Genegenome

To confirm the cell cycle effect, Western blotting was used to investigate the expression change of the cell cycle proteins p27, cyclinD3 and CDK2. HL60 cells (1 × 10⁶ cells/well of 6-well plates) were treated with HAA₂₀₂₀ (250 nM, 500 nM and 1000 nM), dinaciclib (5 nM, 10 nM and 20 nM) and their combination for 6 h (Figure.7). The total proteins were isolated after the complete lysis of cells by a lysis buffer. The concentration of the total protein was determined by the Bradford method. The loading protein samples were electrophoresed on a polyacrylamide gel and transferred to a membrane. The membrane was incubated with p27, cyclinD3 and CDK2 antibodies (Cell signalling) for 2 h at room temperature and the secondary antibody GAPDH for 1 h at room temperature. The immunoreactivity was visualized by chemiluminescence using horseradish peroxidase (HRP)-conjugated secondary antibodies, and their image was detected by a scanner (GeneGenome, Syngene BioImaging) [61 (link)].

Western blotting was used to determine the expression change of ERK, p-ERK, AKT, p-AKT, EGFR, and p21. HT29 or HT29-5FU cells (0.5–1 × 10⁶ cells/well) were treated with vehicle, 5FU (0.25 µM), HAA₂₀₂₀ (3 µM), or their combination for 72 h. Lysis buffer was used to isolate the total proteins, and their concentration was determined by the Bradford method. The loading protein samples were electrophoresed on a polyacrylamide gel and transferred to a membrane. The membrane was incubated with ERK, p-ERK, AKT, p-AKT, EGFR, and p21 antibodies (Cell signaling, Boston, MA, USA) for 2 h at room temperature and the secondary antibody GAPDH for 1 h at room temperature. The immunoreactivity was visualized by chemiluminescence using horseradish peroxidase (HRP)-conjugated secondary antibodies, and their image was detected by a scanner (GeneGenome, Syngene BioImaging) [64 (link)].

Identification of the expression change of cell cycle proteins (AKT, pAKT, CyclinD1, and GAPDH) was confirmed by immunoblotting assay. MCF7 cells (1 × 10⁶ cells/well of 6 well plate) were treated with LY, TAM, and their combinations for 24 h. Lysis buffer was used to isolate total proteins. The Bradford Method was used to determine the concentration of total proteins, which were electrophoresed using a polyacrylamide gel and transferred to membrane. The membrane was incubated with AKT, pAKT cyclin D1 antibodies (Cell signalling) for 2 h at room temperature and secondary antibody GAPDH for 1 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used to visualize the immunoreactivity by chemiluminescence, and images were captured by a scanner (GeneGenome, Syngene Bioimaging, Cambridge, CB4 1TF, United Kingdom) [51 (link)].