Figure 6.

ACP5 Regulated the Expression of SMAD3 by p53

(A) Knockdown of the expression of ACP5 attenuated the levels of SMAD3 and p-SMAD3. Upper panel: Representative western blot results for SMAD3, p-SMAD3, SMAD2, and p-SMAD2 at different time points of TGF-β1 stimulation. Lower panel: Graphs showing the data with three replications. (B) Quantitative real-time PCR results for SMAD3 and SMAD2 in ACP5-silenced A549 cells after TGF-β1 stimulation. (C) p53 inhibited expression of SMAD3. Upper panel: mRNA levels of SMAD3 in A549 cells after knockdown of the expression of p53. Lower panel: mRNA levels of SMAD3 in A549 cells after overexpression of p53. (D) Schematic illustrations of the three binding sites (the blue boxes) of p53 (site a, −116 to −99 bp; site b, −28 to −10 bp; site c, +16 to +27 bp, with the SMAD3 transcription start site as +1) to the SMAD3 promoter as predicted by the Lasagna databases (https://biogrid-lasagna.engr.uconn.edu/lasagna_search/). (E) ChIP-PCR assays using antibody specific for p53 to prove that p53 binds to SMAD3 promoter. (F) Results for SMAD3 promoter luciferase reporter assays. Upper panel: Schematic results showing the mutant plasmids (MUT1–MUT3) of these three binding sites. Each of the mutant plasmids (MUT1–MUT3) of these three binding sites maintained one normal binding site (blue box) and two mutant binding sites (red boxes), and all binding sites were deleted in MUT4 (blue boxes with red cross). Lower panel: Result for promoter reporter assays. (G) After silencing ACP5 for 24 h, we transfected SMAD3 plasmid into the A549 cells for 24 h and then stimulated cells with TGF-β (10 ng/mL) for 24 h and collected the cells for western blot, and overexpression of SMAD3 reversed the process of EMT after silencing ACP5 in A549 cells. The results are summarized as the mean ± SEM of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by independent Student’s t test.