Cl caspase3

Metformin and 5-Fu were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Cell Cycle Detection Kit, Cell Counting Kit-8, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit, TUNEL Apoptosis Detection Kit and BCA protein assay kit were purchased from KeyGen Biotech Co., Ltd (Nanjing, China).
Antibodies against caspase3, cleaved-caspase3 (cl-caspase3), PARP, cleaved-PARP (cl-PARP), Bax, Bcl-2, p-YAP, YAP, p-AMPK, AMPK, Akt, p-Akt, PTEN, HIF-1a, p21, c-myc and GAPDH were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), the P-gp and MRP1 antibodies were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA), and the cyclin D1 antibody was purchased from Abgent (San Diego, CA, USA).

Protein was extracted from lung tissue or AECs-II using RIPA lysis buffer (Beyotime), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose membranes (Millipore, Boston, MA, USA), and then immunoblotted with primary antibodies against HO-1, STAT3, p-STAT3 (Tyr705), p-Akt (Ser473), Akt (all from Cell Signaling Technology Inc., Danvers, MA, USA), Bcl-2, Bcl-xL, Bax, caspase-3, cl-caspase-3 (all from Santa Cruz), and β-actin (Sigma); followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Sigma). Detection was performed with an enhanced chemiluminescence assay kit (Pierce, Rockford, IL, USA). All experiments were performed in triplicate.