naofumi Kagara, Kelly T. Huynh, Christine Kuo, Hideyuki Okano, Myung Shin Sim, David Elashoff, Kelly Chong, Armando E. Giuliano, Dave S.B. Hoon 
The American Journal of Pathology 
Volume 181, Issue 1, Pages (July 2012)
DOI: /j.ajpath
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
Pearson's correlation coefficient: mRNA expression versus individual CpG island methylation. Pearson's correlation analysis demonstrating the most significant inverse correlation between methylation status and mRNA expression at CpG site 3 of CD44 (−0.925), CpG site 25 of CD133 (−0.792), and CpG site 9 of Musashi-1 (−0.875). The promoter region of CD24 was uniformly unmethylated in all cell lines, regardless of mRNA expression levels.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
Epigrams demonstrating methylation of individual CpG sites in breast cancer cell lines before and after 5-aza treatment. Demethylation of T47D and MDA-MB-231 BCa cell lines with 5-aza is associated with increased hypomethylation of CD44 CpG 3 (A), CD133 CpG 25 (B), and Musashi-1 CpG 9 (C), as demonstrated by MassARRAY. Open circles, sites without CpG islands.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
Assessment of BCSC gene activation after 5-aza treatment. A: The protein expression of CD44 and CD133 on T47D cells untreated or treated with 5-aza is evaluated by FACS analysis. Dotted, untreated cells; bold line, 5-aza–treated cells; and gray area, isotype controls. B: Musashi-1 expression is evaluated by IHC of MDA-MB-231 cells before and after 5-aza treatment.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
IHC analysis of BCSC genes in clinical breast cancer specimens. The clinical samples with the lowest and highest methylation rate of CD44, CD133, and Musashi-1 were evaluated by IHC with each Ab. The numerical value of the methylation status of each gene promoter is represented in each photograph, with the MassARRAY methylation results of the corresponding CpG site represented. Representative tumors are shown that were hypomethylated (A) and demonstrated strongly positive staining of CD44, CD133, and Musashi-1 (Spearman rank correlation analysis: P < , P = 0.02, and P = 0.03, respectively) and hypermethylated (B) and showed negative or weak staining. Scale bar = 100 μm.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
MassARRAY analyses of BCSC genes in breast cancer tissues. The methylation status of CD44, CD133, Musashi-1, and combined was evaluated by MassARRAY and is represented in the TN and non-TN groups (A) and in four subtypes (B) based on HR and HER2 status (HR+/HER2−, HR+/HER2+, HR−/HER2+, and HR−/HER2−). Vertical lines under each subtype represent those subtypes with statistically significant differences in the methylation status for each individual CSC gene and then as a combination of all three genes by Tukey's test. Bars and dotted bars indicate the mean value and the SD, respectively. *P < 0.05.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

7. Figure 6
ROC analysis of the BCSC genes for patients with TNBC. ROC curves toward TNBC showing a combination of all three BCSC genes (CD44, CD133, and Musashi-1) (A) and in individual genes CD44 (B), CD133 (C), and Musashi-1 (D). The area under the curve (AUC) and P values for each BCSC individually and in combination are shown.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2012 American Society for Investigative Pathology Terms and Conditions