Recombinant human latent tgf β1 protein

The knockdown or overexpression of Arkadia was confirmed by western blot analysis after transfecting MCs with mouse-derived Arkadia siRNA (Integrated DNA Technologies, Inc., Coralville, IA, USA) or with an overexpression plasmid (GFP-Arkadia/RNF111; Addgene plasmid #112228; http://n2t.net/addgene:112228; RRID: Addgene_112228), respectively. Briefly, cells were seeded in six-well plates and transfected with Arkadia siRNA/plasmid or a negative control mixed with Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) for up to 24 h according to the manufacturer's instructions. The cells with knockdown or overexpression of Arkadia were then treated with recombinant human latent TGF-β1 protein (20 ng/mL; R&D Systems) under low (5.5 mM) or high (35 mM) D-glucose conditions for up to 6 h, and subsequently harvested for western blot analysis.

Mouse mesangial cells (MCs) and tubular epithelial cells (mTECs) were from Prof. Hui-Yao Lan's lab and cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% FBS at 37 °C under an atmosphere of 5% CO₂. Cells were seeded in six-well plates at a density of 2×10⁵ cells/well, and then cultured in FBS-free medium for 24 h and stimulated with recombinant human latent TGF-β1 protein (20 ng/ml; R&D Systems, Minneapolis, MN, USA) or latency-associated peptide (15 ng/ml, R&D Systems, Minneapolis, MN, USA) under low (5.5 mM) or high (35 mM) D-glucose conditions for up to 6 h; D-mannitol (29.5 mM) was used as an osmotic control. The cells were harvested to detect the levels of fibronectin, Collagen I, Collagen IV, IL-1β, phospho-NF-κB/p65, phospho-Smad3, Arkadia, and Smad7 protein, as well as the mRNA expression of fibronectin, TNF-α, and Arkadia. All experiments were repeated independently at least three times.