Fig 2: Upregulated expression of Ror1 in MKN45 cells is required for their ability to form tumors stimulated by co–injection of UE6E7T-12 cells. A, MKN45 cells stably expressing either ctrl or Ror1 shRNA were obtained as described in Section 2. Expression levels of Ror1 mRNA and Ror1 protein in these cells were assessed by quantitative RT-PCR and western blotting, respectively. B, The shRNA-expressing MKN45 cells were injected subcutaneously into nude mice with or without UE6E7T-12 cells, and tumor volumes were measured at 7, 14, 21 and 28 d after injection, as described in Section 2 (left panel). Data are expressed as mean ± SD (n = 5 mice per each group). **P < 0.01, Tukey's HSD test. Representative images of xenograft tumors isolated at 28 d after tumor cell injection are shown (right panel). Scale bar, 5 mm. C, The shRNA-expressing MKN45 cells were injected orthotopically into the stomach walls of nude mice with or without UE6E7T-12 cells, and the stomachs were removed at 28 d after injection (left panel). An arrowhead indicates the tumor. Scale bar, 5 mm. Tumor volumes were measured as described in Section 2 (right panel). Data are presented as a box-and-whisker plot. n = 6 mice per each group. *P < 0.05, Fisher's protected least significant difference (PLSD) post–hoc tests test. D, MKN45 cells were injected subcutaneously into nude mice with or without UE6E7T-12 cells. At 7 days after injection, anti–CXCL16 antibody or control IgG were administrated around the tumor. Tumor volume was measured at the indicated time points (left panel). Data are expressed as mean ± SD (n = 4 mice per each group). *P < 0.05, Tukey's HSD test. Representative images of xenograft tumors isolated at 14 days after tumor cell injection were shown (right panel). Scale bar, 5 mm

Fig 3: UE6E7T-12 cell-derived CXCL16 induces activation of STAT3 in co–cultured MKN45 cells to promote their proliferation and migration. A, MKN45 cells were treated with CXCL16 (10 ng/mL) for the indicated periods, and the levels of phosphorylated (pSTAT3 S727) and total STAT3 were analyzed by western blotting. The histograms indicate the relative band intensities of pSTAT3 normalized by those of total STAT3. Data are expressed as mean ± SD (n = 3). *P < 0.05, Tukey's honest significance difference (HSD) test. B, C, MKN45 cells were either monocultured or co–cultured with UE6E7T-12 cells for 24 h. In (C), UE6E7T-12 cells were transfected with either ctrl or CXCL16 siRNA 72 h before being subjected to co–culture experiments. The levels of pSTAT3 S727 and total STAT3 in MKN45 cells were analyzed by western blotting. The histograms indicate the relative band intensities of pSTAT3 normalized by those of total STAT3. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01 t test (B) or Tukey's-HSD test (C). D, MKN45 cells transfected with either ctrl or STAT3 siRNA were monocultured or co–cultured with UE6E7T-12 cells, as indicated, and proliferation and migration of MKN45 cells were assessed as described in Section 2. Data are expressed as mean ± SD (n = 3). **P < 0.01, Tukey's HSD test

Fig 4: CXCL16 derived from UE6E7T-12 cells upregulates expression of Ror1 in co–cultured MKN45 cells through the STAT3 pathway. A, B, MKN45 cells were monocultured or co–cultured with UE6E7T-12 cells for 24 h. Expression levels of Ror1 mRNA (A) and Ror1 protein (B) in MKN45 cells were analyzed by quantitative RT-PCR and western blotting, respectively. Data are expressed as mean ± SD (n = 3). * P < 0.05, t test. C, D, UE6E7T-12 cells were transfected with ctrl, Ror2 or CXCL16 siRNA, as indicated. MKN45 cells were monocultured or co–cultured with siRNA-transfected UE6E7T-12 cells for 24 h. Expression levels of Ror1 mRNA (C) and Ror1 protein (D) in MKN45 cells were analyzed by quantitative RT-PCR and western blotting, respectively. The histograms in (D) indicate the relative band intensities of Ror1 normalized by those of a-tubulin. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, Tukey's honest significance difference (HSD) test. E, F, MKN45 cells, either untransfected (E) or transfected with ctrl or CXCR6 siRNA (F), were treated with CXCL16 (10 ng/mL) for the indicated periods. Expression levels of Ror1 and a-tubulin were analyzed by western blotting. Knockdown efficiency of si-CXCR6 was assessed by quantitative RT-PCR (F, right panel). G, H, MKN45 cells were transfected with either ctrl or STAT3 siRNA and treated with CXCL16 (10 ng/mL) for the indicated periods (G) or 60 min (H). Expression levels of Ror1 mRNA (G) and Ror1, STAT3 and a-tubulin proteins (H) were analyzed by quantitative RT-PCR and western blotting, respectively. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, Tukey's HSD test. I, MKN45 cells were pretreated with either vehicle alone (DMSO) or STAT3 inhibitor VII (6 µmol/L) for 60 min and then treated with CXCL16 (10 ng/mL) for the indicated periods. Expression levels of Ror1 mRNA were analyzed by quantitative RT-PCR. Data are expressed as mean ± SD (n = 3). **P < 0.01, Tukey's HSD test