Fig. 7.

Overexpression of c-Cbl reverses HER2-mediated tamoxifen resistance. a c-Cbl protein level was detected in BT474 and T47D cells. And then, BT474 cells were transfected with 3 × flag-CMV-9-c-Cbl (OE-c-Cbl) or 3 × flag-CMV-9 vector(Vector), and then examined the c-Cbl level by immunoblot analysis. b Immunoprecipitation after overexpression c-Cbl 48 h in BT474. c BT474 cells were transfected with control vector or c-Cbl overexpression plasmids, 24 h later, followed by vehicle, estrogen(10 nmol/L), and tamoxifen (1 μmol/L) or combination treatment for 4 h. Cell lysates were examined by immunoblot analysis using the indicated antibodies. d BT474 cells were transfected with plasmids expressing c-Cbl for 24 h, and then exposed to vehicle, estrogen(10 nmol/L), or tamoxifen(1 μmol/L) treatment for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. (*p < 0.05) (e) Colony formation assays in BT474 cells transfected with vector or plasmids expressing c-Cbl. BT474 cells were transfected with vector or plasmids expressing c-Cbl for 24 h and then treated with E2 (10 nmol/L) and/or TAM (1 μmol/L) for 14 d. On day 14, colonies were fixed and stained with Giemsa. *P < 0.05