Ectopic expression of mutant p53 R163H cooperates with p53-KD to alter cyst morphology.

<p><b>A</b>, Generation of MCF-10A cell lines in which siRNA-resistant mutant p53-R163H was expressed along with knockdown of endogenous wild-type p53. The levels of wide-type p53 and mutant p53-R163H were determined by Western blotting. <b>B</b>, The level of wild-type p53 transcripts was determined by RT-PCR. <b>C</b>, Representative images of MDCK cells or MDCK cells with p53-KD-R163H in 2-D culture. <b>D</b>, Representative images of MDCK cells or MDCK cells with wild-type p53-KD and overexpression of mutant p53-R163H in 3-D culture for 12 d. Scale bar: 100 µM. <b>E</b>, Top panel: colony formation assay was performed with MDCK cells or MDCK cells with p53-KD and overexpression of R163H. Bottom panel: the number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>F</b>, Wound healing assay was performed with MDCK cells, MDCK cells with p53-KD, or MDCK cells with p53-KD and overexpression of R163H. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. Bottom panel: the time required for wound closure was measured and presented as mean ± SD from three separate experiments.