Fig 2: BEND5 eliminates Notch signaling-induced BC cell proliferation, migration and invasion. (A) GSEA was conducted to predict the function of BEND5 in BC. (B) MDA-MB-231 cells were transfected with empty vector or FLAG-tagged BEND5 and treated with/without Notch signaling activator DLL4 (10 ng/ml). Immunoblot was used to detect Notch pathway downstream targets and EMT-related proteins. (C and D) CCK8 assays and colony formation assays for MDA-MB-231 cells transfected and treated as in (B). (E and F) Wound-healing assays and transwell assays for MDA-MB-231 cells transfected and treated as in (B). Data shown are mean ± SD of triplicate measurements with similar results (*P < 0.05, **P < 0.01).

Fig 3: BEND5 suppresses proliferation, migration and invasion in MDA-MB-231 cells. (A) MDA-MB-231 cells were transfected with FLAG-tagged BEND5 or empty vector and cultured for a specified time. CCK8 assays were used to detect cell numbers, and immunoblot was used to detect the expression of BEND5 in MDA-MB-231 cells. ß-actin was used as a loading control. (B) Colony formation assays for MDA-MB-231 cells transfected as in (A). (C and D) Wound-healing assays and transwell assays for MDA-MB-231 cells transfected as in (A). (E) MDA-MB-231 cells were transfected with control shRNA, BEND5 shRNA or BEND5 shRNA plus shRNA-resistant BEND5 (BEND5-R). CCK8 assay was used to detect cell numbers, and immunoblot was used to detect the expression of BEND5 in MDA-MB-231 cells. (F) Colony formation assays for MDA-MB-231 cells transfected as in (E). (G and H) Wound-healing assays and transwell assays for MDA-MB-231 cells transfected as in (E). Data shown are mean ± SD of triplicate measurements with similar results (*P < 0.05, **P < 0.01 versus empty vector).

Fig 4: BEND5 inhibits Notch signaling via binding to RBPJ. (A) MDA-MB-231 or ZR75-1 cells were immunoprecipitated with anti-BEND5 or normal IgG, and the precipitates were analyzed by immunoblot with the indicated antibodies. (B) HEK293T cells were co-transfected with MYC-RBPJ and FLAG-BEND5 or FLAG-BEND5 ?BEN as indicated. Cell lysates were immunoprecipitated with anti-FLAG, followed by immunoblot with anti-MYC. (C) HEK293T cells were co-transfected with FLAG-BEND5 and MYC-RBPJ, MYC-RBPJ ?CTD, MYC-RBPJ ?BTD, or MYC-RBPJ ?NTD. Cell lysates were immunoprecipitated with anti-MYC, followed by immunoblot with anti-FLAG. (D) Co-IP analysis of MDA-MB-231 cells and ZR75-1 cells transfected with FLAG-tagged BEND5 and MYC-tagged RBPJ. Cell lysates were immunoprecipitated with anti-MYC, followed by immunoblot with the indicated antibodies. (E) Immunoblot was used to detect Notch pathway downstream targets and EMT-related proteins in MDA-MB-231 and ZR75-1 cells transfected with empty vector, FLAG-BEND5, or FLAG-BEND5 ?BEN. Data shown are mean ± SD of triplicate measurements with similar results (*P < 0.05, **P < 0.01).

Fig 5: Knockdown of BEND5 promotes BC tumor growth and metastasis in mice. (A) MDA-MB-231 cells stably transfected with BEND5 shRNA or control shRNA were injected subcutaneously in the right flank of nude mice, and tumor volume was measured with vernier-caliper at the indicated times. (B) Immunoblot analysis of representative excised tumor tissues from (A). (C) Representative bioluminescence image at 30 days of nude mice injected by tail vein with MDA-MB-231 cells expressing firefly luciferase and the indicated constructs (n = 6). The luminescence signal is represented by an overlaid false-color image with the signal intensity indicated by the scale (right panel). (D) Representative lung tissues and H&E-stained sections of the lung tissues from (C). The number of tumor nodules are shown (right panel). Data are shown as mean ± SD (n = 6) (*P < 0.05, **P < 0.01 versus control shRNA). (E) MDA-MB-231 cells stably transfected with BEND5 shRNA or control shRNA were injected into mammary fat pad on the right side of nude mice (n = 6). Representative bioluminescence images are shown one month after injection. Red arrow indicates tumor growth and green arrow tumor metastasis. Tumor growth was compared between MDA-MB-231 cells expressing BEND5 shRNA and control shRNA (**P < 0.01 versus control shRNA). (F) A proposed model for BEND5 modulation of BC growth and metastasis. BEND5 inhibits Notch signaling by disrupting MAML-RBPJ interaction via competing with MAML for binding RBPJ.