ERK inactivation upregulates Slug via the miR-204-SPDEF axis. (A-B) Alteration of SPDEF expression in NSCLC cells after ERKi treatment. A549, HCC827 cell lines and PDX72 primary tumor cells were treated with BVD or SCH at 2.5 µM for 2 days. The SPDEF mRNA level was examined using qRT-PCR (A). The SPDEF protein level in A549 and HCC827 cells were determined using immunoblotting (B). Arrow: non-specific band. (C) Effect of SPDEF knockdown on Slug expression. A549 and HCC827 cells were transfected with two different SPDEF shRNA for 2 days, the expression of SPDEF and Slug were determined using immunoblotting. Arrow: non-specific band. (D) Effect of ERKi treatment on expression of miR-204. A549 and HCC827 cells were treated with BVD or SCH at 2.5 µM for 2 days. miR-204 expression was determined using qRT-PCR. (E-F) Effect of miR-204 inhibition on BVD-induced downregulation of SPDEF and upregulation of SNAI2. A549 and HCC827 cells were transfected with either negative control miRNA inhibitors (NC-I) or miR-204 inhibitors (miR-204-I), treated with DMSO or BVD for 2 days. The mRNA levels of SPDEF (E) and SNAI2 (F) were determined using qRT-PCR. N = 3, bar: SD, *: P < 0.05; **: P < 0.01.