Fig. 1

BMP3 with a hypermethylation status is downregulated in CRC. a BMP3 protein levels in normal, adenoma, and carcinoma tissues were assessed by immunohistochemistry (Bar, 200 μm). Enlarged images (lower panel, bar, 72 μm) and red arrows show that BMP3 is located in the cytoplasm with strong staining in normal tissue (1), moderate staining in adenoma (2), and weak staining in carcinoma. (3). b qMSP analysis of BMP3 methylation status in CRC tissues (Cancer) and paired adjacent normal tissues (Adjacent normal) (n = 80). Methylation percentages are 51.89% for cancer tissues and 5.06% for normal tissues. c ROC curve for BMP3 methylation levels in CRC versus paired adjacent normal tissues. d Upper panel: Western blot analysis of BMP3 expression in CRC tissues (T) and paired normal tissues (N). Pt: patient (n = 11). Middle and lower panels: Detection of BMP3 methylation status by MSP in the same paired normal and CRC tissue samples (n = 11), in addition to one negative control and one positive control. Lanes U and M indicate unmethylated and methylated MSP products of BMP3 gene, respectively. e Left panel: Relative protein expression level of BMP3 in a total of 36 sample pairs, 13 sample pairs without methylation in CRC (T-Unmethylated), and 23 sample pairs with methylation in CRC (T-methylated). Right panel: Dot histogram showing the protein expression level in 23 CRC tissues with methylation (T-methylated) and without methylation (T-unmethylated) (GraphPad Prism). BMP3 protein expression was significantly reduced in methylated group compared to that of unmethylated group (p < 0.01). f Western blot and MSP analyses in six CRC cell lines. g Reactivated expression of BMP3 mRNA in CRC cell lines after treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) and Trichostatin (TSA) (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001