Fig 2: CXCR7 activates YAP through the Gαq/11-ROCK-LATS axis in gastric cancer. A, B CXCR7 activation induced YAP dephosphorylation through Gaq/11. MGC803 and Hs746T cells were transiently transfected with control, Gaq/11, or Gas siRNAs. MGC803 and Hs746T cells were treated with TC. YAP, phosphorylated YAP, Gaq/11 and Gas were determined by immunoblotting. C CXCR7 activation induces YAP nuclear localization through Gaq/11. MGC803 cells were transiently transfected with control, Gaq/11 or Gas siRNAs. MGC803 cells were treated with TC. Endogenous YAP (green) and nuclei (blue) were stained with specific antibodies and DAPI, respectively; scale bar, 20 mm. Quantifications of YAP subcellular localization from at least 100 randomly selected cells. C, cytoplasm; N, nucleus. D Nucleoplasm separation experiments by immunoblotting confirmed that Gαq/11 silencing could block the YAP nuclear accumulation caused by CXCR7 activation in MGC 803 cells. E CXCR7 activation via TC decreases LATS1 activity. MGC803 and Hs746T cells were stimulated with TC. LATS1 was immunoprecipitated. Phosphorylation of YAP by LATS1 was determined by a phospho-YAP antibody. F Ectopic expression of LATS1 blocks YAP dephosphorylation induced by CXCR activation. MGC803 and Hs746T cells were transiently transfected with control, LATS1 wild type (WT), or kinase dead mutant (K/R). MGC803 and Hs746T cells were treated with TC for 1 h. Phosphorylation and protein levels of YAP were determined by immunoblotting. G Rho GTPase is involved in YAP dephosphorylation induced by CXCR activation. MGC803 and Hs746T cells were transiently transfected with control, Myc-Rho-L63, or C3. MGC803 and Hs746T cells were treated with TC. Total YAP and phosphorylated YAP protein levels were determined by immunoblotting. H ROCK is required for CXCR7-induced YAP activation. Serum-starved MGC803 and Hs746T cells were pretreated with GSK429286 (1 mmol/L) or Y27632 (1 mmol/L) for 4 h, followed by treatment with TC. Total YAP and phosphorylated YAP protein levels were determined by immunoblotting

Fig 3: Drugs targeting CXCR7 restrain gastric cancer progression. A, B ACT antagonist treatment against CXCR7 inhibited the proliferation of gastric cancer cells. MGC803 and Hs746T cells were treated with different doses of ACT. After 24 h, CCK-8 assays were used to determine the metabolic activity of the cells at the indicated time points after transfection. Experiments were performed in triplicate. Comparison of cell growth, *P < 0.05, **P < 0.01, ***P < 0.001. C, D Wound healing assay of MGC803 and Hs746T cells treated with an antagonist against CXCR7. Wound closure was quantified for the indicated time points. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). E, F ACT antagonist treatment against CXCR7 inhibited the migration of MGC803 and Hs746T gastric cancer cells. MGC803 and Hs746T cells were transfected with different doses of ACT. After 24 h, the migration was assessed by Transwell assays. Cell numbers were determined, and data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). G, H ACT antagonist treatment against CXCR7 promoted apoptosis of MGC803 and Hs746T cells. MGC803 and Hs746T cells were transfected with different doses of ACT. After 24 h, the cells were stained with PI and Annexin V, and then, FACS analysis was performed on the cells to determine the proportion of apoptotic cells. Each group was analysed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison. I, J ACT antagonist treatment against CXCR7 inhibited the colony-forming ability of MGC803 and Hs746T gastric cancer cells. MGC803 and Hs746T cells were transfected with different doses of ACT. Quantification of colony formation is shown at the indicated time points. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). K, L Cell cycle analysis was performed to assess the effect of treatment with the CXCR7 antagonist ACT on MGC803 cells and Hs746T cells. MGC803 and Hs746T cells were transfected with different doses of ACT. After 24 h, the cells were harvested, fixed in 70% ethanol, and stained with propidium iodide. Cells were subjected to FACS analysis. Experiments were performed in triplicate. Comparison of cell proportions, *P < 0.05, **P < 0.01, ***P < 0.001. Representative histograms and cell cycle phase distribution plots are shown in Fig. 3K and L, respectively. M–O ACT antagonist treatment against CXCR7 inhibited gastric tumour growth in vivo. MGC803 cells (2 × 106) were injected into the right dorsal side of 4-week-old female BALB/c nude mice. Tumour formation in nude mice treated with vehicle or ACT at the indicated concentrations was monitored over a period of 4 weeks. Tumour volume was calculated using the following formula: tumour volume = 0.5 × length × width2. Five weeks after tumour cell injection, mice were sacrificed. Tumour growth curves, weights and photographs are shown in Panels M, N and O, respectively. P, Q In the patient-derived explant (PDEx) assay, ACT treatment inhibited the proliferation potential of gastric tumours. The gastric tumour samples were cultured ex vivo on sponges for 48 h with 10% FBS medium. The gastric tumour explants were treated with vehicle or 4 µM ACT. The samples were fixed and stained with YAP, CXCR7 and Ki67 via IHC. The Ki67-positive cells were counted for analysis

Fig 4: Drugs activating CXCR7 promote gastric cancer progression. A, B CXCR7 activation via TC promoted the proliferation of gastric cancer cells. MGC803 and Hs746T cells were treated with different doses of TC. After 24 h, CCK-8 assays were used to determine the metabolic activity of the cells at the indicated time points after transfection. Experiments were performed in triplicate. Comparison of cell growth, *P < 0.05, **P < 0.01, ***P < 0.001. C, D Wound healing assay of MGC803 and Hs746T cells with CXCR7 activation via TC. Wound closure was quantified for the indicated time points. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). E, F CXCR7 activation via TC promotes the migration of MGC803 and Hs746T gastric cancer cells. MGC803 and Hs746T cells were transfected with different doses of TC. After 24 h, the migration was assessed by Transwell assays. Cell numbers were determined, and data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). G, H CXCR7 activation via TC inhibited apoptosis of MGC803 and Hs746T cells. MGC803 and Hs746T cells were transfected with different doses of TC. After 24 h, the cells were stained with PI and Annexin V, and then, FACS analysis was performed on the cells to determine the proportion of apoptotic cells. Each group was analysed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison. I, J CXCR7 activation via TC promotes the colony-forming ability of MGC803 and Hs746T gastric cancer cells. MGC803 and Hs746T cells were transfected with different doses of TC. Quantification of colony formation is shown at the indicated time points. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). K, L Cell cycle analysis was performed to assess the effect of CXCR7 activation via TC on MGC803 cells and Hs746T cells. MGC803 and Hs746T cells were transfected with different doses of TC. After 24 h, the cells were harvested, fixed in 70% ethanol and stained with propidium iodide. Cells were subjected to FACS analysis. Experiments were performed in triplicate. Comparison of cell proportions, *P < 0.05, **P < 0.01, ***P < 0.001. Representative histograms and cell cycle phase distribution plots are shown in Fig. 4 K and L, respectively

Fig 5: CXCR7 is required for gastric cancer cell progression. A, B Depletion of CXCR7 inhibited the proliferation of gastric cancer cells. MGC803 and Hs746T cells were transfected with siControl or siCXCR7. Two different siRNAs were used. After 24 h, CCK-8 assays were used to determine the metabolic activity of the cells at the indicated time points after transfection. Experiments were performed in triplicate. Comparison of cell growth, *P < 0.05, **P < 0.01, ***P < 0.001.C, D Wound healing assay of MGC803 and Hs746T cells transfected with siCXCR7 or siControl. Wound closure was quantified for the indicated time points. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). E, F Depletion of CXCR7 inhibited the migration of MGC803 and Hs746T gastric cancer cells. MGC803 and Hs746T cells were transfected with siControl or siCXCR7. After 24 h, the migration was assessed by Transwell assays. Cell numbers were determined, and data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). G, H Depletion of CXCR7 promoted apoptosis of MGC803 and Hs746T cells. MGC803 and Hs746T cells were transfected with siCXCR7 and siControl. After 24 h, the cells were stained with PI and Annexin V, and then, FACS analysis was performed on the cells to determine the proportion of apoptotic cells. Each group was analysed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison. I, J Depletion of CXCR7 inhibited the colony-forming ability of MGC803 and Hs746T gastric cancer cells. MGC803 and Hs746T cells were transfected with siCXCR7 and siControl. Quantification of colony formation is shown at the indicated time points. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001 (Student's t test). K, L Cell cycle analysis was performed to assess the effect of CXCR7 silencing on MGC803 cells and Hs746T cells. MGC803 and Hs746T cells were transfected with siCXCR7 or siControl. After 24 h, the cells were harvested, fixed in 70% ethanol and stained with propidium iodide. Cells were subjected to FACS analysis. Experiments were performed in triplicate. Comparison of cell proportions, *P < 0.05, **P < 0.01, ***P < 0.001. Representative histograms and cell cycle phase distribution plots are shown in Fig. 2 K and L, respectively. M–O Depletion of CXCR7 inhibited gastric tumour growth in vivo. MGC803 cells were stably transduced by a lentiviral vector expressing either control shRNA or CXCR7 shRNA. These MGC803 cells (2 × 106) were injected into the right dorsal side of 4-week-old female BALB/c nude mice. Tumour formation in nude mice was monitored over a period of 4 weeks. Tumour volume was calculated using the following formula: tumour volume = 0.5 × length × width2. Five weeks after tumour cell injection, the mice were sacrificed. Tumour growth curves, weights and photographs are shown in Panels M, N and O, respectively. P Immunohistochemical analysis showed that CXCR7 depletion decreased the expression of Ki67 in xenograft tumours