Cilengitide

Live Cell Imaging solution, Fluo4-AM, CellTracker Deep Red, pHrodo iFL Red STP Ester, NucBlue live cell nuclear stain, NucGreen dead cell nuclear stain, and LIVE/DEAD fixable aqua stain were obtained from Invitrogen. DAPI was from Sigma, and paraformaldehyde (4% in PBS) was obtained from Alfa Aesar. Human annexin V protein was from BD Biosciences, human C5a protein from Peprotech, human TGFβ1 from Miltenyi Biotec, and recombinant human IFNγ and recombinant truncated human vitronectin from Gibco. Cytochalasin D was from Cayman Chemicals, Bafilomycin A1 from Abcam, and Jasplakinolide from Santa Cruz. PSB0739, MRS2179, MRS2211, and cilengitide were all obtained from Tocris. ADP, BSA, and crystal violet were from Sigma.

The rat neonatal H9c2 cardiac myoblast cell line was obtained from the Korea Cell Line Bank (Seoul, Korea). The cells were cultured in Dulbecco’s modified Eagles medium (DMEM)/high glucose with 10% fetal bovine serum and 1% penicillin-streptomycin (Corning/Mediatech, Inc., Manassas, VA, USA,).
DOX was purchased from Tocris Bioscience (Cat#2252, Bristol, UK). LY294002 (Cat#1130), the PI3K inhibitors, cilengitide (Cat#5870), and losartan potassium (Cat#3798), the integrin inhibitors α_vβ₃ and α_vβ₅, and the angiotensin II type 1 receptor (AT1R) inhibitors respectively, were purchased from Tocris Bioscience. The ILK inhibitor Cpd22 (Cat#407331) was obtained from EMD Millipore Corp. (Darmstadt, Germany). The survivin inhibitor YM-155 was purchased from MedChem Express (Cat#HY-10194, Monmouth Junction, NJ, USA).

Recombinant suPAR was purchased from R&D Systems (Minneapolis, MN, USA) and was added to cell culture media at a final concentration of 10 ng/mL. FMLF and cilengitide were obtained from Tocris (Minneapolis, MN, USA). Both were used at a final concentration of 1 µM. Pertussis toxin (PTX) was obtained from Sigma-Aldrich (Saint Louis, MO, USA). WRW4 was obtained from Abcam (Boston, MA, USA). Agonists were applied for 24 h or for 60 min. Nearly all previous studies on suPAR signaling in podocytes have utilized a 24 h exposure [9 (link),20 (link),21 (link)]. By contrast, GPCR signaling through G proteins (as opposed to arrestins) almost always operates over a much shorter time scale often in the range of seconds to minutes. We utilized 60 min exposures to allow sufficient time for ROS accumulation in the cytosol.