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. 2019 Mar 25;10(4):282. doi: 10.1038/s41419-019-1521-5

Fig. 3. EGFR-stabilizing DYRK1A is negatively regulated by p53.

Fig. 3

a Epidermal growth factor receptor (EGFR) is downregulated by the depletion or inhibition of dual-specificity tyrosine-phosphorylated and tyrosine-regulated kinase 1A (DYRK1A) in U2OS cells. Top, EGFR is downregulated by the depletion of DYRK1A in U2OS cells, as measured by western blot. Bottom, U2OS cells were treated with the indicated concentration of harmine (DYRK1A inhibitor) for 72 h. b DYRK1A small interfering RNA (siRNA) and harmine, respectively, induced senescence in U2OS cells. Cellular senescence was examined 7 days after RNA interference (RNAi) of DYRK1A or harmine (20 μM) treatment in U2OS cells. c p53 negatively regulates DYRK1A. Top, the protein levels of DYRK1A and p53 in U2OS cells treated with the indicated concentration of Nut3a for 72 h. Bottom, ectopic of p53 expression downregulates DYRK1A. At 48 h after transfection, cell extracts were examined by western blot for the determination of Flag, p53, and DYRK1A. d Nut3a failed to downregulate DYRK1A in p53-knockdown cells. The protein levels of DYRK1A 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. e Kinetics of DYRK1A and EGFR downregulation by p53 activation. The protein levels of DYRK1A, EGFR and p53 in U2OS cells were determined at different time points after Nut3a treatment. f Ectopic expression of DYRK1A attenuates Nut3a-induced downregulation of EGFR, as measured by western blot. pEZ-M77-DYRK1A expression vector was transfected into U2OS cells using Lipofectamine 2000, and cells transfected with pEZ-M77-EV (empty vector) were as control. Cells with stable ectopic expression of DYRK1A were obtained after G418 selection. Cell extracts obtained 72 h after Nut3a treatment was subjected to western blot. g Ectopic expression of DYRK1A attenuates Nut3a-induced downregulation of EGFR, as measured by flow cytometry. Cells were treated as in f. Representative fluorescence intensity curves and histograms were shown. h Ectopic expression of DYRK1A attenuates Nut3a-induced cellular senescence. Cellular senescence was examined 7 days after Nut3a treatment. i EGFR is downregulated by the depletion of DYRK1A in U87 cells. j p53 activation negatively regulates DYRK1A and EGFR in U87 cells. k p53 activation negatively regulates DYRK1A in HT1080 and A172 cells. * p < 0.05, **p < 0.01, ***p < 0.001 vs. control