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. 2014 Jun 26;5(14):5602–5614. doi: 10.18632/oncotarget.2136

Fig.5. NUMB-1 induces ESCC cell cycle arrest in M phase by interacting with Aurora-A and suppressing its activity.

Fig.5

(A) Overexpression of NUMB-1 inhibits the Notch pathway. Immunoblots showed overexpression of Numb isoform 1 in KYSE150 downregulated the NICD as well as its downstream protein Hes-1. NUMB-1 was tagged with HA sequence (pAd-HA-NUMB1), and the protein of Numb-1 wih HA was detected by immunoblots between 72 and 95KD. (B) Immunoblots showing the expression of various cell-cycle markers in control and Numb-1 overexpressing cell lines. Increased Numb-1 expression upregulated cyclin B1, cdc2 and p21 levels but did not affect cyclin D1. (C) Increased Numb-1 expression upregulated cyclin B1 and cdc2 in vivo. Immunohistochemistry (IHC) was performed in xenograft samples from mice with Ad-Null and Ad-NUMB-1 tumor. IHC showed higher expression of cyclinB1 and cdc2 in the cytoplasma and nucleus in Numb-1 overexpression samples. (D) Overexpression of Numb-1 in KYSE30 cells did not affect Aurora-A expression but significantly suppressed its activity, as indicated by the decrease in p-Aurora-A, possibly leading to G2/M arrest consistent with previous studies. (E) Lysates of KYSE30 cells were immunoprecipitated (IP) with either preimmune IgG (negative control) or anti-Aurora A, anti-p53 (positive control), or anti-NUMB-1 antibodies followed by immunoblotting (IB) with the antibodies indicated to show co-IP of Aurora-A and Numb-1.