Fig 2: Optimization of anti-MCM2, anti-MCM4, and anti-MCM6 monoclonal antibodies used in IHC.Specificity of the anti-MCM2, anti-MCM4, and anti-MCM6 monoclonal antibodies is confirmed using normal colonic mucosa and normal tonsil, respectively, as positive control and normal cerebral cortex as negative control based on the expression data in the Human Protein Atlas database [37]. Isotype control antibodies are used to estimate the nonspecific binding of target primary antibodies due to Fc receptor binding or other protein–protein interaction. An isotype control antibody should have the same immunoglobulin type and be used at the same concentration as the test antibody.

Fig 3: Low expression of MCM2, MCM4, MCM6, and MKI67 is associated with increased probability of relapse-free survival [using the online survival analysis tool Kaplan-Meier Plotter (Gyorffy et al., 2010)].The association between MCM2, MCM4, MCM6, and MKI67 expression and relapse-free survival of breast cancer patients was studied using the online survival analysis tool Kaplan-Meier Plotter. MCM2, MCM4, MCM6, and MKI67 mRNA expression was stratified into high or low expression using the median mRNA expression level as the cutoff point. Kaplan-Meier survival curve for MCM expression and the P value for log-rank test are shown alongside for each of the studied transcript expression (P < .0001).

Fig 4: Patterns of MCM2, MCM4, MCM6, and Ki-67 expression in normal breast tissue.(A) A tissue core showing normal breast tissue and stained with HPS. (B) MCM2, MCM4, and MCM6 nuclear labeling in only a few scattered luminal cells lining the terminal duct lobular units was noticed in 57%, 57%, and 48% of normal breast tissue samples, respectively. This pattern of expression was also found in 100% of normal breast tissues using Ki67. (C) A proportion of normal breast tissues demonstrated a strong MCM2, MCM4, and MCM6 nuclear labeling in most if not all of the luminal cells lining the normal breast ducts (43%, 43%, and 52% of normal breast tissue samples, respectively).

Fig 5: Overexpression of MCM2, MCM4, and MCM6 is associated with high histological grade of breast cancer.(A) Protein overexpression of MCM2, MCM4, MCM6, and Ki-67 is associated with high histological grade of breast cancer. Levels of MCM2, MCM4, and MCM6 expression exceed that of Ki-67 in different grades of breast cancer (the same tissue cores were stained with the four antibodies in consecutive sections prepared from the tissue microarray). The whole tissue cores are shown, and a magnified area is shown inside the red square (magnification 30×). (B) Histograms displaying the percentage of patients with low and high MCM2, MCM4, MCM6, and Ki-67 expression in breast cancer of different histological grades (cutoff values are 35%, 40%, 55%, and 14% for MCM2, MCM4, MCM6, and Ki-67, respectively). Most of grade I breast cancer patients expressed low levels of MCM2, MCM4, MCM6, and Ki-67. In contrast, high levels of MCM2, MCM4, MCM6, and Ki-67 expression were detected in the majority of grade III patients. (C) In silico analysis of MCM2, MCM4, MCM6, and MKI67 mRNA expression in the three SBR grades, as represented by bc-GenExMiner database v4.0.