Fig. 2. Suppression of EGFR signaling induces Notch1 expression through p53.
A : HKCs were transfected with siRNAs specific for p53 plus/minus siRNAs for EGFR in parallel with scrambled siRNA control. Cells were analyzed 48 hours after transfection. “Knock down” of p53 expression was confirmed by real time RT-PCR and immunoblot analysis (left panels). Hes1 and Notch1 expression was assessed by real time RT-PCR as for the previous experiments. B : HKCs were infected with the lentiviral reporter vectors pTRH4-N1-2.4 (N1-2.4) and pTRH4-N1-0.4 (N1-0.4), carrying an internal luciferase gene driven by either a 2.4 or 0.4 kb region of the human Notch1 promoter (nucleotides −2472 to −1 and −392 to −1 from the initiation codon, respectively), which contains and lacks, respectively, the mapped p53 binding sites2, 17. The pTRH4 vector, carrying the luciferase gene devoid of exogenous promoter, was used as control. HKCs stably infected with these viruses were transfected with siRNAs specific for p53 or scrambled siRNAs, followed by treatment (for 24 hours) with AG1478 (2 μM) or DMSO control, as indicated. For each set of cells, values are expressed as relative luciferase activity after protein normalization. C : HKCs were treated with AG1478 (2 μM) or DMSO control for 24 hours, followed by real time RT-PCR analysis of p21WAF1/Cip1 and Gadd45α expression. D : HKCs treated with AG1478 or DMSO as in the previous experiments, were analyzed by immunoblotting with antibodies against Mdm2, and γ-Tubulin as equal loading control. E : HKCs were treated with AG1478 and Nutlin (2 μM) for 24 hours, individually and in combination, followed by immunoblot analysis of p53 and γ-Tubulin expression. F : HKCs were treated with AG1478, transfected with EGFR-specific siRNA, or stimulated with EGF as in the previous experiments, and analyzed, in parallel with the corresponding controls, for levels of p53 mRNA expression by real time RT-PCR. G : HKCs were treated with AG1478 or DMSO control for 24 hours. Cells were then processed for chromatin immunoprecipitation analysis (ChIP) with an antibody against c-Jun and purified rabbit IgG as non-immune control. Real-time PCR of a distinct region of the human p53 gene promoter containing several conserved AP-1 binding sites (around position −2.6 kb, Site A in the map above) was performed along with PCR of a more downstream region devoid of such sites (around position −0.3 kb, Site B). The primers used to amplify these p53 promoter regions are listed in Supplemental Table II. H : cells were co-transfected with the p53n-Luc reporter plasmid22, carrying a luciferase gene driven by a 2 kb region of the human p53 promoter, together with an expression vector for human c-Jun (c-Jun-pCMV,42) or empty vector control (left panel), or with siRNAs against c-Jun and scrambled siRNAs control. Luciferase activity was determined 48h later. For each set of cells, values are expressed as relative luciferase activity after protein normalization. I : HKCs were transfected with siRNAs against c-Jun in parallel with scrambled siRNA control (as in Fig. 1F, G) followed by measurement of p53 expression by real time RT-PCR. J : HKCs were transfected with siRNA against c-Jun either alone or in combination with siRNA against p53, in parallel with scrambled siRNA control. Notch1 expression was determined by real time RT-PCR as before.