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. 2021 Jan 6;8:597673. doi: 10.3389/fcell.2020.597673

FIGURE 4.

FIGURE 4

Association of EpCAM with BC radioresistance. (A) Kaplan Meier graph showing the overall survival difference between the EpCAM high and EpCAM low groups of 904 patients who underwent radiotherapy in the METABRIC dataset. (B) Immunofluorescence photomicrograph showing EpCAM expression (red) in ZR-75-1, ZR-75-1FR, and ZR-75-1EpCAM cells, cross-stained with DAPI (blue) (Scale bar-10 μm). (C) Semi-quantitative Western blots showing the level of EpCAM. For comparison of EpCAM level between MCF-7 and MCF-7FR as well as ZR-75-1 and ZR-75-1EpCAM, 40 μg of protein was loaded. While in case of ZR-75-1 and ZR-75-1FR 150 μg of protein was loaded. (D) Comparison of survival fraction between ZR-75-1, ZR-75-1FR, and ZR-75-1EpCAM cells against different doses of radiation. Inset table indicates radio-resistivity index (D0 value) for each. Dose modifying factor(DMF) of ZR-75-1EpCAM cells is shown in inset (E,F) Immunofluorescence photomicrograph and graphical representation of γH2AX (red) time kinetic in ZR-75-1 and ZR-75-1EpCAM cells (n = 45) after 2Gy radiation exposure. The nucleus is cross-stained with DAPI (blue) (Scale bar-5 μm). Statistical significance between the two groups in (F) is determined by Anova. (G) Graph showing mean fluorescence intensity of γH2AX foci in ZR-75-1 and ZR-75-1EpCAM cells (n = 45) 24 h after treatment with DNA damaging chemotherapeutic drugs, i.e., Doxorubicin (10 μg/ml), Cisplatin (15 μg/ml), and Gemcitabine (60 μg/ml). (H) Graph showing the proliferation fraction of ZR-75-1, ZR-75-1FR, and ZR-75-1EpCAM cells before and after 10Gy irradiation as determined by EdU proliferation assay. Statistical significance in (G,H) is determined by Student’s t-test. (I) The table represents data derived from the EdU assay as the percentage reduction in cell proliferation in 10Gy irradiated cells over unexposed control. (J) Semi-quantitative Western blot showing expression of important molecules of DNA repair pathways involved in untreated and 2Gy radiation treated ZR-75-1 and ZR-75-1EpCAM cells. *P < 0.05, ***P < 0.001, and ****P < 0.0001.