Figure 5.

E2F1 Downregulation Is Required for the Transcriptional Inhibition of RRM1 and RRM2 Induced by the Combination of Everolimus and ERK Inhibitor

(A–C) Caki-1 and 786-O cells treated with everolimus alone or in combination with SCH772984 for 72 h were subjected to qRT-PCR analysis to elucidate the mRNA levels of RRM1 (A), RRM2 (B), and E2F1 (C). (D) Phospho-p70S6k, phospho-Erk1/2, and E2F1 protein levels were detected by immunoblotting analysis when Caki-1 and 786-O cells were treated with everolimus and SCH772984 for the indicated time points. (E) Caki-1 and 786-O cells were transfected with E2F1 overexpression plasmid or empty plasmid (mock) for 48 h, followed by everolimus-SCH772984 combination treatment for another 24 h. The overexpression efficiency and the alterations of RRM1 and RRM2 were detected by immunoblotting analysis. Also, the proliferation of cells was measured by CCK-8 assay. ***p < 0.001. (F) Caki-1 and 786-O cells were subjected to ChIP assay using E2F1 antibody, followed by qRT-PCR analysis using primers targeting the promoter regions of RRM1 and RRM2. ***p < 0.001. Error bars represent mean ± SD of three independent experiments.