Fig. 5: CREPT modulates G2/M arrest by regulating Cyclin B1.

a Cyclin B1 protein level is increased in CREPT overexpressed MGC803 cells and decreased in CREPT deletion MGC803 cells. Exogenous (Exo-CREPT), endogenous (Endo-CREPT) CREPT and Cyclin B1 levels were examined by western blot. b CREPT regulates the expression of Cyclin B1 at mRNA level. Relative mRNA levels of Cyclin B1 were examined using a quantitative real-time PCR from CREPT overexpression (HA-CREPT) or deletion (CREPT–/–) MGC803 cells. The results represent the mean ±  SD from 3 independent experiments; ***p < 0.001, **p < 0.01. c Exogenously expressed CREPT restores the decrease of Cyclin B1 in CREPT deletion cells. The level of Cyclin B1 mRNA was examined by a real-time PCR analysis; ***p < 0.001, **p < 0.01. d CREPT participates in the transcription of CCNB1. An 1171 bp sequence for the CCNB1 promoter was cloned to drive the expression of luciferase reporter into pGL3-Basic vector (top). Luciferase activities were examined from cells with overexpression of CREPT (HA-CREPT) or deletion of CREPT (CREPT–/–). HA-CREPT was induced to rescue the effect of deletion. The activity was expressed as fold changes, normalized by an internal control (Renilla). Results were from three independent repeats and presented as means ±  SD; ***p < 0.01, **p < 0.01. e CREPT occupies at the promoter of CCNB1. Primers at the CCNB1 gene are shown. E1 represents exon 1. Numbers indicate the nucleotides counting from the primary translation site as +1 (top). ChIP experiments were performed by immunoprecipitation (IP) with an antibody against CREPT (3E10) and by PCR amplification of the fragments (Bottom). f Exogenous expression of Cyclin B1 partially rescues the CREPT-induced mitosis arrest. HA-Cyclin B1 was transiently transfected into CREPT deletion MGC803 cells (CREPT–/–). After 48 h, cells were harvested and stained with PI and H3ser10 antibody and Alexa Fluor® 488 conjugate secondary antibody. The mitotic index was measured by FACS. Three independent experiments were performed and the results were presented as mean ± SD; *p < 0.05. g A presentative FACS analysis for the population of mitotic cells. h–j CREPT correlates with Cyclin B1 expression. Spearman's correlation (Rho) was calculated with 450 gastric cancer samples from the TCGA database (h) and a tissue microarray containing 357 gastric cancer samples (i). The tissue microarray was stained with anti-CREPT or anti-Cyclin B1 antibody respectively. The expression levels of Cyclin B1 and CREPT were classified as low, moderate, and high according to a DAB staining result. Representative immunohistochemical staining is shown. Scale bars, 100 μm (j)