Fig 1: Effect of 5E5F6 mAb on PAN-induced podocyte apoptosis.A Flow cytometry analysis of apoptosis in podocytes incubated with different media and quantification of these results (n = 3). B Cell viability of podocytes was analyzed using an MTT assay following different treatments (n = 3). C Western blot assay showed the expression of ANGPTL3 at different time points in human podocytes after PAN treatment. *p < 0.05, **p < 0.01 compared with the Ctrl group. D Relative expression of ANGPTL3, BAX, Bcl2, caspase 8, cleaved caspase 9 and cleaved caspase3 in different groups (n = 3). *p < 0.05, **p < 0.01 compared with the PAN group.
Fig 2: Immunohistochemistry of ANGPTL3 in mouse renal tissue. a At the first time point, the ANGPTL3 staining in the glomerulus along the loops of the blood vessels was significantly strengthened in the WT + HF group. b Compared with the WT group, the IHC intensity scores of ANGPTL3 in the WT + HF group were significantly different at each time point. (Bars = 20 µm)
Fig 3: Lack of ANGPTL3 inhibited the decreased expression of ACTN4 in podocytes after high-fat feeding. a The staining intensity of ACTN4 is shown in the four groups; b the staining intensity gradually decreased in the WT + HF group compared to the WT group. In contrast, the staining intensity did not decline in KO + HF mice. (Bars = 20 µm)
Fig 4: In ADR-induced nephropathy mice, anti-ANGPTL3-FLD monoclonal antibody (5E5F6) preserve podocytopathy.A Schematic figure of experimental design and 5E5F6 dosing plan. B Evaluation of proteinuria at 0 to 8th weeks. Figures show individual data of each animal and the mean ± SEM of the different groups at each time point (n = 6 per group). C The expression of Angptl3 in glomeruli of mice was detected by immunohistochemistry staining. D TUNEL staining in different groups. A total of 50 glomeruli per kidney were calculated. White arrow: Both TUNEL and WT1-positive staining cells. Original magnification 400×. E Representative immunohistochemistry images against podocyte marker Wilms tumor protein1 (WT1; brown stained nuclei) for control mice, ADR nephropathy mice, and 5E5F6 mAb-treated ADR nephropathy mice (ADR + mAb) Magnification ×400. Bars = 20 µm. F Representative images of light microscopy of kidney sections of control mice, ADR nephropathy mice (ADR), and 5E5F6 mAb-treated ADR nephropathy mice (ADR + mAb), stained with periodic acid–Schiff (PAS) in 8w. Original magnification: ×400, Bar = 20 µm. Arrows indicate tuft adhesion. G Average WT1-positive cell numbers per glomeruli, the width of foot process detected by transmission electron microscopy (TEM), TUNEL-positive and WT1-positive cells per 50 glomeruli were quantified. Figures show individual data of each animal and the mean ± SEM of the different groups (60 glomeruli per group in immunobiological staining; n = 6, *p < 0.05, **p < 0.01). H Representative images of the TEM analysis show significant damage to podocyte foot processes in ADR nephropathy mice, whereas relatively normal foot processes were present in the 5E5F6 mAb-treated ADR nephropathy mice (ADR + mAb). Upper row, scale Bar 2 µm, original magnification 3400. Bottom row, scale Bar 1 µm, original magnification 6800.
Fig 5: 5E5F6 mAb reduced integrin ß3 activation and ROS production in the podocyte.A The ribbon structure of the human ANGPTL3-FLD and integrin ß3-b1 domain complex, human ANGPTL3-FLD and 5E5F6-Fab complex, human ANGPTL3-FLD, 5E5F6-Fab and integrin ß3-b1 domain based on the computer-guided modeling and docking methods. B Analysis of the activity of 5E5F6 blocking the binding of ANGPTL3-FLD to Integrin avß3 by competitive ELISA assay, the EC50 is 1.57 ug/ml. C Immunofluorescence analysis of kidney tissue in different treatment groups. Kidney tissue were stained with AP5 antibody that recognizes the active form of integrin ß3 and WT1 antibody to mark podocytes. Magnification in ×400. D Representative images showing AP5 staining in podocytes from different groups. E Fresh renal cortical tissues were obtained from the different treated mice and prepared as frozen cryostat sections for staining DCFH-DA (green), a marker of ROS. F Representative images of podocytes stained with DCFH-DA. G Average fluorescence intensity representing the level of integrin ß3 activation detected. Data represent measurements of >50 cells and are plotted as means ± SD (n = 3). H Average fluorescence intensity representing the level of DCFH-DA detected. Data represent measurements of >50 cells and are plotted as means ± SD (n = 3). *p < 0.05, **p < 0.01 compared with the PAN group.
Supplier Page from Abcam for Anti-ANGPTL3 antibody [EPR7319]