Fig. 3

Phosphorylation of TGIF2 by EGFR/ERK signaling is necessary for TGIF2-enhanced EMT and metastasis in LUAD. (A) Western blot results of E-cadherin, Vimentin, TGIF2, ERK, and p-ERK expression in H1299 cells in the presence or absence of EGF and SCH772984. Cells were pre-incubated with 1 μM SCH772984 for 1 h and exposed to 50 ng/mL EGF for 5 min or 48 h. (B) Western blot results of E-cadherin, Vimentin, TGIF2, ERK, and p-ERK expression in TGIF2 knockdown and control H1299 cells in the presence or absence of EGF (50 ng/mL). (C) Schematic of the phosphorylation-deficient mutations (TGIF2^AA) of the two MAPK sites in the TGIF2 coding sequence. (D) Western blots for Flag-tagged TGIF2^WT and TGIF2^AA in H1299 cells treated with the indicated dose of EGF for 5 min or 48 h. (E) Western blot results of TGIF2, p-TGIF2, E-cadherin, and Vimentin expression in H1299 cells overexpressing TGIF2^WT or TGIF2^AA. (F) Immunofluorescence analysis of E-cadherin and Vimentin in H1299 cells with ectopic expression of TGIF2^WT or TGIF2^AA. Scale bars, 20 μm. (G) Phase-contrast images showing the morphology of H1299 cells with ectopic expression of TGIF2^WT or TGIF2^AA. Scale bars, 50 μm. (H) Transwell assay of H1299 cell migration according to the ectopic expression of TGIF2^WT or TGIF2^AA. Scale bars, 100 μm. (I, J) Tumor presence (I) and growth curve (J) derived from different groups; 1.5 × 10⁶ H1299-vector, H1299-TGIF2^WT, or H1299-TGIF2^AA cells were subcutaneously injected into each mouse. Tumors were excised and analyzed 35 d later. Data are presented as the mean ± SEM (n = 5 mice per group). (K) Representative lung images and number of metastatic foci in the different groups. **, p < 0.01; ns, not significant. All data shown are representative of three repeated experiments