FAT10 expression at the transcript level and promoter is cell-cycle regulation in HCT116 cells. (A) Cell-cycle profiles of HCT116 cells synchronized at the various cell-cycle stages: C – Asynchronous cells; G1-Cells synchronized to G1-phase with L-minosine; S-Cells syncrhonized to S-phase with Thymidine and G2/M-Cells synchronized to G2/M phase with nocodazole. (B) FAT10 protein expression normalized against β-actin protein levels. Top panel: Western blot of a representative experiment. Bottom panel: mean and standard deviation of results quantitated from Western blots from three independent experiments. *p < 0.05. (C) FAT10 mRNA transcript levels normalized against β-actin mRNA levels. Results shown are the mean and standard deviation from three independent experiments. *p < 0.05 and **p < 0.01. (D) Promoter-reporter construct used to assay FAT10 promoter activity. Various lengths of the FAT10 promoter drive the β-galactosidase reporter gene while the constitutive CMV promoter drives the EGFP to normalize for differences in transfection efficiencies. (E) Left panel – FAT10 promoter activity in HCT116 cells expressed as normalized β-galactosidase activity from 3 independent experiments. Middle Panel – Pictorial representation of the various regions of DNA upstream and downstream the transcription start site of the FAT10 gene that is cloned before the β-galactosidase gene. Right Panel – FAT10 promoter activity in HCT116 cells synchronized to the various cell-cycle stages (white: G2/M; black: S and Grey: G1) in HCT116 cells. Results shown are the mean and standard deviation from three independent experiments.