Fig. 1

SMURF1 depletion inhibits ER alpha positive breast cancer cell proliferation in vitro and in vivo. a and b Estrogen stimulates the expression of endogenous SMURF1 in breast cancer cells. MCF-7cells are treated with 10 nM estradiol. After 24 h, SMURF1 mRNA and protein levels were determined by Western blot analysis. Actin was used as internal control. Experiments were done in triplicates. *P < 0.05; ** P < 0.01; ***P < 0.001 for SMURF1 mRNA level comparison. c SMURF1 depletion inhibits the cell proliferation in breast cancer cells. MCF-7 cells were transfected with 50 nM SMURF1 siRNA (mix of #1 and #2) or 50 nM control siRNA. After 24 h, the WST assay was used to determine the cellar metabolic activity at indicated time points after transfection. Cells are treated for indicated times with 10 nM E2 or vehicle. Experiments were done in triplicates. *P < 0.05; ** P < 0.01; ***P < 0.001 for cell growth comparison. d, e and f MCF-7 cells were stably transfected with lentivirus carrying scrambe shRNA or SMURF1 shRNA. Female NOD scid gamma (NSG) mice were estrogen-supplemented by implantation of slow-release 17β-estradiol pellets (0.72 mg/90-d release; Innovative Research of America) 1 day before MCF-7 tumor cell injection into the mammary fat pad (2 × 10⁶ MCF-7 cells suspended in 100ul Matrigel solution). MCF-7 tumor xenografts were measured every 3~5 days and the tumor volume were calculated by length × width² /2. The mice were sacrificed at 2 month after transplant, and the tumors were weighted. The tumor growth curve, photograph and tumor weight were shown in Figure (d), (e) and (f) respectively. g Wound healing assay of MCF-7 transfected with the indicated siRNA. Quantification of wound closure at the indicated time points. Data are presented as ± SD. **, P < 0.01, ***, P < 0.001 (student’s t-test). h Clone formation assay of MCF-7 cells transfected with indicated siRNA. Quantification of clone formation is shown at the indicated time points. Data are presented as ± SD. **, P < 0.01, ***, P < 0.001 (student’s t-test)