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. Author manuscript; available in PMC: 2011 Nov 1.
Published in final edited form as: Mol Cancer Ther. 2010 Oct 26;9(11):2893–2902. doi: 10.1158/1535-7163.MCT-10-0635

Figure 4. PUMA mediates the apoptotic and anticancer activities of UCN-01 through the mitochondrial pathway.

Figure 4

HCT116 cells with different genotypes, including p21-KO, p21-KO/p53 binding site-KO (BS-KO), and p21-KO/PUMA-KO, were treated with 0–3 μM UCN-01 for 48 hr. Apoptosis was analyzed using several methods. A. Apoptosis was measured by nuclear fragmentation assay following staining with Hoechst 33258. B. Activation of caspase 3 and caspase 9 in the cells treated with 3 μM UCN-01 was analyzed by Western blotting. C. Cytosolic fractions were isolated from the cells treated with 3 μM UCN-01 and probed for cytochrome c by Western blotting. Cytochrome oxidase subunit IV (Cox IV), which is a mitochondrial marker, was analyzed as the control for fractionation. α-tubulin was used as a control for loading. D. Colony formation assay was performed by seeding a equal number of UCN-01 (2 μM)-treated p21-KO and p21-KO/PUMA-KO cells in 12-well plates, and then staining attached cells with crystal violet 14 days later. Left: Representative pictures of colonies. Right: Quantification of colony numbers. The results were averages of 3 experiments ± standard deviation.