Cell viability and cell migration assays

Cell viability was assessed using the CellTiter 96 Non-Radioactive Cell Proliferation Assay MTT (Sigma-Aldrich). 8×10³ cells were seeded on 96-well plates in DMEM-F12 medium. After stimulation, cell survival was quantified by adding MTT tetrazolium solution according to the manufacturer’s protocol. Absorbance was measured at 540 nm in an ELx800TM Absorbance Microplate Reader (BIOTek).

VSMC migration was examined using a 6.5 mm Transwell chamber with an 8 μm pore size (Corning Costar Inc., New York, NY, USA). 3×10⁴ cells were serum-starved in the upper compartment of each chamber for 16 h; inhibitors were added to the upper chamber and the stimuli (AngII and/or IL-1β) were added to the bottom chamber. Cells were allowed to migrate 24 h and cells of the upper membrane surface were removed with a cotton swab. Then, the membrane was washed with PBS and migrating cells were fixed in 4% (v/v) paraformaldehyde. Migration values were determined by counting three fields per chamber after staining the migrated cells with Hoechst 33342 or DAPI (Life Technologies).

Cell migration and proliferation in response to physical damage was determined using a wound healing assay. VSMC monolayers were wounded using a sterile 10 μl pipette tip. Phase contrast images were taken immediately after wounding and at 24 h post-stimulation using a Nikon microscope (Tokyo, Japan) connected to a video camera (Sony Corporation, Tokyo, Japan). To measure migration, wound area was quantified using Adobe Photoshop and expressed as fold increase of wound closure in control cells.