Fig. 1: EGFR was downregulated by p53 activation in a subset of cancer cell lines.

a, b Negative regulation of epidermal growth factor receptor (EGFR) by Nutlin-3a (Nut3a) in U87 cells. The protein levels of EGFR were determined by western blot (a) or by flow cytometry (b, 8 μM for 72 h). Right, quantitative summary of EGFR expression by flow cytometry was shown. Representative results of three independent experiments were shown. c, d Negative regulation of EGFR by Nut3a in U2OS cells. c Western blot analysis of EGFR. d Flow cytometry analysis of EGFR in cells treated with Nut3a (8 μM) for 72 h. Right, quantitative summary of EGFR expression by flow cytometry. e Reduction of EGFR by Nut3a requires p53 function. Left, the protein levels of EGFR and p53 in U2OS cells transfected with pLKO.1-Puro-shNeg lentiviral vector or pLKO.1-Puro-shp53 lentiviral vector were measured 72 h after treatment with Nut3a. Right, U87 cells were transfected with small interfering RNA (siRNA) duplexes (200 nM) specific to p53 or negative oligo in serum-free medium for 4 h, and then were incubated with complete medium for 48 h. The protein levels of EGFR and p53 in U87 (siNeg and sip53) were measured 72 h after treatment with Nut3a (8 μM). f Downregulation of EGFR by ectopic expression of p53. p53 expression vectors were transfected into U2OS cells using Lipofectamine 2000, and cells transfected with empty vectors were as control. After 48 h, the cells extracts were examined by western blot for the determination of Flag, p53, EGFR. g, h EGFR was downregulated by Nut3a in HT1080, A172, and A2780 cells. The position of protein markers (in kDa) is indicated in each case. Immunoblots are representative of at least two independent experiments with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as a protein loading control. **p < 0.01 vs. control, and ***p < 0.001 vs. control