c-Myc suppression mediates downregulation of hTERT expression by osimertinib. (A) Putative transcriptional factors were predicted with PROMO program. (B) Alterations of transcriptional factors in RNA-seq data generated from both PC-9 and HCC827 cells treated with DMSO or 100 nM osimertinib (Osim) for 14 h presented in the heatmap (triplicate treatments). (C and D) PC-9 cells were exposed to 200 nM for the indicated times (C) or 24 h (D), (E) The indicated cell lines were transfected with the indicated siRNAs for 48 h. (F) The indicated cell lines were infected with lentiviruses carrying hTERT shRNA followed by puromycin selection. (G) The indicated cell lines expressing ectopic vector (V), WT, and mutant c-Myc genes, respectively, were exposed to DMSO or 200 nM osimertinib (Osim) for 16 h. After the aforementioned treatments, the proteins of interest were detected with western blotting (C and E–G) or IF (D). (H) Reporter constructs harboring the core hTERT promoter region and deleted regions. (I) The indicated cell lines were transfected with the given reporter constructs for 24 h followed by 200 nM osimertinib for another 16 h. Cells were then harvested for luciferase assay. The data are the means ± SD of four replicate determinations. Statistical differences were assessed with two-sided unpaired Student’s t test. Source data are available for this figure: SourceData F2.