Fig 2: Downregulation of SFN promotes HER2-induced human breast cancer tumorigenesis.(a) SFN expression level is anti-correlated with the protein expression level of multiple members of the HER2 pathway in human breast cancer cell lines. Presented are the 50 most negatively associated proteins, the HER2 pathway members are labelled in red. IC, information coefficient. (b) Low expression of SFN and high expression of HER2 are associated with worse prognosis in HER2-enriched subtype tumours. Presented are Kaplan–Meier plots of the patients' overall survival based on a limited cohort of 89 HER2-enriched subtype patients46. (c) Immunoblot analysis of SFN protein levels in stable MCF10A cell lines overexpressing an empty vector (MCF10A-Vec; non-tumorigenic), HER2 alone (MCF10A-ERBB2; non-tumorigenic), 14-3-3? alone (MCF10A-14-3-3?; non-tumorigenic) or both (MCF10A-ERBB2/14-3-3?; tumorigenic). Expression of 14-3-3? results in significant reduction of SFN expression. (d) Immunoblot analysis of SFN protein levels in transformed MCF10A cell lines. Overexpression of the SFN open reading frame restores SFN protein expression. (e) Decreased migration of transformed MCF10A cells following the restoration of SFN expression, as evaluated by a transwell migration assay. * P=1.5 × 10-5(Student's t-test). (f) Decreased invasion of transformed MCF10A cells following the restoration of SFN expression, as evaluated by a transwell invasion assay. *P=0.04 (Student's t-test). (g) Decreased colony formation of transformed MCF10A cells following the restoration of SFN expression, as evaluated by a soft-agar assay. Upper panel: images of colonies. Lower panel: quantification of the number of colonies in each condition. *P=0.04 (Student's t-test). Bar plots represent the mean±s.d. Experiments were performed in triplicates. See also Supplementary Figs 10–13.

Fig 3: SMG7 14-3-3-like domain mediates its interaction with Ser15-phosphorylated p53. (a) Schematic illustrating various SMG7 fragments and point mutants used in (b,c). FH represents a Flag and HA tag at the 5' end of all constructs. (b) p53 binding to SMG7 in vitro. GST or GST-p53 fusion proteins were used in pulldown assays with purified FH-SMG7 (full length or fragments). (c) Cell extracts and a-Flag (SMG7) immunoprecipitates from U2OS cells stably expressing FH-SMG7 or mutants treated with etoposide (20 µM, 6 hours) were analyzed by western blot using antibodies as indicated. Red arrows denote various SMG7 fragment expression sizes which were identified using SMG7 and HA antibodies. (d) H1299 cells co-transfected with p53 and F-SMG7 or F-SMG7-K66E constructs were analyzed as in (c). (e) Cell extracts and a-p53 immunoprecipitates from control and irradiated cells were analyzed by western blot using antibodies as indicated (long and short exposures shown).