Guava cell nexin reagent

Dulbecco’s Modified Eagle Medium (DMEM), trypsin and penicillin-streptomycin were supplied from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS), RIPA buffer, protease inhibitors and Coomassie Plus™ Protein Assay Reagent were obtained from Thermo Scientific Company (Waltham, MA, USA). Guava Cell Nexin Reagent was purchased from Guava Technologies (Darmstadt, Germany). Nitrocellulose membrane and ECL reagent were supplied from GE Healthcare (Little Chalfont, UK). Gelatin, propidium iodide (PI) and 3-Methyladenine (3-MA) were obtained from Sigma (St. Louis, MO, USA). Antibodies specific to COX-2, and cyclin -D1 were purchased from Millipore (Darmstadt, Germany). Antibodies specific to β-actin, uPA, urokinase-type plasminogen activator receptor (uPAR), poly (ADP-ribose) polymerase (PARP), and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for the detection of ERK1/2, p38, JNK, c-Jun, and p65 were purchased from Cell Signaling Technology (Danvers, MA, USA). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA).

A549 cells were incubated with or without various concentrations of PRFR (0–50 µg/mL) for 24 h. The cell suspension was then prepared and stained with annexin V and 7-amino actinomycin D (Guava Cell Nexin Reagent; Guava Technologies) for 20 min according to the Guava Nexin Assay protocol. Apoptosis was determined on a Guava PCA Instrument using Guava® Viacount^TM Software (Merck Ltd., Darmstadt, Germany).