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. 2019 Mar 1;7(4):783–802. doi: 10.1016/j.jcmgh.2019.02.003

Figure 3.

Figure 3

p53 activation suppresses paracrine induction of EMT. (A) Western blot analyses of indicated proteins in SW480/pRTR-p53-VSV cells, treated with vehicle (p53 off) or with DOX (p53 on) for 48 hours. (B) Cell number of SW480/pRTR-p53-VSV cells, treated with vehicle (p53 off) or with DOX (p53 on) at the indicated time points. (C) Phase-contrast images showing cell morphology of DLD1, HCT15, HCT116, and LoVo cells treated with CM from SW480/pRTR-p53-VSV cells for 96 hours, which were treated with vehicle (p53 off) or DOX (p53 on) for 72 hours. (D) qPCR analyses of indicated EMT-related genes in DLD1, HCT15, HCT116, and LoVo cells treated with CM from control or SW480/pRTR-p53-VSV cells for 96 hours, which were treated with vehicle (p53 off) or DOX (p53 on) for 72 hours. (E) Immunofluorescence detections of E-cadherin in DLD1, HCT15, HCT116, and LoVo cells treated with CM from SW480/pRTR-p53-VSV cells for 96 hours, which were treated with vehicle (p53 off) or DOX (p53 on) for 72 hours. Nuclear DNA was detected by staining with 4′,6-diamidino-2-phenylindole. Scale bar: 50 μm. (F) Wound healing assay of DLD1 and LoVo cells treated for 30 hours with CM collected from SW480/pRTR-p53-VSV cells, which were treated with vehicle (p53 off) or DOX (p53 on) for 72 hours. The width of scratches in 2 independent wells was analyzed for each state. Results represent the average (%) of wound closure. (F) Invasion assay in modified Boyden chambers. DLD1, HCT15, HCT116, and LoVo cells were seeded on Matrigel-coated filters with CM from SW480/pRTR-p53-VSV cells, which were treated with vehicle (p53 off) or DOX (p53 on), used as a chemoattractant in the lower well. After 48 hours, cells that invaded through the Matrigel were counted after 4′,6-diamidino-2-phenylindole staining. (B and D) Means ± SD (n = 3 biological replicates), and (F and G) means ± SD (n = 2 biological replicates; each n = 3 technical replicates) are provided. Significance was determined using 1-way analysis of variance with the Tukey multiple comparison post-test; *P < .05; **P < .01; ***P < .001.