Akt antibody

The human colon cancer cell lines HT-29, HCT-116, and HCT8/S11 were maintained in DMEM (Hyclone, Cramlington, UK) supplemented with antibiotics (penicillin: 50 U/mL; streptomycin: 50 µg/mL) (Hyclone, Cramlington, UK) and with 10% fetal bovine serum (FBS; Biowest, Nouaille, France). In all experiments, the cell viability was higher than 99% using trypan blue dye exclusion. Frondoside A, oxaliplatin, and 5-FU were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA). Antibodies to phospho-AKT, phospho-p44/42 MAPK (ERK1/2), cleaved caspase-3 (Asp175), and cleaved poly (ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology (Cell Signaling, Beverly, MA, USA). The AKT antibody was obtained from Abcam (Abcam, Cambridge, UK). The antibody to phospho-histone H2AX was obtained from Millipore (Millipore, Hayward, CA, USA). Antibodies to ERK2 and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Melanoma tissue and paracancerous tissue were collected and fixed in paraformaldehyde at room temperature, rinsed with PBS for 3 times, and embedded in paraffin and sectioned. The sections were dewaxed with xylene-ethanol solution, followed by sodium citrate buffer (pH 6.0) for antigen repair, and rinsed with PBS for 3 times. They were put in 30% hydrogen peroxide solution and reacted for 30 minutes in dark at room temperature. They were rinsed with PBS for 3 times. They were blocked with 3% BSA at room temperature for 20 min, then, Axl antibody (Abcam) or Akt antibody (Abcam) was added and incubated overnight in the refrigerator at 4°C. They were rinsed with PBS for 3 times, and HRP-labeled secondary antibody (Abcam) was added at the appropriate concentration and incubated at room temperature for 30 min. They were rinsed with PBS 3 times, 5 minutes each time. DAB chromo-developing solution (Solarbio®, Life Science) was added for staining for 2 min, and sections were rinsed with running water. The hematoxylin solution was redyed for 2 min and rinsed with PBS for 15 min. The slides were dehydrated in ethanol, then, 80% glycerin was added to the slides, and the cover glass was sealed. Finally, microscope observation was performed and photographs were taken (magnification: ×200).

The human liver cancer cell line HepG2 was originally obtained from the American Type Culture Collection (Manassas, VA, USA). Hep3B and Huh7 cells were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids, and antibiotics. In all experiments using the trypan blue dye exclusion method, the cell survival rate was higher than 99%. Oxaliplatin and doxorubicin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Phospho-AKT and phospho-ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cleaved caspase-3 (Asp175) and cleaved poly (ADP-ribose) polymerase (PARP) were purchased from Bioworld (Nanjing, Jiangsu, China), AKT antibody from Abcam, phospho-histone H2AX antibody from Millipore, and the ERK2 and β-actin antibodies from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Diaporine was extracted from endophytic fungi; prior to each experiment, it was dissolved in dimethyl sulfoxide (DMSO).