Fig 2: Addition of C3a to the medium only partially mimics the effect of overexpression of C3a in HPC cells. HPC cells were divided into three groups: HPC, HPC+SB+C3a and HPC+C3a. Morphological changes were observed under a phase contrast microscope. (A) Reverse transcription-PCR analysis of the expression of focal adhesion-associated genes. *P<0.05, **P<0.01 vs HPC group; #P<0.05, ##P<0.01 vs HPC+C3a group. (B) Immunofluorescence staining for VCL. Scale bar, 50 µM. (C) Representative images taken under phase contrast microscopy demonstrated cell morphology. Scale bar, 100 µM. (D) Results of adhesion analysis. COL1A1, collagen I α1; FN1, fibronectin 1; LAMA1, laminin α1; LAMB1, laminin β1; LAMC1, laminin γ1; ITGA1, integrin α1; ITGA2, integrin α2; ITGB1, integrin β1; PTK2, protein tyrosine kinase 2; NRP1, neuropilin 1; TLN2, talin 2; FERMT2, fermitin family member 2; VCL, vinculin; C3a, human C3a anaphylatoxin; HPC, human podocyte cell; SB, SB290157.

Fig 3: Immunohistochemistry analysis of five membrane glycoproteins (carcinoembryonic antigen 5 [CEA], integrin beta 1 [ITGB1], mucin 16 [MUC16], prostate stem cell antigen [PSCA], glucose transporter type 1 [GLUT1]) identified by LC‐MS/MS analysis of rBC2LCN‐precipitated fractions in pancreatic ductal adenocarcinoma (PDAC) tissues (n = 19) and normal pancreatic tissues (n = 22). A, Representative images of immunohistochemical staining of five membrane glycoproteins (CEA, ITGB1, MUC16, PSCA, GLUT1) in PDAC tissues and normal pancreatic tissues. Black arrow: pancreatic duct. Scale bars, 100 μm. B, The percentage of cells with high expression of each membrane glycoprotein was quantified in PDAC tissues (T; n = 19) and normal pancreatic tissues (N; n = 22). NS: not significant, *P < .05, ***P < .0001

Fig 4: Expression levels of the genes encoding integrins A3 (ITGA3) and B1 (ITGB1) were suppressed by transfection of PC3 prostate cancer cells with si‐ITGA3 and si‐ITGB1. (a, b) ITGA3 and ITGB1 mRNA expression levels were evaluated by quantitative RT‐PCR 72 h after transfection with si‐ITGA3 and si‐ITGB1 (10 nM). ACTB was used as an internal control. *P < 0.0005. ITGA3 and ITGB1 expression levels were evaluated by Western blotting 72 h after transfection with si‐ITGA3 and si‐ITGB1 (10 nM). GAPDH was used as a loading control. (c–e) Effects of ITGA3 silencing on PC3 cells. (c) Cell proliferation was determined by XTT assays. (d) Cell migration activity was determined by wound‐healing assays. (e) Cell invasion activity was determined by Matrigel invasion assays. (f–h) Effects of silencing ITGB1 on PC3 cells. (f) Cell proliferation was determined by XTT assays. (g) Cell migration activity was determined by wound‐healing assays. (h) Cell invasion activity was determined by Matrigel invasion assays. *P < 0.0001.

Fig 5: Expression of the gene encoding integrin B1 (ITGB1) was suppressed by transfection of prostate cancer cell lines PC3 and PC3M with miR‐223. (a) ITGB1 mRNA expression was evaluated by quantitative RT‐PCR 72 h after transfection with miR‐223 (10 nM). ACTB was used as an internal control. *P < 0.005. (b) ITGB1 protein expression was evaluated by Western blotting 72 h after transfection with miR‐223 (10 nM). GAPDH was used as a loading control. (c) miR‐223 binding site in the 3′‐UTR of ITGB1 mRNA. (d) Luciferase reporter assays in PC3 cells using vectors encoding a putative miR‐223 target site at position 255–261 of the ITGB1 3′‐UTR. Renilla luciferase values were normalized to firefly luciferase values. *P < 0.0001.