Figure 5. Restoration of TP53 function in BRAF^V600E/TP53^Null lung tumor cells results in a G1 cell cycle arrest.

A. Phase contrast photomicrographs of BRAF^V600E/TP53^Null cells infected with either a control (BPC) or a TP53:ER (BPER-D) encoding pBabePuro retrovirus vector. Cells were then treated with 4-hydroxytamoxifen (4-HT) to activate TP53:ER in the absence or presence of Nutlin-3a for 48 hours as indicated. The expression of TP53:ER (green) was assessed by IF.

B. BPC or BPER-D cells were treated with vehicle or 4-HT in the absence or presence of nutlin-3a as indicated with cell proliferation assessed using Crystal Violet staining or by cell counting (lower panels). BRAF^V600E/INK4A-ARF^Null (BIA) lung tumor cells were treated with vehicle or nutlin-3a as indicated with cell proliferation assessed using Crystal Violet staining or by cell counting. A 2-sided t-test was performed to assess significance (* and ** indicates p < 0.05 and p<0.001 respectively).

C. Immunoblot analysis of BPER-D cell lysates treated with ethanol (ET) as vehicle control, nutlin-3a (Nut), 4-HT or the combination as indicated.

D. Analysis of BrdU incorporation by flow cytometry in BPER-D or BIA cells after 48 hours of treatment with the indicated agents.

E. Crystal violet staining of BIA cells replated and cultured for 5 days following treatment with either vehicle or nutlin-3a for 48 hours.