Isolation and Characterization of Mouse Aortic Smooth Muscle Cells

Mouse aortic SMCs were isolated as the following steps: Briefly, mouse aortas were separated and the peri‐aortic adipose tissue was cleaned as much as could be to avoid possible contamination. The adventitia was digested with 2 mg mL⁻¹ collagenase type‐II (LS004177; Worthington Biochemical Corp.) at 37 °C in water bath for 10 min. Aortas were then cut into small pieces and digested in 1 mg mL⁻¹ collagenase type‐II at 37 °C for 45 min with shaking every 15 min. After digestion, 10 mL of DMEM were added containing 10% fetal bovine serum (FBS) to stop. After centrifugation, cells were resuspended with fresh media (DMEM with 10% FBS and 1% penicillin and streptomycin) and plated on a 60 mm culture dish. Media was changed every 2 d. When the SMCs became confluent, the cells were transferred to new cell culture plates. Cells will be ready to use after passage 4. SMCs were pretreated with or without EOS lysates from WT mice at 1 × 10⁶ EOS mL⁻¹, ILC2 lysates from WT Il13^−/−and Il5^−/− mice at 2 × 10⁴ ILC2 mL⁻¹, and different concentrations of mouse recombinant IL5 (10, 100, and 200 ng mL⁻¹, 405‐ML, R&D Systems, Minneapolis, MN) and mouse recombinant IL13 (10 ng mL⁻¹, 575904, BioLegend) for 24 h. SMCs were then incubated with 10 ng mL⁻¹ TGF‐β, 20% FBS, and PDTC respectively. Immunoblot analysis tested the expression of p‐Smad2 (1:1000, 3108S, Cell Signaling Technology), p‐Smad3 (1:1000, ab52903, Abcam), Smad2 (1:1000, 5339S, Cell Signaling Technology), Smad3 (1:500, 9523S, Cell Signaling Technology), p‐AKT (1:1000, 9271S, Cell Signaling Technology), cleaved caspase‐3 (1:500, 9661L, Cell Signaling Technology), and GAPDH (1:3000, 2118S, Cell Signaling Technology). Immunoblots were quantified by gel density analysis using the Image J software.