nescence fluorometer using specific excitation and emission wavelengths for each peptide. Enzyme activity was determined as initial velocities and expressed as relative intensity /min/mg protein. The experiments were performed in triplicates for each experimental condition. ” Cell viability and apoptosis analysis by Annexin V/7-AAD staining HGT-1 cells were trypsinized and harvested as described above. After washes in PBS, cells were stained with Annexin V-FITC and 7-AAD according to the manufacturer’s instructions. Cells were then analyzed by flow cytometry. Cell analysis and data processing were carried out on 10 000 cells using the same device and software as above. Three independent experiments were performed. Reactive oxygen species detection Production of ROS was assessed by flow cytometry using dihydroethidium probe to detect superoxide anion as previously described. HGT-1 cells were harvested as described above, washed in PBS, and labeled with 5 mM DHE. Fluorescence emission of oxidized DHE was analyzed by flow cytometry for 10 000 cells. Menadione was used “7069713 as a positive control. Statistical analysis Results are expressed as the mean 6 standard deviation. Significance was determined by Student’s t test for paired samples. The significance is shown as follows: P,0.05; P,0.01. March 2012 | Volume 7 | Issue 3 | e31892 Fermented Milk-Induced Apoptosis of HGT-1 Cells Supporting Information bacterial metabolite or by camptothecin is detected by 3 methods. HGT-1 were treated as in Dairy propionibacteria strains and their origin. Acknowledgments We would like to thank Dr. C. Laboisse for the generous gift of the HGT-1 cell line. M.B. Maillard is acknowledged for her technical assistance in HPLC analysis, M.N. Madec for confocal microscopy and J. Fauquant for milk 
ultrafiltrate preparation. Author Halofuginone site Contributions Conceived and designed the experiments: FJC MTDB LC GJ. Performed the experiments: FJC SJL. Analyzed the data: FJC SJL. Contributed reagents/materials/analysis tools: FJC SJL. Wrote the paper: FJC SJL MTDB LC GJ. 5. 6. 11 March 2012 | Volume 7 | Issue 3 | e31892 Fermented Milk-Induced Apoptosis of HGT-1 Cells 12. Bordonaro M, Lazarova DL, Sartorelli AC Butyrate and Wnt signaling A possible solution to the puzzle of dietary fiber and colon cancer risk Cell Cycle 7: 11781183. 13. Matthews GM, Howarth GS, Butler RN Short-chain fatty acid modulation of apoptosis in the Kato III human gastric carcinoma cell line. Canc Biol Ther 6: 10511057. 14. Tsai LC, Hung MW, Chang GG, Chang TC Apoptosis induced by the sodium butyrate in human gastric cancer TMK-1 cells. Anticancer Res 20: 24412448. 15. Yan J, Xu YH Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest. World J Gastroenterol 9: 660664. 16. Elmore S Apoptosis: A review of programmed cell death. Toxicol Pathol 35: 495516. 17. Kurosaka K, Takahashi M, Watanabe N, Kobayashi Y Silent cleanup of very early apoptotic cells by macrophages. J Immunol 171: 46724679. 18. Savill J, Fadok V Corpse clearance defines the meaning of cell death. Nature 407: 784788. 19. Jan G, Belzacq AS, Haouzi D, Rouault A, Metivier D, et al. Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria. Cell Death Differ 9: 179188. 20. Lan A, Lagadic-Gossmann D, Lemaire C, Brenner C, Jan G Acidic extracellular pH shifts colorectal cancer cell death from apoptosis to necrosis upon exposure to propionate and ace
