Fig 2: CXCR7 protein expression in the human brain.Staining with 11G8 or ab72100 was performed on different human tissues, using either a tissue array containing samples from multiple normal organs or additional brain sections as described in the methods. Images from the multi-organ array are shown in the top panels (A to C; scale bars: 200 µm). Neuronal CXCR7 expression was observed in the gray matter (A) while no staining is observed in normal breast tissue (B), as expected. CXCR7 was also expressed in the kidney (C). Additionally, serial sections from human frontal cortex were stained with: (D) rabbit polyclonal antibody ab72100, (E) 11G8, or (F) H&E (details in methods). Similar neuronal staining was observed with both CXCR7 antibodies. No staining was detected when the primary antibody was omitted (G) or replaced by the isotype control (H). Images in insets are higher magnification of D, E, and F. Scale bars: 400 µm (D–H), 50 µm for insets. NC = negative control.

Fig 3: CXCR7 expression in prostate-cancer cells. (A) Western blot of 1, 2, and 4 μg of LNCaP total lysate with pAbCXCR7 antibody in the presence of the non-competitive AR peptide (a.a. 299–315, left panel) or the CXCR7 blocking peptide (a.a. 348–362, right panel) as detailed in Materials and Methods section. The CXCR7 bands are indicated by arrowheads. (B)AR, CXCR7, CXCR4, and PSA gene expressions in LNCaP cells transfected with AR, CXCR7, or scrambled control siRNA. RNA was isolated 72 hrs post-transfection and measured by qPCR. Student’s t-test was used to calculate significant differences (*p ≤ 0.05, n = 3) between control and experimental cells. (C) Western blot (left panel) of whole cell lysates from LNCaP cells transfected with control or two experimentally-validated CXCR7 siRNAs (CXCR7 #1 or #2) for 72 hrs using antibodies to pAbCXCR7 and GAPDH. Silver-stained gel demonstrated equal protein loading across samples (right panel). The densitometry values were labeled below the blot and normalized to the control transfected cells loaded with the same amount of total proteins. (D) Light microscopy of LNCaP cells transfected with control or CXCR7 siRNA for 72 hrs.

Fig 4: CXCR7 functionally interacts and colocalizes with AR. (A) Western blot of LNCaP cells transfected with the indicated siRNA combinations: control (50 nM), AR (50 nM), CXCR7 (50 nM), AR/control (25 nM/25 nM), CXCR7/control (25 nM/25 nM), or AR/CXCR7 (25 nM/25 nM) for 72 hrs. Western blot was performed using AR, CXCR7, and PSA antibodies. Silver staining demonstrates equivalent loading across the samples. The densitometry values were normalized to control siRNA transfected cells and labeled below the blots. (B) Immunofluorescence analysis of CXCR7 and AR in LNCaP cells under AR or CXCR7 knockdown conditions. Cells were transfected with control (I-I to I-III), AR (II-I to II-III), or CXCR7 (III-I to III-III) siRNA, stained with antibodies against AR and CXCR7, and treated with DAPI.

Fig 5: YAP1 inhibitor suppresses CXCR7 induced tumor progression and metastasis. A Western blot analysis of the expression of DCLK1 or Vimentin normalized to β-actin in HCT116 and SW620 cells treated with CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM) and Verteporfin (3 µM) for 48 h. B Representative images of livers and lungs and H&E-stained sections of liver metastatic nodules in nude mice inoculated with HCT116LV−CXCR7 and HCT116Control cells via tail veins with or without the treatment of Verteporfin (10 mg/kg, n = 3 per group). The arrows point out the visible metastatic nodules of liver. C Representative images of colons from AOM/DSS-treated WT, Villin-CXCR7 mice and Villin-CXCR7 mice treated with Verteporfin (10 mg/kg). Average size of colon polyps was analyzed in different groups (n = 5). D RT-qPCR analysis of expression levels of miR-124-3p and miR-188-5p in colon cancer tissues from above-mentioned C57BL/6 mice. E Representative IHC staining of Vimentin in AOM/DSS-induced colon adenocarcinoma tissues from wild type C57BL/6 mice and Villin-CXCR7 mice administered with Verteporfin (10 mg/kg) or vehicle control via intraperitoneal injection daily. F Western blot analysis of DCLK1 and Vimentin expression in colon cancer tissues from these mice. GAPDH was used as loading control and statistical analysis was performed. *P < 0.05, **P < 0.01, ***P < 0.001