Fig. 5. EGFR-ERK pathway activates GRHL1 and promotes its nuclear translocation.

A pGL3-enhancer vector containing RAD21 promoter fragment was transfected into A549-control cells and A549-shGRHL1 cells, co-transfected with Renilla control plasmid. After serum-free treatment for 12 h, EGF (100 ng/ml) was added to two groups to stimulate for 24 h. The relative levels of luciferase activity were normalized to the levels of luciferase activity of the Renilla control plasmid. Data represent the average of three independent experiments (mean ± SD). **P < 0.01. B After serum-free treatment for 12 h of A549-control cells and A549-shGRHL1 cells, EGF (100 ng/ml) was added to two experimental groups. Twenty-four hours later, the cells were lysed. Protein expression was assessed by western blotting using the indicated antibodies. C After 24 h of stimulation with EGF (100 ng/ml), the nuclear and cytoplasmic fractions were separated and the location of GRHL1 was detected by western blot in A549 cells. TBP: TATA binding protein, VDAC: mitochondrial outer membrane protein porin. D After serum-free treatment for 12 h of A549 cells, LY3214996 (ERK inhibitor, 300 nM) was added. 12 h later, EGF (100 ng/ml) was added to both two experimental groups. After 24 h, the nuclear and cytoplasmic fractions were separated and the location of GRHL1 was detected by western blot. E, F After serum-free treatment for 12 h of A549 and H1299 cells, LY3214996 (ERK inhibitor, 300 nM) was added. 12 h later, EGF (100 ng/ml) was added to both two experimental groups. After 24 h. The location of GRHL1 was detected by immunofluorescence staining. Cells were stained with anti-GRHL1 (left panel, red) and DAPI (middle panel, blue). The merged images are shown in the right panel. Scale bar = 25 µm, magnification: ×400.