Fig. 7. Inducible-TFF3 expression in HMEC-hTERT cells increased TFF3 expression and stimulated oncogenic phenotype in 3D Matrigel and capacity for anchorage-independent growth.

a Western blot analysis was performed to assess the levels of TFF3, pSTAT3, CCND1, BCL2, and STAT3 in HMEC-hTERT-TetON-Dual2-TFF3 cells after exposure to increasing concentration of DOX (0.01–100 µg/ml). Soluble whole cellular extracts were run on an SDS-PAGE and immunoblotted as described in Materials and methods. β-actin was used as an input control for cell lysate. The sizes of detected protein bands in kDa are shown on the right side. b Confocal laser scanning microscopic cross-sections of the mammary acinar structured formed by HMEC-hTERT-TetON-Dual2-TFF3 and HMEC-hTERT-TetON-Dual2 cells cultured three-dimensional Matrigel on exposure to increasing concentration of DOX 0.01–100 µg/ml. Green colour denotes ZsGreen1, and red colour denotes mCherry. Consistent with western blot results obtained herein, elevated levels of TFF3 expression was observed after exposure to DOX. Capacity for anchorage-independent of HMEC-hTERT-TetON-Dual2-TFF3 cells and HMEC-hTERT-TetON-Dual2 cultured in complete medium with or without DOX (1 µg/ml) demonstrated using c soft agar colony formation and d foci formation. Soft agar colony formation evaluated (upper) and images (lower) of colonies was captured after cultured in the complete medium over a period of 18 days. Images were captured under ×100 magnification using a bright field microscope. All assays were performed as described in Material and methods. The column is mean of triplicate experiments; bars, ±SD. **P < 0.001, *P < 0.05