Figure 3.

ATG7 Overexpression Inhibited p27 mRNA Transcription in Human BC Cells

(A) UMUC3(shATG7#1), UMUC3(shATG7#2), and UMUC3(Nonsense) cells were cultured in 6-well plates until the cell density reached 70%–80%. Following synchronization for 12 hr, the medium was replaced with 10% FBS DMEM for another 12 hr. Then the cells were extracted for total RNA with TRIzol reagent. RT-PCR was used to determine p27 mRNA expression, whereas β-actin was used as an internal control. (B) Schematic representation of the p27 promoter region p27 KPNI and its deletion fragment SACII. (C) p27 promoter transcription activity was evaluated by using the two p27 promoter-driven luciferase reporters shown in (B). The results were normalized by internal TK activity, and the bars show mean ± SD from three independent experiments. The asterisk indicates a significant increase in comparison with vector control transfectant (*p < 0.05), whereas double asterisks indicate a significant decrease in p27 SACII transfectant in comparison with p27 KPNI transfectant (**p < 0.05). (D) Potential transcriptional factor binding sites in the p27 promoter region (–1324 to +162) were analyzed by using the TRANSFAC 8.3 engine online. (E and F) The indicated cell extracts were subjected to western blot for determination of the expression of ATG7, E2F1, FOXO1, c-Fos, c-Jun, p65, and Sp1. GAPDH was used as a protein loading control. The result represented one of three independent experiments.