Piperine causes DNA damage and modulates G1 cell cycle regulatory proteins.

<p>SK MEL 28 (A) and B16 F0 (B) cells were treated with different concentrations of piperine for 48 h. Cells were lysed and total lysate was prepared as described under <i>Materials and Methods</i> and analyzed by western blotting. Representative immunoblots show the effect of piperine on the phosphorylation of H2A.X (Ser139), ATR (Ser428), Chk1 (Ser296) and p-Rb (Ser795), and the protein levels of DNA Polymerase β, p53, p21, Cyclin D1 and E2F1. Each blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading. (C)Representative immunofluorescence images of p. Chk1 (Ser 296) in control and 150 µM piperine treated SK MEL 28 cells. Alexafluor 594 (Red) represents p.Chk1, Alexafluor 488(green) represents β-actin and DAPI (blue) represents nucleus. Each experiment was performed at least three times independently and the results were comparable.