Pe annexin v 7 aad kit

ART was purchased from Guilin Pharmaceutical (Shanghai, China), dissolved in dimethyl sulfoxide (DMSO) and stored at − 80 °C. The concentration of DMSO in all experiments was 0.1%. The FAK inhibitor PF562271 was purchased from Selleck Chemicals (Houston, TX, USA). CCK-8 was obtained from BioSharp (Wuhan, China). The PE-Annexin V/7-AAD kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). TRIzol reagent was purchased form Invitrogen/Thermo Fisher Scientific (Carlsbad, CA, USA). Prime Script RT reagent Kit and SYBR® Premix Ex Taq™ were purchased from Takara (Tokyo, Japan). The BCA protein assay kit was from Pierce/Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies against FAK (# 3285), p-FAK (# 3283), Akt (# 4691), p-Akt (# 4060), GSK-3β (# 9315), p-GSK-3β (# 9336), β-catenin (# 9562), Bax (# 2772), Bcl-2 (# 3498) and GAPDH (# 2118) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-rabbit secondary antibody IgG H + L (# 5151) was purchased from Cell Signaling Technology Inc.

Once HeLa and HEK 293 cells reached the confluency of 70%, they were treated with recombinant p28 peptide at the concentrations of 0.5, 1, and 2 µM. After 24 hours, the apoptosis level in the treated cells was evaluated using PE-AnnexinV/7AAD kit (BD Biosciences; San Jose, California, USA) according to the manufacturer’s instructions.‎

Cells were seeded into 6-well plates (2 × 10⁵ cells/well) and incubated with different concentrations of ART (0, 12.5, 25 and 50 μg/ml) at 37 °C for 24 h. Floating and adherent cells were collected, washed twice with cold phosphate-buffered saline (PBS), and assessed for apoptosis using the PE-Annexin V/7-AAD kit, followed by flow cytometry (BD Biosciences, San Jose, CA, USA). Cells staining positive for PE-annexin V and negative for 7-AAD were considered to be in early apoptosis, while those positive for both PE-annexin V and 7-AAD were considered as late apoptotic cells.