Fig. 4.

Lentiviral shRNA silencing of FGFR1 inhibited FGF9-induced ERK1/2 phosphorylation, cell proliferation, EMT markers, migration and invasion in LLC cells. (A) Western blot analysis and quantification of gene silencing verification at protein level in LLC cells transfected with a specific shRNA against FGFR1-4. Two different shRNA targeted sequences were used for each FGF receptor (Table S3). Cells transfected with a non-silencing shRNA sequence (scrambled sequence) in the PLKO.TRC1-puro (Scr1) or PLKO.TRC2-puro (Scr2) vectors were used as control as indicated. (B) The FGFR1 knockdown (shFGFR1) LLC cells and their scrambled control were treated with 0 (Control) or 50 ng/ml FGF9 for 0.25 h. The protein expression levels of ERK1/2 and p-ERK1/2 were analyzed by Western blot assay. (C) MTT assay for cell proliferation of shFGFR1 LLC cells treated with 0 (Control) or 50 ng/ml FGF9 for 24 h. (D) Western blot analysis for the expression of E-cadherin and N-cadherin in shFGFR1 LLC cells treated with 0 (Control) or 50 ng/ml FGF9 for 24 h. (E) Western blot analysis for the expression of MMP2, MMP3, MMP9 and MMP13 in shFGFR1 LLC cells treated with 0 (Control) or 50 ng/ml FGF9for 24 h. (C) The migration and (D) matrigel invasion assays with 0 (Control) or 50 ng/ml FGF9 treatment in shFGFR1-LLC cells and their scramble control (Scr1) using transwell system. All values are represented as the mean ± SEM; (B) n=6; (C) and (D) n=4. The data were analyzed by two-way ANOVA with Sidak's multiple comparisons post-tests; *p<0.05, **p<0.01 and ***p<0.001 vs. the Control group.