Figure 2: Proof of concept: non-carcinogenic compound pifithrin-afacilitates DMBA carcinogenesis. A) Schematic representation ofthe experiment. B) FACS analysis of the proliferative status ofpifithrin-a-treated cells. C) Phenotype of pifithrin-a-treated cells.D) Growth curve of cells treated with pifithrin-only (Pif, ) cells,DMBA alone (DMBA, ) or pifithrin-a + DMBA (DMBA + Pif, ). E)104 MEFs were seeded in a 10 cm plate and were treated with 10 Mpifithrin-a and 10 M DMBA. After 7-10 days the plate was fixedand stained with crystal violet to identify clones. F) Individualclones were subcultivated independently and the presence of p53was analyzed by Western blot. Cells expressing mutant p53 (175H)were used as control. G) Photo of a colony of MEFs treated withpifithrin-a and DMBA growing in soft agar. *: p<0.05, **: p<0.01,***: p<0.001, Student’s T test. H) Number of soft agar growingcolonies in 6 different clones.