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. 2010 Sep 20;5(9):e12847. doi: 10.1371/journal.pone.0012847

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Chang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Immunohistochemical analysis of RASSF1A and BLU proteins in paraffin-embedded sections of tumor and normal specimens of representative NSCLC patients. The RASSF1A protein expression status for three samples are marked as panel (a), (b), and (c) and the BLU protein expression status for the other three samples are marked as panel (d), (e), and (f). The cells that exhibited brown color staining in cytoplasm were considered positive for protein expression (patients a and d). Negative immunoreactivity of RASSF1A and BLU was found in two patients (b and e). The histologies in (a) and (d) are adenocarcinomas, (b) and (e) are squamous cell carcinomas, and (c) and (f) are normal lung tissues. Original magnification x200. (B) Representative examples of semi-quantitative multiplex RT-PCR analysis of the RASSF1A and BLU genes in lung cancer patients. GAPDH was used as the internal control for the analysis. N and T represent the paired normal and tumor lung cells from the same patient. (C) Methylation status of RASSF1A and BLU genes were assessed by MSP in tumor (T) and matched normal (N) lung samples. (D) Concordance analysis between protein expression, mRNA expression, and methylation status of RASSF1A and BLU genes. Y-axis represents the percentage of cases; X-axis represents the type of comparison. Concordance analysis for RASSF1A and BLU was depicted as percentage. “+” indicated positive protein expression, positive mRNA expression, and DNA hypermethylation, as opposed to “–”, which indicated a negative result. Numbers above the bars indicated the percentage in the total concordant group (gray column) and discordant group (white column). P ≤0.05 was considered to be statistically significant and was labeled with *. (E) Correlation analysis of methylation status between RASSF1A and BLU. Numbers above the bars indicated the percentage in the total concordant group (gray column) and discordant group (white column).