P pi3k

A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, Manassas). Dulbecco’s Modified Eagle’s medium (DMEM), fetal calf serum, and pancreatin were purchased from Invitrogen (Invitrogen, Waltham, MA, USA). Bicinchoninic acid (BCA) protein quantification kit was purchased from Pierce (PIERCE, Illinois, USA). Matrigel^TM basement membrane matrix, Transwell chamber and Matrigel and FACS Vantage SE flow cytometer were purchased from BD Biosciences (BD Biosciences, NJ, USA). Antibodies for Survivin, PCNA, Caspase3, Caspase9, PI3K, p-PI3K, AKT and p-AKT were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). HRP-labeled goat anti-rat secondary antibody was purchased from Santa Cruz (Santa Cruz Biotechnology Dallas, TX, USA). Apoptosis detection kit (Annexin PE/7-AAD) was purchased from Kaiji (Nanjing Kaiji Biological Co., Ltd., China). High speed refrigerated centrifuge was from Beckman (Beckman, Coulter, CA, USA). CO₂ incubator was from Thermo (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoresis chamber and Trans-Blot Turbo were purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). Gel imaging system GDS-800 was purchased from UVP (1UVP, LLC, Upland, CA, USA).

Lysed by radioimmunoprecipitation assay buffer (Takara, Tokyo, Japan), the cell protein (30 µg) was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 1% skim milk powder. After being probed with the primary antibody, the PVDF membrane was re-probed with the secondary antibody, followed by development with enhanced chemiluminescent and exposure under the Chemiluminescence Imaging System (Bio-Rad, Hercules, CA, USA). ImageJ software was utilized for evaluating the optical density of protein bands. The primary antibodies included TIMP3 (ab276134, 1:1000, Abcam, Cambridge, UK), PI3K (#4249, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), AKT (#9272, 1:1000, Cell Signaling Technologies), GAPDH (#5174, 1:800, Cell Signaling Technologies), p-PI3K (1:100, Sigma-Aldrich), p-AKT (ab38449, 1:800, Abcam), and goat anti-rabbit secondary antibody (ab205718, 1:5000, Abcam).