Cell migration assay

Fibroblast migration ability was measured using wound healing and Transwell migration assays. Briefly, fibroblasts were cultured to 60–70% confluence in 3.5-cm diameter dishes. Cells were starved of serum for 12 h, then divided into the following groups: i) Control; ii) AngII; iii) IPA-3; and iv) AngII + IPA-3 or i) NC; ii) AngII; iii) PAK1-shRNA; and iv) AngII + PAK1-shRNA for wound healing and Transwell migration assay. The multiplicity of infection of PAK1-shRNA package by lentiviral particles in the present study was difficult to determine because human adult cardiac fibroblasts can proliferate. In order to ensure the efficiency of virus interference, confluence of the cells was decreased for PAK1-shRNA silencing. In order to determine the wound area, a thin scratch (wound) was made in the central area of the dish using 1-ml pipette tips. Following washing with fibroblast medium-2 to remove detached cells, fibroblasts were cultured with fresh medium with 5% fetal bovine serum. Images of the scratch wounds were captured with a light microscope (magnification, ×4) at 0 and 24 h, and the wound width was calculated using the following formula: (T0 - T24/T0) × 100%, where T0 and T24 indicated the width of the wound area at the start time (0 h) and end point (24 h), respectively. For the Transwell migration assay, fibroblasts (density, 3×10⁴) were rehydrated in 100 µl fibroblast medium-2 supplemented with 0.1% bovine serum albumin. Following digestion and resuspension in serum-free fibroblast medium-2, fibroblasts were placed into a Costar Transwell chamber with 8.0-µm pore size membrane (Corning, Inc.). Fibroblasts were seeded into 6-well plates containing 200 µl fibroblast medium-2 supplemented with 5% fetal bovine serum at 37°C with 5% CO₂ for 12 h. Subsequently, fibroblasts on the inner side of the membrane were removed with a cotton bud; migrated cells on the outer side of the membrane were stained with 0.5% crystal violet for 10 min at room temperature, then observed with a light microscope (magnification, ×4) and counted manually.