shRNA knockdown of p53 expression reverses dinaciclib-induced apoptosis.

<p>A) Knockdown of p53 levels using a lentiviral shRNA construct. WM35 melanoma cells were infected with a lentivirus encoding for shRNA against p53 and were selected by flow cytometric sorting for GFP (designated WM35 shp53). Western blot shows knockdown of p53 protein. Actin shows equal protein loading. B) WM35 cells expressing p53 shRNA show diminished cell accumulation in the sub-G1 phase of the cell cycle following dinaciclib treatment. Cells were treated with 30 nM of dinaciclib for either 12 hours or 24 hours, before being fixed and stained for DNA content with propidium iodide. C) Apoptosis in a wild-type p53 melanoma cell line is dependent upon p53. WM35 and WM35 shp53 cells were treated with 30 nM dinaciclib for 48 hrs and were assessed for apoptosis via Annexin-V staining. WM35 shp53 cells exhibited a dramatic reduction of apoptosis when compared to WM35. D) The knockdown of p53 led to decreased pRB (ser807/811) and a decrease in cleaved PARP in cells treated with dinaciclib. WM35 and WM35 shp53 cells were treated with 30 nM of dinaciclib for 8, 16, 24, and 48 hrs, after which protein were assessed for the levels of p53, pRBser807/811, PARP, and Mcl-1. Actin was used as loading control.