Our gratitude to professor Jianguo Cao (Department of Pharmaceutical Science, Hunan Normal University, Changsha, Hunan, China) for helpful advices and revision of the manuscript. Notes Chen J, Zhong J, Liu Y, et?al. (5\FU) or NVP\BZE235 resulted in a synergistic antitumor effect via PUMA induction in HCT\116 cells. VB1 significantly suppressed the cell proliferation of wild\type (WT) HCT\116 and LoVo cells in vitro and tumor growth in vivo. The results indicate that p53/PUMA/Bax axis plays a critical role in VB1\induced apoptosis and VB1 may have valuable clinical applications in cancer therapy as a novel anticancer agent used alone or in combination with other chemotherapeutic drugs. mice (Vital River Lab Animal Technology Co. Ltd., Beijing, China, Certificate No. SYXK2013\0001) were housed in sterile microisolator cages (five per cage) with free access to water and food ad libitum. All animal experiments were carried out followed the protocols approved by Central South University Animal Use and Care Committee (Changsha, Hunan, China). 1??106 cells were injected s.c. into both flanks of mice. Mice were administered by i.p. injection of VB1 40?mg/kg every other day for 2?weeks when tumors were measurable, whereas the same Balofloxacin volumes of normal saline (NS) were used as vehicle control. Mice were euthanized when tumors reached ~1.0?cm3 (1000?mg) in size. Tissues of tumors were collected and examined. The protein was extracted using a Total Protein Extraction kit (Chemicon International, Temecula, CA, USA) and analyzed by Western Blotting. 2.5. Analysis of cell viability and apoptosis Cells were cultured in 96\well microplate at a density of 5??103 cells/well for 24?hours. Cell viability was assessed with Balofloxacin Cell Counting Kit\8 (CCK\8) (7Sea Biotech, Shanghai, China) at indicated time post\treatment following the manufacturer’s instructions. The absorbance value at 450?nm (OD450) was read with a 96\well plate reader (DG5032, Hua Dong, Nanjing, China), to determine the cell viability. For colony formation assay, cells were cultured in 6\well plate at a density of 5??104 cells/well for 24?hours. The cells were then treated with indicated concentrations of drugs and medium (control) for 24?hours. Medium was changed Balofloxacin every 2?days. Colonies were visualized with crystal violet staining at Day 14. For analysis of apoptosis by nuclear staining, cells were cultured in a 3.5\cm dish, rinsed with phosphate\buffered saline (PBS) twice and then 500?L DMEM containing Rabbit Polyclonal to POLE4 5?g Hoechst 33342 was added into the plates and incubated for 15?minutes in an incubator. Apoptosis was assessed through microscopic visualization of condensed chromatin and micronucleation. Apoptosis indices were calculated as the percentage of apoptotic cells among one hundred cells in a randomly selected portion. The positive rate of apoptotic cells was calculated by GD\10.0 image analysis system. 2.6. Flow cytometry HCT\116 and LoVo cells were suspended in 1??106 cells/mL, and 5?L of Annexin V and propidium iodide staining solution were added to 300?L of the cell suspension. After the cells were incubated at room temperature for 15?min in the dark, stained cells were assayed and quantified using a FACSort Flow Cytometer (Beckman Coulter, Brea, CA, USA). Cell debris was excluded from the analysis by an appropriate forward light scatter threshold setting. Compensation was used wherever necessary. 2.7. Co\immunoprecipitation HCT\116 cells were cultured in 10\cm dish at a density of 8??106 cells/dish for 24?hours. Cells were then treated with 10?mol/L VB1 for 24?hours, and the same volume of medium was used as control. Cells were harvested and lysed with lysis buffer (25?mmol/L HEPES, 125?mmol/L K\acetate, 2.5?mmol/L\acetate, 2?mmol/L DTT, 0.4% Tx\100, 2X Phosphatase Inhibitor, Protease Inhibitor, Na Orthovanadate 400?mol/L, pH?=?7.2). To detect the interaction between PUMA and Bax, anti\PUMA antibodies (~4?L) were firstly added to 400?L cell lysates and mixed on a.