Fig 2: The effects of TBL1XR1 on tumour cell proliferation and apoptosis. (a) Western blot analyses of TBL1XR1 expression in NCI-N87 and SGC7901 cells underwent transient transfection of TBL1XR1 shRNA (sh#1, sh#2, sh#3 and sh#4). (b) TBL1XR1 protein expression in NCI-N87 and SGC7901 cells transfected with TBL1XR1-shRNA (sh#3) was confirmed by western blot analysis. (c) TBL1XR1 protein expression in BGC823 and MKN45 cells transfected with TBL1XR1-constructed plasmid. (d) The effect of TBL1XR1 knockdown on NCI-N87 and SGC7901 cell proliferation was analysed by CCK8 assay. (e) The effect of TBL1XR1 overexpression on BGC823 and MKN45 cell proliferation was analysed by CCK8 assay. (f) and (g) Knockdown of TBL1XR1 induced the apoptosis of NCI-N87 cells. (h) and (i) Overexpression of TBL1XR1 promoted the clone formation of BGC823 cells. Data are representative of three independent experiments (mean±s.d.). *P<0.05, **P<0.01.

Fig 3: The activation of ERK1/2 induced by TBL1XR1 is mediated via the ß-catenin signalling pathway. (a) The phosphorylation levels of ß-catenin (p-ß-catenin) and ERK1/2 in NCI-N87/TBL1XR1-shRNA and SGC7901/TBL1XR1-shRNA cells were detected by western blot analysis. (b) The phosphorylation levels of ß-catenin and ERK1/2 in BGC823/TBL1XR1 and MKN45/TBL1XR1 cells were detected by western blot analysis. (c) The phosphorylation level of ERK1/2 and EMT in BGC823/TBL1XR1 and MKN45/TBL1XR1 cells treated with XAV939 were detected by western blot analysis (10 µM). (d) The effect of U0126 (20 µM) on phosphorylation of ß-catenin in NCI-N87 and SGC7901 cells was analysed by western blot analysis. Densitometry shows relative protein expression.

Fig 4: The activation of ERK1/2 induced by TBL1XR1 is mediated via the ß-catenin/MMP7/EGFR signalling pathway. (a) and (b) The expression levels of TBL1XR1, p-ß-catenin, MMP7, pEGFR and pERK1/2 in NCI-N87/TBL1XR1-shRNA cells (a) and BGC823/TBL1XR1 cells (b) were detected by western blot analysis. (c) and (d) The expression levels of TBL1XR1, p-ß-catenin, MMP7, pEGFR and pERK1/2 in NCI-N87 cells (c) and BGC823/TBL1XR1 cells (d) in the presence of XAV939 (10 µM), Batimastat (10 µM), Afatinib (5 µM) and U0126 (20 µM) were detected by western blot analysis. Densitometry shows relative protein expression.

Fig 5: TBL1XR1 enhances the migratory and invasive ability of GC cells. (a), (c) and (e) Effects of knockdown and overexpression of TBL1XR1 on GC cell wound healing, migration and invasion was measured and representative images of distance (mm) of wound healing, migrated and invaded cells (magnification: × 100) are shown. (b), (d) and (f) Histogram shows the relative distance (mm) (magnification: × 100) of wound healing and the number of migrated and invaded cells. Five random fields were selected for statistical analysis. (g) and (i) The effects of knockdown and overexpression of TBL1XR1 on EMT markers was determined by western blot analysis. Densitometry shows relative protein expression normalized for GAPDH. (h) and (j) Densitometric analysis shows the effects of knockdown and overexpression of TBL1XR1 on EMT markers of GC cells. Data are shown as mean±s.d. of three independent experiments. *P<0.05, **P<0.01.