Application of MMC for 5 minutes induces apoptosis in human fibroblasts.

<p>(A) Cell viability after different treatments was detected by using the CCK-8 assay. MMC-induced growth inhibition occurred in a dose- and time-dependent manner. (B) Apoptosis analysis was assessed via Annexin V/propidium iodide double staining. Cells were exposed to 0.4 mg/ml MMC for 5 minutes and then incubated for the corresponding indicated times. Apoptosis rates were determined via flow cytometry analysis. The cells shown in the bottom right quadrant were Annexin V-FITC positive and propidium iodide negative, indicating an early stage of apoptosis. The cells in the top right quadrant stained positively for Annexin V-FITC and propidium iodide, indicating that they consisted of secondary late apoptotic/necrotic cells. The total percentage of cell death is shown in bold. Statistical analysis of the total recorded apoptotic cells was performed, and the results are shown in the bar graphs. (C) Cell cycle analysis was conducted to detect the changes in the cell cycle at different time points after MMC treatment (0.4 mg/ml, 5 minutes). (D) Cells were TUNEL stained 48 hours after MMC treatment and then observed using a fluorescence microscope. Nuclei are shown in blue, and TUNEL staining is shown in green. (E) Western blots revealed that MMC induces cleavage of caspase-3 from a size of 35 kDa to 17 kDa and the degradation of poly ADP-ribose polymerase (PARP) by the cleaved caspase-3 into 85 to 90 kDa fragments. β-actin was included as a control. Gels were run in triplicate. The histograms in panels A, B, C and D represent the mean ±SD of three independent experiments. *P<0.05, **P<0.01 versus 0 or the control group.