Fig 2: pSTAT5, but not NS1, interacts with the MCM complex.(A) Immunoprecipitation (IP) assay. Cell lysates of NS1Flag-expressing UT7/Epo-S1 cells were prepared for pull-down assays with either anti-Flag-conjugated beads or control beads. Immunoprecipitated proteins were examined for the presence of MCM2 by Western blotting. Blots were reprobed with rabbit anti-pSTAT5(Y694), anti-E2F5, and anti-Flag antibodies. Detection of E2F5 was used as a positive control for NS1 IP. (B) Co-IP assay. UT7/Epo-S1 cells were collected, washed, and lysed with RIPA buffer. After centrifugation, the supernatant was incubated with either rabbit anti-pSTAT5(Y694) or control IgG antibody. Immunoprecipitated proteins were blotted for the presence of the MCM complex with an anti-MCM5 antibody and for pSTAT5 with rabbit anti-pSTAT5(Y694). (C) Reverse Co-IP assay. Reverse Co-IP was performed with an anti-MCM5 antibody. Immunoprecipitated proteins were examined for pSTAT5, MCM2, and MCM5, respectively. (D) Co-IP of lysates treated with DNase. UT7/Epo-S1 cell lysates, either treated or untreated with DNase (750 units of Benzonase) were incubated with anti-pSTAT5(Y694) or control IgG antibodies for Co-IP assay, and immunoprecipitated proteins were examined for MCM2 by Western blot analysis. (E-H) Immunofluorescence analysis. (E&F) Mock- or B19V-infected CD36+ EPCs were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies, followed by (E) incubation with respective secondary antibodies, or by (F) proximal ligation assay, which produces amplified signal for labeled molecules in close proximity. (G) CD36+ EPCs were incubated with either DMSO or pimozide (at 30 µM) for 2 days. And then the cells were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies for proximity ligation assay. (H) Infected EPCs were stained with an anti-capsid antibody. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.

Fig 3: 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 4: Correlation analysis of MCM2, MCM4, MCM6, and MKI67 in breast cancer patients.(A) Correlation diagrams using MiSTIC visualization tool to correlate MCM2, MCM4, and MCM6 with MKI67 and ESR1 in breast cancers based on RNA-sequencing data derived from TCGA. A strong positive correlation could be detected between each of MCM2, MCM4, and MCM6 and MKI67 (r = 0.73, 0.66, and 0.73, respectively), whereas a negative correlation was observed between ESR1 and each of MCM2, MCM4, MCM6, and MKI67(r = -0.33, -0.34, -0.44, and -0.38, respectively). (B) Targeted correlation analysis of MCM2, MCM4, MCM6, and MKI67 in all breast cancer patients and within molecular subtypes as represented by bc-GenExMiner database v4.0. The correlation values were different within each molecular subtype.

Fig 5: Increased expression of MCM2, MCM4, and MCM6 is associated with luminal B, HER2-positive, and triple-negative breast cancer.(A) Elevated levels of protein expression of MCM2, MCM4, MCM6, and Ki-67 were detected in luminal B, HER2-positive, and triple-negative breast cancer. Levels of MCM2, MCM4, and MCM6 protein expression exceed that of Ki-67 in the different molecular subtypes 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×). Histograms displaying the percentage of patients with low and high MCM2, MCM4, MCM6, and Ki-67 expression in breast cancer of different molecular subtypes (cutoff values are 35%, 40%, 55%, and 14% for MCM2, MCM4, MCM6, and Ki-67, respectively). Most of luminal A 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 Her2-positive and triple-negative subtypes. (B) Significant differences (P < .0001) were detected between these three subtypes and luminal A breast cancer.