Figure 4.
FOXO1 Is an ATG7-Regulated Transcription Factor Mediating p27 Downregulation in Human BCs In Vitro and In Vivo
(A–C) FOXO1 knockdown constructs were stably transfected into UMUC3 (A), T24 (B), and UMUC3(shATG7#1) cells (C). The FOXO1 knockdown efficiency and its downstream p27 expression were assessed by western blotting. GAPDH or β-actin was used as a protein loading control. (D) The stable transfectants as indicated were subjected to an anchorage-independent soft agar growth assay. Representative images of colonies were photographed under an Olympus DP71. (E and F) The number of colonies was counted with more than 32 cells of each colony, and the results are presented as colonies per 104 cells. The bars show mean ± SD from three independent experiments. The asterisk indicates a significant increase in comparison with nonsense transfectants as indicated (*p < 0.05). (G) A ChIP assay was carried out using anti-FOXO1 antibody to detect the interaction of FOXO1 with the p27 promoter. (H and I) IHC staining was performed to evaluate FOXO1 expression in the indicated xenograft tumors. The IHC images were captured using the AxioVision Rel.4.6 computerized image system, and protein expression levels were analyzed by calculating the integrated IOD/area using Image-Pro Plus version 6.0. Results are presented as the mean ± SD of five mice in each group. Student’s t test was utilized to determine the p value (*p < 0.05).