Altered expression of key cell cycle regulators, silencing of Cdc2 and inhibition of p53 in SCC25 cells by the PCT analogs.

<p>(A): Immunoblotting showed mild dose-dependent (50 and 100 nM) reduction in the expression of cyclin D1, moderate induction of cyclin E, and no or negligible effect on cyclin B. Both PCT analogs did not cause any change in the levels of Cdk2 and Cdk4, but induced the expression of Cdc2 (Cdk1) and Cdc25C. The analogs significantly augmented phosphorylation of Cdc2 at the Tyr15 residue, thus upregulating the level of phospho-Cdc2(Tyr15), which is an inactive form of Cdc2. Induction was also observed in the expression of phospho-Cdc25C(Ser216) in samples treated with TM-025/TM-026. Equal loading was confirmed by <i>β</i>-actin. Quantifications are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125322#pone.0125322.s004" target="_blank">S4 Fig</a> Part B. (B): siRNA mediated silencing of Cdc2. Cells were transfected with Si-Cdc2 and si-Con (control), and 24h later 50 nM of PCT analogs were added and incubated for 24h and harvested for protein extraction. Immunoblots showed knock down efficiency of the Cdc2 siRNA. Briefly, the gel showed untransfected cells, Cdc2 siRNA-transfected cells, Cdc2 siRNA or control siRNA-transfected cells treated with 50 nM PCT analogs. (C): Bar graph shows % S-phase cells after cell cycle analysis of propidium iodide (PI) stained cells. NS: not significant. (D): Pharmacological inhibition of p53 by PFT. Cells were treated with vehicle, PCT analogs, and PCT analogs with multiple doses of 50 nM PFT. Western blots showing inhibition of p53 by PFT at 72 h in TM-025 and TM-026 (50 nM) treated SCC25 cells. (E) Bar graph shows % S-phase cells after cell cycle analysis of PI-stained cells. Results showing significant changes are denoted by <b>*</b> (P<0.05) and not significant denoted as ns. β-Actin was used as loading control for A, B and D.