Research Article

Human Embryonic Stem Cells Acquire Responsiveness to TRAIL upon Exposure to Cisplatin

Figure 2

Cisplatin sensitizes hESC towards TRAIL-induced apoptosis. Cells were cultured in the presence of 1 μM and 2 μM doses of cisplatin for 24 hours and then they were treated with recombinant human TRAIL (200 ng/ml) for 24 hours. (a) Morphology of hESC upon the given treatments as observed by light microscopy (10x, bar 50 μm). Both CCTL12 and CCTL14 lines of hESC were used ( for CCTL12; for CCTL14); a representative picture is shown. (b) Graphs showing cell death incidence as determined by flow cytometric analysis after double staining with Annexin-V and propidium iodide. At least 10,000 cells were analyzed per sample. Living (Annexin-/PI-), apoptotic (Annexin+/PI-), necrotic (Annexin-/PI+), and secondary necrotic cells (Annexin+/PI+) are reported as a percentage of the total cell count. The CCTL14 line of hESC was used (). (c) Activation of the caspase cascade and PARP cleavage upon the given treatments as determined by western blot. Inactive full length caspases include procaspase 3 (~35 kDa), procaspase 8 (~55 kDa), and procaspase 10 (~60 kDa); their active forms include cleaved caspase 3 (~17/12 kDa), cleaved caspase 8 (~18 kDa), and cleaved caspase 10 (~20 kDa). Staining with 0.1% amidoblack was used to evaluate the protein loading. Both CCTL12 and CCTL14 lines of hESC were used ().