Fig. 1: Forced expression of TFF3 in immortalized-HMECs stimulates phenotypic changes and cell proliferation. | Cell Death & Disease

Fig. 1: Forced expression of TFF3 in immortalized-HMECs stimulates phenotypic changes and cell proliferation.

From: Expression of two non-mutated genetic elements is sufficient to stimulate oncogenic transformation of human mammary epithelial cells

Fig. 1

HMEC-hTERT, MCF10A and MCF12A cells were transfected with the pIRESneo3-TFF3 expression construct to generate the corresponding stable cell lines with forced expression of TFF3; a pIRESneo3 construct was used as vector control as described in Materials and methods7,8. Stable clones were designated as HMEC-hTERT-vector and HMEC-hTERT-TFF3; MCF10A-vector and MCF10A-TFF3; and MCF12A-vector and MCF12A-TFF3. The levels of TFF3 expression in immortalized-HMECs with forced expression of TFF3 and their vector control cells was demonstrated using a RT-PCR and b western blot. Soluble whole-cell extracts were run on an SDS-PAGE and immunoblotted using rabbit polyclonal anti-TFF3 antibody as described in Materials and methods. β-actin was used as input control. The sizes of PCR product in base pair (bp) are shown on the left side and detected protein bands size in kDa are shown on the right side. Among immortalized-HMECs, HMEC-hTERT cells exhibit-deficient endogenous levels of TFF3 mRNA and protein, whereas, endogenous expression of TFF3 was not detected in MCF10A and MCF12A cells by RT-PCR and western blot. c Representative phase-contrast microscopic images of immortalized-HMECs with forced expression of TFF3 and their vector control cells on monolayer culture. Images were captured under ×200 magnification. d Representative confocal laser scanning microscopy images of immortalized-HMECs with forced expression of TFF3 and their vector control cells cultured on Matrigel (2D). Images were captured under ×100 magnification. e Total cell number, f BrdU incorporation, and g apoptotic cell death of immortalized-HMECs with forced expression of TFF3 and their vector control cells. Forced expression of TFF3 in immortalized-HMECs significantly abrogated apoptotic cell death as a consequence of serum deprivation when compared to their vector control cells. All assays were performed as described in Material and Methods. Column or chart point is mean of triplicate experiments; bars, ±SD. **P < 0.001, *P < 0.05

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