Fig 1: Potential involvement of Ca2+/Mg2+-dependent endonuclease in DNA fragmentation. A: We observed no obvious reduction in full-length ICAD levels during apoptosis of miPS-LLCcm. miPS-LLCcm cells were treated with 100 nmol/L of daunorubicin for the indicated periods and analyzed by western blotting analyses; B: detection of processed caspases in attached or floating cells; C: apoptotic cells in the presence of BAPTA-AM were detected by annexin V staining. Cells were pretreated with 10 µmol/L of BAPTA-AM for 1 h, treated with 100 nmol/L daunorubicin, and analyzed by FACS; D: Ca2+ chelator did not affect caspase levels during apoptosis of miPS-LLCcm cells; E: suppression of DNA fragmentation by treatment with a Ca2+ chelator. DNA fragmentation in the cells treated with daunorubicin in the presence of 10 µmol/L of BAPTA-AM was analyzed
Fig 2: Comparison of apoptotic features among L1210, miPS-LLCcm, and Hela cells. A: Mouse leukemia L1210 cells were treated with 1 µmol/L daunorubicin for the indicated periods. Protein levels of activated caspase-3 and its substrate ICAD were determined by western blotting; B: DNA fragmentation in L1210 cells was analyzed. Cells were treated with daunorubicin for 24 or 36 h; C: DNA fragmentation in miPS-LLCcm and L1210 cells treated with staurosporine. Cells were treated with various concentrations of staurosporine for 3 h (miPS-LLCcm) or 12 h (L1210), after which DNA fragmentation was observed; D: Western blot analysis of PARP-1 and ICAD levels in staurosporine-treated cells (3 h for miPS-LLCcm and 12 h for L1210); E: ICAD levels in Hela cells treated with 1 µmol/L daunorubicin for the indicated periods were analyzed by western blotting; F: DNA fragmentation in Hela cells treated with 1 µmol/L daunorubicin
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