Fig 2: Activation of ß-catenin is involved in the effect of TFPI on the expression of CXCR7 and the CXCL12-mediated TEM of CLL cells. (A) Western blotting and (B) quantification of the active ß-catenin expression, relative to ß-actin, after treating the CLL cells from patients with 200 ng/mL rTFPI for 0, 15, 60, 180 and 360 min. n = 3 individuals; *P < 0.05 (C) Quantitative PCR was used to detect the mRNA expression of CXCR7, relative to TBP, after CLL cells were pre-treated with IWP4 (5 µM) for 30 min prior to 24 h treatment of 200 ng/mL rTFPI, with DMSO as a control. n = 4 individuals; *P < 0.05, **P < 0.01. (D) Western blotting and (E) quantification of CXCR7 expression, relative to ß-actin, after CLL cells were pre-treated with 5 µM IWP4 for 30 min prior to 24 h treatment of 200 ng/mL rTFPI, with DMSO as a control. n = 3 individuals; *P < 0.05. (F) CLL cells were pre-treated with 5 µM IWP4 for 30 min prior to 24 h treatment of 200 ng/mL rTFPI, with DMSO as a control. Then CLL cells were washed and applied to TEM assay. TEM results were presented as we described in Fig. 2A. n = 10 individuals; **P < 0.01.

Fig 3: Plasma TFPI concentration increased in patients with CLL and related to CLL progression. Increased plasma TFPI level was observed in CLL patients compared to healthy controls. Compared to the patients with Binet stage A and B, the patients with Binet stage C showed the highest TFPI concentration in the plasma. Binet stages represent the severity of organ infiltration. Data are presented as dot plots and columns represent median levels. Concentrations are expressed as ng/mL. p values in the graph represent differences vs. controls. *p < 0.05, **p < 0.01. All statistical comparisons were adjusted for age using UNIANOVA with Bonferroni adjustment on log transformed TFPI values.

Fig 4: CXCR7 plays a key role in the effect of TFPI on the CXCL12-mediated TEM of CLL cells. (A) Western blotting and (B, C) quantification of CXCR7 and CXCR4 expression, relative to ß-actin, after treating CLL cells with different doses of rTFPI for 24 h. n = 3 individuals, *P < 0.05. (D) Western blotting and (E, F) quantification of CXCR7 and CXCR4 expression, relative to ß-actin, after treating the CLL cells with 200 ng/mL rTFPI for 0, 15, 60, 180, 360 and 960 min. n = 4 individuals, **P < 0.01. (G, H) mRNA expression of CXCR7 and CXCR4 was detected by quantitative PCR, relative to TBP, after treating the CLL cells with 200 ng/mL rTFPI for 0, 15, 60, 180, 360 and 960 min. n = 4 individuals; *P < 0.05. (I) After 24 h exposure of 200 ng/mL rTFPI, CLL cells were treated with 2 µM CCX771 for 1 h before they were used in the TEM assay. DMSO was used as control. n = 6 individuals; *P < 0.05, **P < 0.01. (J) After 24 h exposure of 200 ng/mL rTFPI, CLL cells were treated with 5 µg/mL AMD3100 for 1 h before they were applied to the transwells for TEM assay. For panels (I, J), results were presented as described in Fig. 1A. n = 6 individuals; *P < 0.05.

Fig 5: TFPI binds to GPC3 on the membrane of CLL cells. (A) Western blotting and (B) quantification of TFPI protein in the cell lysates of CLL patients, relative to ß-actin, after 0, 50, 100 and 200 ng/mL rTFPI treatment. n = 3 individuals; *P < 0.05. (C) After 24 h treatment of 200 ng/mL rTFPI, co-localization of TFPI (red) and GPC3 (green) on the surface of human CLL cell line HG3 was assessed by immunofluorescence staining. Nuclei were stained with DAPI (blue). (D) Co-IP was performed in the HG3 and K562 cells. Immunoblots showed the bait protein TFPI and the pull-down of the prey protein GPC3. (E) Western blotting and (F) quantification of CXCR7 expression, relative to ß-actin, after CLL cells were pre-treated with GPC3 antibody HS20 (100 µg/mL) for 1 h prior to 24 h treatment of 200 ng/mL rTFPI. Human IgG was used as control since HS20 is isolated from human serum. n = 4 individuals; *P < 0.05, **P < 0.01. (G) After CLL cells were pre-treated with HS20 before rTFPI treatment, the cells were washed and applied to TEM assay. Human IgG was used as control. Results were presented as we described in Fig. 2A. n = 10 individuals; **P < 0.01.