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. 2012 May 22;20(8):1540–1549. doi: 10.1038/mt.2012.102

Figure 3.

Figure 3

Cell-permeable p18INK4c induces apoptosis. (a) Biomarker expression. HCT116 cells were treated with the indicated proteins (10 µmol/l) for hour and cell extracts were immunoblotted with antibodies against phospho-Rb (Ser780), phospho-p53 (Ser15), phospho-ATM (Ser1981), p21Waf1/Cip1 and β-actin. (b) Kinetics of apoptosis induction. HCT116 cells, treated with 10 µmol/l HM103E, Hp18, and HM103p18 for the indicated times, were stained with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide and analyzed by flow cytometry. The percentage of annexin V positive cells (±SD of triplicate experiments) is plotted. *Significant differences (P < 0.05) between HM103p18 as compared to vehicle, HM103E or Hp18 as determined by one-tailed Student's t-tests. (c) Kinetics of Caspase-3 activation. HCT116 cells were treated as before with 10 µmol/l HM103E, Hp18, and HM103p18. Caspase-3 assays measured production (±SD of triplicate experiments) of cleaved Caspase-3 substrate (Clonetech, Mountain View, CA) by increased absorbance at 405 nm after 1 hour at 37 °C. *Significant differences (P < 0.05) between HM103p18 and other samples as determined by one-tailed Student's t-tests.