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Figure 2. Daunorubicin induces apoptotic cell death in miPS-LLCcm cells with DNA fractionation. A: Morphology of miPS-LLCcm cells undergoing cell death. Cells were treated with daunorubicin at a concentration of 100 nmol/L. Images were captured after 12 h of treatment. Bar: 50 mm. Red arrows show dying miPS-LLCcm cells, and blue arrows show non-apoptotic cells; B: detection of apoptotic cells by annexin-V staining. miPS-LLCcm were treated with 100 nmol/L daunorubicin for 3 h, harvested, and stained with annexin-V APC. Flow cytometry was performed twice, and the representative results are shown; C: detection of oligonucleosomal DNA ladder in bulk of miPS-LLCcm and in stem cells. DNA was prepared from miPS-LLCcm bulk (left) or puromycin-treated (right) cells subsequently treated with 100 nmol/L daunorubicin for the indicated periods and subjected to agarose gel electrophoresis. Nuclear accumulation of p53 protein following daunorubicin treatment; D: miPS-LLCcm cells were treated with daunorubicin for the indicated periods. Whole cell lysates prepared and analyzed by western blotting using p53 antibody. β-actin levels were used as internal control; E: nuclear accumulation of p53 proteins at the indicated periods was fractionated and subjected to western blotting. M: Mitochondrial, C: cytoplasmic, and N: nuclear. β-tubulin and Histone H3 were detected for fractionation markers; F: expression of p53-regulated genes in daunorubicin-treated miPS-LLCcm cells. RT-qPCR was performed using cDNA prepared from cells treated with 100 nmol/L daunorubicin for the indicated periods (n = 3); G: suppression of Nanog gene expression was confirmed by RT-qPCR. Error bars in Figure 2E and F are shown as SD; H: reduction in Nanog protein levels after daunorubicin treatment. Whole-cell lysates prepared from daunorubicin-treated cells were subjected to western blot analysis