Figure 5. Effect of KDT-11 on cell cycle progression.

The cells indicated were treated with vehicle (0.1% DMSO) or KDT-11 (10 μM) for 24 hours. They were then incubated with 10 μM EdU for 90 minutes to label S-phase cells, followed by labeling the newly incorporated DNA with Alexa647-azide and staining with propidium iodide for total DNA content. The cells were then analyzed by flow cytometry. Bar graphs represent the mean of triplicate readings, error bars represent standard deviation of these readings. (A) MM1.R cell cycle profile (B) HEK293T cell cycle profile (C) SK-MEL-5 cell cycle profile. The data show that KDT-11 blocks entry into S phase rapidly and almost completely in the two cancer cell lines but has almost no effect on the fraction of HEK293T cells in S phase. See Supplemental Figure 5 for additional SK-MEL-5 data. (D) MM.1R cells were treated with KDT-11 (10 μM) or vehicle (DMSO) for 24 hours. P21 and p27 levels (relative to GAPDH) were then assessed by SDS-PAGE and Western blotting. Quantitation of the Western blots using ImageJ. Error bars represent the standard deviation of triplicate readings. Protein was quantified using ImageJ analysis and Excel. A two-tailed Student’s t-test was performed between DMSO and KDT-11. p - value for p27 levels = 0.0001. p-value for p21 = 0.0249.