YF454A plus erlotinib significantly suppresses the receptor tyrosine kinase signalling pathways in EGFR‐TKI‐resistant NSCLC cells. (A) Effects of either YF454A or erlotinib alone or in combination on the protein and mRNA expression of EGFR, Her2, AXL, c‐Met and IGF‐1R. PC9/ER cells were treated with YF454A (0.2 μM), erlotinib (5 μM) or YF454A/erlotinib for 24 h. Cell lysates and total mRNA were prepared, and Western blotting assays (i) and real‐time PCR analysis (ii) were further carried out. (B) The combination of YF454A and erlotinib time‐dependently suppressed protein expression (i) and mRNA expression (ii) of EGFR, HER2, AXL, MET and IGF‐1R. (C, D) YF454A/erlotinib down‐regulated the phosphorylation of Akt and ERK in PC9/ER cells (C) and in HCC827/ER cells (D). PC9/ER and HCC827/ER cells were treated with YF454A (0.2 μM) and erlotinib (5 μM) as a single agent alone or in combination for 24 h. The whole‐cell lysates were prepared and probed with specific Akt and ERK antibodies. Western blotting and real‐time PCR assays were performed three independent times, and representative images are shown.