HK‐2 cells (American Type Culture Collection, Manassas, VA) and THP‐1 cells (Riken BRC) were maintained as described previously 38 , 39 . After the starvation of HK‐2 cells with SF‐hMSC‐CM (with or without TSG‐6 siRNA transfection), 10%hMSC‐CM, or serum‐free DMEM for 24 hours, the cells were exposed to 1 μM human angiotensin‐II (Ang‐II; Sigma–Aldrich) for 12 hours, 5 ng/ml recombinant human TGF‐β1 (R&D Systems, Minneapolis, MN) for 12 hours, or 10 ng/ml TGF‐β1 for 1 hours or 48 hours. Whole cell lysates were prepared in RIPA buffer (Sigma–Aldrich) and analyzed. Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) and subjected to PCR.
THP‐1 cells were differentiated into M1 macrophages by treatment with phorbol 12‐myristate 13‐acetate (160 nM; Sigma–Aldrich) for 48 hours and then stimulated for a further 24 hours with recombinant human interferon‐γ (20 ng/ml; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). A Transwell cell coculture system (Corning, Corning, NY) was used to examine the phenotypic change of macrophages by MSCs without direct cell contact. Upper inserts (pore size, 1.0 μm; Corning) with cultured SF‐hMSCs, 10%hMSCs, or SF‐hMSCs transfected with TSG‐6 siRNA (5 × 104 cells/insert) were dipped into the basal plate of cultured THP‐1 macrophages. After 48 hours, the surface antigens of THP‐1 macrophages were determined by flow cytometry.
THP‐1 cells were differentiated into M1 macrophages by treatment with phorbol 12‐myristate 13‐acetate (160 nM; Sigma–Aldrich) for 48 hours and then stimulated for a further 24 hours with recombinant human interferon‐γ (20 ng/ml; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). A Transwell cell coculture system (Corning, Corning, NY) was used to examine the phenotypic change of macrophages by MSCs without direct cell contact. Upper inserts (pore size, 1.0 μm; Corning) with cultured SF‐hMSCs, 10%hMSCs, or SF‐hMSCs transfected with TSG‐6 siRNA (5 × 104 cells/insert) were dipped into the basal plate of cultured THP‐1 macrophages. After 48 hours, the surface antigens of THP‐1 macrophages were determined by flow cytometry.