Fig 2: Analysis of RASSF1A and RASSF1C proteins in ccRCC by western blot analysis. Semi-quantitative analysis of RASSF1A and RASSF1C proteins in tumor (T), control kidney (C ) and metastasized (M) samples normalized to GAPDH protein level. Lines 1 and 2 represent biopsies from patient characterized by TNM 3 and Fuhrman's 2 grade, whereas lines 3–5 represent biopsies from patient with TNM 4, Fuhrman's 3 grade.

Fig 3: Correlation plots between either RASSF1A promother methylation, methylation and mRNA (A) or mRNA and protein levels of (B) RASSF1A and (C) RASSF1C. Details in the text.

Fig 4: Immuhistochemical localization of RASSF1A and RASSF1C proteins in ccRCC. Immuhistochemical localization of (A–D) RASSF1A and RASSF1C (F–I) proteins in ccRCC. Normal kidney (A and F), tumor kidney of TNM 3 and Fuhrman's grade 2 (B and G) or TNM 4, Fuhrman's grade 4 (C and H) or metastasized lymph node (D and I) of two ccRCC patients (according to Fig. 5) are shown. Strong reaction was observed for RASSF1C in all samples, RASSF1A is characterized by strong presence in epithelial cells of control kidney; weak expression was observed in tumor and mestastized samples as compared to negative control (primary antibody was omitted) of (J) either tumor or (E) metastasized lymph node samples. Magnification, ×20.

Fig 5: Quantitative comparison between RASSF1A mRNA levels in tumor and metastasized samples divided by methylation of the RASSF1A gene promoter. Impact of RASSF1A promoter methylation on the gene expression. mRNA results of kidney tumor and metastasized samples (T, M) were divided according to DNA methylation; 25% DNA methylation was treated as threshold. Mann-Whitney U test was applied: *P<0.05; **P<0.01 between groups.