Fig 2: TCF7L2 promotes the GC process in vivo. a Knockdown of TCF7L2 inhibited the growth of subcutaneous xenograft tumors in nude mice. Bioluminescence images of the nude mice subcutaneous tumors were displayed through a small animal in vivo imaging system, and the tumor growth curve was measured for 24 days. b Gross specimen and average weight of the tumor after animals were sacrificed. Knockdown of TCF7L2 inhibited the volume and weight of subcutaneous xenograft tumors in nude mice. c Histological characteristics and Ki67 expression of the tumor were examined via H&E staining and IHC, respectively. Knockdown of TCF7L2 inhibited the Ki67 expression in subcutaneous xenograft tumors. d Knockdown of TCF7L2 inhibited lung metastasis. The bioluminescence image of lung metastases and the count of lung metastatic nodules was obtained 28 days after tail veins were injected with GC cells. The white arrows represent the lung metastasis nodule. e Lung metastases were confirmed via H&E staining (left); knockdown of TCF7L2 inhibited PLAUR expression in lung metastases evaluated by IHC (right). Compared with the control group, * P < 0.001.

Fig 3: Loss of the SxxSS motif in TCF-4 isoforms increases expression of HIF-as under hypoxia.(A and B) Immunoblot analysis of empty vector-transfected (EV), TCF-4J-overexpressing (J), and TCF-4K-overexpressing cells (K). The cells were treated with 0 and 150 µM CoCl2 (A) or cultured under 1% oxygen tension for 48 hr (B). Cell lysates were subjected to detect HIF-as and Myc-tag TCF-4 isoform protein expression. Expression levels were plotted as a ratio to actin (right panel). (C) Confocal microscopy for nuclear localization of HIF-2a protein. Cells were treated with 0 µM (Normoxia) or 150 µM (Hypoxia) CoCl2 and stained with an anti-HIF-2a antibody (green). DAPI was employed for nuclear staining. (D) Immunoblot analysis of total cell lysates from cells treated with 0 or 150 µM CoCl2 for 48 hr. A and B represent HAK-1A cells overexpressing TCF-4A (“short form” of TCF-4K) and TCF-4B (“short form” of TCF-4J), respectively (see Figure 2A). Note the robust increase in expressions of HIF-1a and HIF-2a in B cells.

Fig 4: Tag-density profiles of ChIP-seq datasets‘css’: H3K27Ac ChIP-seq of LNCaP grown in charcoal-stripped serum. ‘dht’: H3K27Ac ChIP-seq of LNCaP exposed to androgen. ‘tcf7l2’: ChIP-seq with anti-TCF7L2 in LNCaP, unstimulated. Top: peak height, is scaled. Middle: peak width, -axis is scaled. Bottom: Peak height vs. width reveals strong correlation.

Fig 5: TCF-4 promotes transcription activity of NF-?B p65 via stimulating p65 translocation from cytosol to nucleus.(a) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with PCMV (0.4 µg/ml) or PCMV-TCF-4 (0.4 µg/ml) plus si-NC (20 nmol/ml) or a siRNA for mouse NF-?B p65 (si-p65, 20 nmol/ml) for 36 hours (n = 5, **P < 0.01). (b) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus pCDNA3.1 (0.4 µg/ml) or pCDNA-p65 (0.4 µg/ml) for 36 hours (n = 5, **P < 0.01). (c) Immunoblotting assay of TCF-4 and p65 in the cytosol or nucleus of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) for 36 hours. (d) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus a reporter construct (0.4 µg/ml) containing the promoter of CCL20 for 36 hours (n = 4, **P < 0.01). The tests from (a–d) were repeated for 3 times, and the representative results were displayed.