Fig 2: Netrin-1 inhibits PDAC growth by decreasing integrin β4 expression(A–B) Real-time RT-PCR (A) and western blotting (B) analyses of integrin β4 expression in control (ctrl) and netrin-1-over-expressing MiaPaCa II cells (ntn1+). GAPDH was used as the internal control for both analyses. (C–D) Real-time RT-PCR (C) and western blotting (D) analyses of integrin β4 expression in MiaPaCa II cells treated with the indicated concentrations of netrin-1 for 48 hr. GAPDH was used as the internal control for both analyses. (E) Western blotting analysis of integrin β4 expression in control GFP RNAi (RiGFP) and ITGB4 RNAi (RiITGB4) MiaPaCa II cells. GAPDH was used as the internal control. (F) Representative xenograft tumors formed by GFP RNAi (asterisk) and ITGB4 RNAi (arrowhead) MiaPaCa II cells in SCID-beige mice (bar, 1 cm). (G) Statistics for the weights of the xenograft tumors formed by the control RiGFP (N = 7) and RiITGB4 (N = 7) MiaPaCa II cells (***P < 0.001) in SCID-beige mice. (H) Growth curves of the RiGFP and RiITGB4 MiaPaCa II xenograft tumors in Balb/c NU/NU mice. The tumor volumes were measured every other day and calculated using the formula v = 0.5 ab2 (a: long diameter; b: short diameter); the total 28-day result is shown (error bars represent the standard deviation of the measurements, *P < 0.05, **P < 0.01).

Fig 3: The expression level of NTN1 was increased in GC specimens. A. NTN1 mRNA expression level in 97 paired GC tissues and adjacent normal tissues were investigated by qRT-PCR. B. Representative results of upregulation of NTN1 protein in GC specimens with or without NI by immunohistochemistry. N indicated nerve. Original magnification, 200×; Scale bar=100μm. C. Quantification of the percentage of cells demonstrating the presence of immunoreactivity for NTN1 (positive staining). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Netrin-1 suppresses ECM adhesion and the 3D growth of MiaPaCa II cells but does not affect proliferation and apoptosis in 2D cultures(A–B) Growth curve analysis of 2D-cultured netrin-1-over-expressing (ntn1+) and control (ctrl) MiaPaCa II cells (A) or MiaPaCa II cells treated with the indicated concentrations (ng/ml) of recombinant netrin-1 (B). No significant differences in cell proliferation were observed following either netrin-1 over-expression or recombinant netrin-1 protein treatment (P > 0.05). (C–D) Analysis of caspase-3 activity (C) and TUNEL assay (D) of the netrin-1-over-expressing (ntn1+) and control MiaPaCa II cells (ctrl). No significant differences in apoptosis were observed in either assay. (E–F) Quantification of the adhesion of the control (ctrl) and netrin-1-over-expressing (ntn1+) MiaPaCa II cells (E) or MiaPaCa II cells pretreated with the indicated concentrations (ng/ml) of recombinant netrin-1 for 24 hr (F) to the designated ECM components (***P < 0.001). (G–H) Micrographs (G) and growth curves (H) of the control (ctrl) and the netrin-1-over-expressing (ntn1+) MiaPaCa II cells cultured in 3D in Matrigel for 8 days (magnification, 200×; bar, 50 μm) (***P < 0.001). (I) Immunohistochemical staining of integrin β4 (ITGB4) and ZO-1 in the mouse xenograft tumor sections from the control (ctrl) and netrin-1-over-expressing (ntn1+) MiaPaCa II cells (magnification, 200×).

Fig 5: Netrin-1 down-regulates integrin β4 expression through the UNC5b receptor and the activation of FAK(A) Real-time PCR analysis for the expression of netrin-1 receptors in MiaPaCa II cells. GAPDH was used as an internal control. (B–C) The anti-UNC5b antibody selectively blocks the integrin β4-suppressing effect of netrin-1. The receptors on the MiaPaCa II cells were blocked by their respective antibodies before the cells were treated with the indicated concentrations netrin-1; integrin β4 expression in the cells was then detected by real-time RT-PCR (***P < 0.001, **P < 0.01) (B) and western blotting (C). GAPDH was used as an internal control. (D) Netrin-1 interacts with UNC5b but not integrin β4 in the myc-netrin-1-over-expressing MiaPaCa II cells. An anti-myc antibody to pull down the myc-tagged netrin protein in the netrin-1-over-expressing MiaPaCa II cells, followed by immunoblotting analysis of the integrin β4 (itgb4), UNC5b and netrin-1 (ntn1) levels in the precipitate. (E) Western blotting analysis of UNC5b expression in the UNC5B RNAi (RiUNC5B) and control GFP RNAi (RiGFP) MiaPaCa II cells. (F) Netrin-1 suppresses integrin β4 expression in the control GFP RNAi cells but not the UNC5B RNAi cells. Unc5b-knockdown and control MiaPaCa II cells were treated with recombinant netrin-1, and the integrin β4 expression was detected before and after netrin-1 treatment by western blotting. (G) Western blotting analysis of the FAK and phospho-FAK levels in the netrin-1-over-expressing MiaPaCa II cells, and the MiaPaCa II cells treated with the indicated concentrations of recombinant netrin-1 for 2 hr. The vector-transfected and the untreated MiaPaCa II cells were used as the respective negative controls, and GAPDH was used as an internal control. (H) UNC5b RNAi abrogates netrin-1-induced FAK phosphorylation. The FAK and phospho-FAK levels in the control and UNC5b RNAi MiaPaCa II cells were detected before and after netrin-1 stimulation by western blotting. (I) FAK inhibition abrogates the netrin-1-induced down-regulation of integrin β4. Western blot analysis of the phospho-FAK and integrin β4 levels in MiaPaCa II cells treated with the indicated concentrations of recombinant netrin-1 in the presence or absence of FAK inhibitor 14. (J) FAK inhibition abrogates the netrin-1-induced growth arrest of MiaPaCa II cells in Matrigel, as analyzed by the 3D growth of the control and netrin-1-over-expressing MiaPaCa II cells in Matrigel in the presence of 20 nM FAK inhibitor 14. Scale bars, 50 μm.