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. 2016 Feb 2;6:20209. doi: 10.1038/srep20209

Figure 3. Ligand-switching reversibly interconverted distinct characteristics of mammary epithelial cells.

Figure 3

(a) Representative projected Z-stack images of the acinus-like structures formed by E-cells and A-cells after a 2-week culture on the reconstituted basement membrane. Cells were visualized by fluorescent phalloidin staining. Yellow arrowheads indicate cell clusters judged as non-acinus. Scale bar: 100 μm. (b) Quantification of acinus-formation efficiency of sequentially generated E-cells and A-cells. The criteria for evaluating the acinus size are shown in Supplementary Fig. 3a. (c) Flow cytometric analysis of CD24 (X-axis) and CD44 (Y-axis) expression in sequentially generated E-cells and A-cells. (d) Western blot analysis of EMT related factors and EGFR in HMT-3522 S1 cells generated by the ligand-switching between EGF (10 ng/mL) and AREG (20 ng/mL). S1-EGF and S1-AREG indicate EGF- and AREG-cultured parental cells, respectively. S1-AREG cells were further cultured in the presence of EGF, generating S1-EGF (2nd). (e) Immunofluorescent images of S1-EGF, S1-AREG and S1-EGF (2nd) cells stained with anti-E-cadherin antibody (green) and anti-EGFR antibody (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar: 40 μm. (f) FACS analysis of the expression of CD44 and CD24 in S1-EGF, S1-AREG and S1-EGF (2nd) cells.