Volume 86, Issue 3, Pages 548-557 (September 2014) Epithelial protein lost in neoplasm modulates platelet-derived growth factor–mediated adhesion and motility of mesangial cells  Haruko Tsurumi, Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Kenji Hayashi, Atsuko Matsunaga, Yuko Kajiho, Shoichiro Kanda, Kenichiro Miura, Takashi Sekine, Akira Oka, Kiyonobu Ishizuka, Shigeru Horita, Motoshi Hattori, Seisuke Hattori, Takashi Igarashi  Kidney International  Volume 86, Issue 3, Pages 548-557 (September 2014) DOI: 10.1038/ki.2014.85 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Expression of epithelial protein lost in neoplasm (EPLIN) in rat and human kidneys. (a) Total RNA (0.5μg) from rat whole kidney, kidney cortex, kidney medulla, and glomeruli was detected using reverse transcriptase (RT)-PCR with specific primers for EPLIN α and β. (b) Sections of rat kidneys were stained with anti-EPLIN antibody (A), anti-EPLIN antibody preabsorbed with recombinant glutathione S-transferase (GST) (B), or GST-EPLIN α (C). Signals were detected in glomeruli (white arrow), proximal and distal tubules (arrowheads), and endothelial cells of the extraglomerular arteries (asterisk). Scale bars=50μm. (c) Sections of human kidneys were stained with anti-EPLIN antibody (A), anti-EPLIN antibody preabsorbed with recombinant GST (B), or GST-EPLINα (C). Similar staining patterns to those in rat kidneys were observed in human kidneys. Scale bars=50μm. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Localization of epithelial protein lost in neoplasm (EPLIN) in mesangial cells. (a) Localization of EPLIN in adult rat glomeruli. Sections were costained for the cell markers intercellular adhesion molecule-2 (ICAM-2), zonula occludens (ZO)-1, or Thy1 (E30) and EPLIN. Merged images are shown in G, H, and I. Higher-magnification images corresponding to squares in G, H, and I are shown in J, K, and L, respectively. Scale bar=50μm. (b) Localization of EPLIN in adult human glomeruli. Sections were costained for zonula occludens-1 (ZO-1) (A) and EPLIN (B). A merged image is shown in C. A higher-magnification image corresponding to the square in C is shown in D. Scale bar=50μm. (c) Immunogold staining for EPLIN detected using immunoelectron microscopy. Immunogold particles for EPLIN were detected in the periphery of the cytoplasmic processes of mesangial cells at the mesangial angles (A, B, arrow). Higher-magnification image corresponding to the square in A is shown in B. The particles were also detected in mesangial cell–cell adhesion sites (C, arrows). En, endothelial cell; GBM, glomerular basement membrane; M, mesangial cell. Scale bars=100nm. (d) EPLIN expression in neonatal rat glomerulus. Section was costained for podocalyxin (A) and EPLIN (B). A merged image is shown in C. Scale bar=20μm. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Reduced expression of epithelial protein lost in neoplasm (EPLIN) in mesangial proliferative glomerulonephritis in rats and humans. (a) Expression of EPLIN and α-smooth muscle actin (α-SMA) after induction of anti-Thy1 glomerulonephritis was shown using double immunostaining for EPLIN (A, B, C, and D) and α-SMA (E, F, G, and H). Merged images are shown in I, J, K, and L. Scale bar=50μm. (b) Western blot analysis of EPLIN in anti-Thy1 glomerulonephritis. Lysates of glomeruli isolated from rats before and after 5, 8, 14, and 28 days of anti-Thy1 antibody injection were immunoblotted with the indicated antibodies. (c, d) EPLIN expression in human glomerulonephritis. Periodic acid–Schiff staining (A, B, and C) and double immunostaining for EPLIN and zonula occludens-1 (ZO-1) in the control case (D) and patients with membranoproliferative glomerulonephritis (MPGN) (in c) and IgA (in d) nephropathy are shown (E, F). Higher-magnification images corresponding to squares in D, E, and F are shown in G, H, and I, and J, K, and L, respectively. Scale bar=50μm. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Colocalization and interaction of epithelial protein lost in neoplasm (EPLIN) with paxillin in mesangial cells. (a) Merged image (A) of cultured human mesangial cell costained for EPLIN (C), F-actin (D), and paxillin (E). Higher-magnification image corresponding to the square in A is shown in B. Scale bar=50μm. (b) Lysates of cultured human mesangial cells were immunoprecipitated with mouse IgG or mouse anti-paxillin IgG, and immunoprecipitates were analyzed by western blotting with the indicated antibodies. (c) In situ proximity ligation assay (PLA) in cultured human mesangial cells. Mouse anti-paxillin mAb and rabbit anti-EPLIN antibodies were combined with secondary PLA probes (Olink Bioscience). The interaction events are visible as red dots. Scale bar=20μm. (d) Merged image (A) of human kidney section was costained for paxillin (C), EPLIN (D), and GLEPP1 (E). Higher-magnification image corresponding to the square in A is shown in B. Scale bar=20μm. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Localization of epithelial protein lost in neoplasm (EPLIN) at focal adhesions and its interaction with paxillin regulated by the platelet-derived growth factor (PDGF)–extracellular signal-regulated kinase (ERK) axis. (a) Serum-starved mesangial cells were stimulated with 50ng/ml PDGF for 30min. Cells were costained for EPLIN, F-actin, and paxillin. Scale bar=50 μm. (b) Serum-starved mesangial cells were stimulated with 50ng/ml PDGF for 30min or pretreated with U0126. Cells were immunostained for EPLIN and paxillin. (c) HEK293T cells were co-transfected with myc-tagged EPLIN α, green fluorescent protein (GFP)-tagged paxillin, constitutively active mitogen-activated protein kinase (MEK), and an empty expression vector. Cell lysates and anti-myc immunoprecipitates were analyzed by western blotting with the indicated antibodies. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Epithelial protein lost in neoplasm (EPLIN) depletion facilitates platelet-derived growth factor (PDGF)–induced focal adhesion disassembly. (a) Cultured mesangial cells were transfected with control small interfering RNA (siRNA) (A) and siRNAs #1 (B) and #2 (C) for EPLIN. Three days after transfection, cells were stained with anti-EPLIN antibody (A330-103A, Bethyl) and 4,6-diamidino-2-phenylindole (DAPI). Scale bar=50μm. (b) Western blot analysis of mesangial cell lysates using anti-EPLIN antibody (A300-225A, Bethyl). (c, d) Cultured mesangial cells transfected with control siRNA or EPLIN siRNA #2 were stimulated with 50ng/ml PDGF for 0 or 30min. Cells were fixed and stained for F-actin and paxillin. The degrees of peripheral membrane ruffling and disassembly of peripheral focal adhesions were categorized into four classes, as illustrated in C. Three blinded observers evaluated the ruffling formations and focal adhesions using the classifications. *P<0.05 using Student’s t-test. Data are shown as the means±s.d. of three independent experiments. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Epithelial protein lost in neoplasm (EPLIN) inhibits platelet-derived growth factor (PDGF)–induced mesangial cell migration. Two days after transfection with control small interfering RNA (siRNA) or EPLIN siRNAs, mesangial cells were subjected to a modified Boyden chamber assay with 30ng/ml PDGF in the lower chamber. *P<0.001 using Student’s t-test. Data are shown as the means±s.d. of three independent experiments. Kidney International 2014 86, 548-557DOI: (10.1038/ki.2014.85) Copyright © 2014 International Society of Nephrology Terms and Conditions