Overexpression of ADAM12L in MCF10A breast epithelial cells induces a mesenchymal phenotype.

<p>MCF10A cells were infected with lentiviruses expressing either GFP-ADAM12L fusion protein (ADAM12L) or the control protein GFP (Control). After antibiotic selection, cells were further enriched using flow cytometry and cultured for 48 hours. (A) Bright field pictures were captured to show cell morphology. (B) Cells were fixed and immunostained for E-cadherin, vimentin or stained with rhodamine-conjugated phalloidin to monitor actin stress fibers cytoskeletal actin. (C) Real-time PCR analyses of epithelial (E-cadherin and β-catenin) and mesenchymal (N-cadherin, vimentin, Zeb1, fibronectin and Twist) markers. Results are expressed as fold changes in ADAM12L-overexpressing cells compared with control cells. (D) Western blot analyses of E-cadherin and vimentin in ADAM12L-overexpressing cells. (E and F) ADAM12-overexpressing MCF10A cells were transfected with 100 nM non-targeted siRNA (siControl) or ADAM12 siRNA (siADAM12). After 48 hours, E-cadherin expression was analyzed by Western blotting (E). The efficiency and specificity of RNA interference in cell extracts was confirmed by ADAM12 blots (F). (G) Western blot analyses of phosphorylated Ser465/Ser467P-Smad2, Ser423/Ser425P-Smad3, Ser473P-Akt, Thr202/Tyr204P-ERK, Thr180/Tyr182P-p38 and Thr183/Thr185P-JNK in ADAM12L-overexpressing cells and control cells, 48 hours post-seeding. Immunoblots for ADAM12L and HSC-70 are shown as control. Left, representative western blots. Right, densitometric analyses of protein amounts in cells. Results are expressed as mean±SD from 3 independent experiments (*, p<0.05; **, p<0.01).