human fstl1  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

doi: 10.1016/j.jbc.2022.102010

Figure Lengend Snippet: FSTL1 enhances canonical Wnt signaling. A , both mFSTL1 and hFSTL1 proteins augmented mWnt3a mediated 8× TOPflash luciferase reporter activity in HEK293T cells. Cells were transfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids. Cells then were treated with and without mWnt3a protein (20 ng/ml) in the absence or the presence of mFSTL1 or hFSTL1 (200 ng/ml) before luciferase assay was performed. B , both mFSTL1 and hFSTL1 proteins promoted mWnt3a-mediated 8× TOPflash luciferase reporter activity in NRK-49F cells. Cells were transfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids. Cells then were treated with and without mWnt3a protein (20 ng/ml) in the presence of increasing amounts of mFSTL1 protein ( left panel ) or treated with and without mWnt3a protein (10 ng/ml) in the absence or the presence of hFSTL1 protein ( right panel ), before luciferase assay was performed. C , transfected FSTL1 promoted mWnt3a-induced 8× TOPflash luciferase reporter activity in a dose-dependent manner. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids and increasing doses of FSTL1-HA plasmid. Cells then were treated with or without mWnt3a protein (15 ng/ml) before luciferase assay was performed ( right panel ). The left panel shows FSTL1 expression in cell culture medium. Albumin was used as loading control. D , transfected FSTL1 potentiated transfected Wnt3a-induced 8× TOPflash luciferase reporter activity. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids, either alone or with FSTL1-HA plasmid, in the absence or the presence of Wnt3a plasmid. About 24 h after transfection, cells were serum starved for 16 h before luciferase assay was performed. E and F , siRNA-mediated inhibition of FSTL1 attenuated Wnt3a-induced 8× TOPflash luciferase reporter activity. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids in the presence of scramble (Ctrl) or two Fstl1 siRNA sequences (siFstl1, 110 nM) either separately ( E ) or in combination ( F ). About 24 h after transfection, cells were incubated with or without Wnt3a protein (30 ng/ml) in serum-free medium for 16 h before cells were harvested for luciferase assay. G , the effects of siRNA-mediated inhibition of FSTL1 on Wnt3a signaling were rescued by FSTL1 overexpression. NRK-49F cells were cotransfected with 8× TOPflash luciferase reporter and pRL-TK Renilla plasmids in the presence of scramble (Ctrl) or Fstl1 siRNAs and with or without FSTL1-HA plasmid. About 24 h after transfection, cells were incubated with or without Wnt3a protein in serum-free medium for 16 h before cells were harvested for luciferase assay. n = 3 to 4. ∗∗∗ p < 0.001. FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; hFSTL1, human FSTL1; mFSTL1, mouse FSTL1; NRK-49F, normal rat kidney 49 fibroblast cell.

Article Snippet: Approximately 24 h after transfection, the medium was replaced with serum-free medium, with or without mFSTL1 (catalog no.: 1738-FN; R&D Systems) or hFSTL1 (catalog no.: 1694-FN; R&D Systems) protein, in the absence or the presence of mWNT3a protein (catalog no.: 1324-WN; R&D Systems).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Control, Inhibition, Incubation, Over Expression