IHC was performed according to standard protocols as described previously12

IHC was performed according to standard protocols as described previously12. Analysis of tumor growth The female BALB/C nude mice (181 g) were obtained from the SPF Biotechology, Co Ltd (Beijing, China). of nude mice (values less than 0.05 were considered significant. All statistical analyses were performed using SPSS 17.0 software. Results EBP50 knockdown promotes cell proliferation and stimualtes c-Myc signaling in MCF-7 cells To investigate how EBP50 blocks cell proliferation, the relationship between EBP50 and c-Myc expression was examined in human clinical breast cancer tissues. The results showed that EBP50 expression was lower in breast cancer higher grades of malignancy and the c-Myc expression was positive correlation with malignancy grade (Figure?1A). SCH 546738 These data suggested that there is a significant correlation between EBP50 and c-Myc expression in breast cancer tissue. We further found that EBP50 knockdown promoted cell proliferation (Figure?1B and ?and1C)1C) and induced DNA synthesis (Figure?1D and Supplementary Figure?S1). The knockdown EBP50 also increased c-Myc expression (Figure?1E) and promoted c-Myc transfer from the cytoplasm to nucleus (Figure?1F). Moreover, silencing of EBP50 induced mRNA expression of Cdc25A, Cyclin E1 and Cyclin A1 (Figure?1G), all of which are transcription products regulated by c-Myc; the protein expression of these elements was also increased (Figure?1H). These data suggest that silencing of EBP50 stimulates c-Myc signaling, which in turn promotes the proliferation of MCF-7 cells. Open in a separate window Figure 1 ARHGAP26 Knockdown of EBP50 promotes cell proliferation and stimulates c-Myc signaling in MCF-7 breast cancer cells. (A) The expression of EBP50 and c-Myc was correlation with malignancy grade in breast cancer tissues. The expression of EBP50 and c-Myc was stained by IHC as indicated in Materials and Methods. Scale bar, 200 m. (B, C) Knockdown of EBP50 promoted the proliferation of MCF-7 cells. Colonies were counted (B) and proliferating cells were measured using SRB assays (C) after MCF-7 cells expressing either control shRNA or EBP50 shRNA were cultured for 7 d. (D) Knockdown of EBP50 increased DNA synthesis in MCF-7 cells. Data are representative images of Edu labelling as indicated in the Materials and methods. (E) Knockdown SCH 546738 of EBP50 increased the expression of c-Myc in MCF-7 cells. MCF-7 cells expressing either control shRNA or EBP50 shRNA were cultured and the cell lysates were detected by Western blotting using c-Myc antibodies. (F) Knockdown of EBP50 increased the nuclear import of c-Myc. Subcellular fractions were extracted, and c-Myc expression was detected by Western blotting. (G) Silencing of EBP50 increased the levels of RNA transcription products of c-Myc. Total RNA was isolated from MCF-7 cells expressing either control shRNA or EBP50 shRNA, and the mRNA levels of Cdc25A, Cyclin A1 and Cyclin E1 were analyzed by fluorescent quantitative RT-PCR as indicated in the Materials and methods. (H) Silencing of EBP50 increased the expression of transcription products of c-Myc. Cell lysates were detected by Western blotting using Cdc25A, Cyclin A1 and Cyclin E1 antibodies. Data are presented as the meanSEM (n=4). EBP50 knockdown promotes cell proliferation by stimulating c-Myc and and and (Figure?5A and ?and5B).5B). Additionally, silencing of EBP50 decreased the interaction between p62 and c-Myc (Figure?5C). Interestingly, overexpression of EBP50 also reduced the binding of p62 and c-Myc, and inhibiting autophagy using CQ promoted the interaction between p62 SCH 546738 and c-Myc (Figure?5D). Furthermore, EBP50 promoted the integration of c-Myc and p62 when the lysosomal pathway was inhibited by CQ (Figure?5E). These data suggest that p62 recognizes c-Myc as a substrate of lysosomal degradation by binding to c-Myc during EBP50-induced lysosomal degradation of c-Myc. Open in a separate window Figure.