Figure 2.
p27 Downregulation Mediates ATG7 Promotion of Human BC Anchorage-Independent Growth
(A and B) The indicated cell extracts were subjected to western blot for determining the expression of ATG7, p53, Cyclin A2, Cyclin B1, CDK2, and p27. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a protein loading control. (C and D) IHC staining was performed to evaluate p27 expression in xenograft tumors obtained from nude mice. 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). (E and F) p27 knockdown constructs were stably transfected into UMUC3(shATG7#1) and T24(shATG7#1) cells. The knockdown efficiency of p27 protein was assessed by western blotting. (G) UMUC3(shATG7#1/shp27#1) cells, UMUC3(shATG7#1/shp27#2) cells versus UMUC3(shATG7#1/Nonsense) cells or T24(shATG7#1/shp27#1) cells, and T24(shATG7#1/shp27#2) cells versus T24(shATG7#1/Nonsense) cells, were subjected to an anchorage-independent soft agar growth assay using the protocol described in Materials and Methods. Representative images of colonies were captured under an Olympus DP71. (H and I) The number of colonies was counted, with the standard being more than 32 cells of each colony, and the results are presented as colonies/104 cells. The bars show mean ± SD from three independent experiments. An asterisk indicates a significant increase in comparison with nonsense transfectants (*p < 0.05).