Figure 2
From: LiCl induces TNF-α and FasL production, thereby stimulating apoptosis in cancer cells
LiCl induces cell death in p53-replete and p53-deficient cells. (A) p53-positive and negative HCT116 cells were treated with 50 mM LiCl. Cells were fixed after 0, 16, 24 and 48 hours, stained with Draq5 and subjected to FACS analysis. Relative numbers of apoptotic cells and of cells in G1-, S- and G2-phase were determined. Mean values and standard deviations of three independent experiments were calculated and plotted. Statistical analysis was performed for alterations in the cell cycle and for induction of apoptosis in comparison to mock-treated cells (*: P < 0.05; **: P < 0.01; ***: P < 0.001). (B) p53-positive and negative HCT116 cells were treated with 50 mM LiCl. for 0, 16, 24 and 48 hours. Cells were stained with PI and FITC-coupled Annexin V, subjected to FACS analysis and relative numbers of apoptotic, necrotic and vital cells were determined. Mean values and standard deviations of three independent experiments were calculated and plotted. Statistical analysis was performed for alterations in the number of apoptotic, necrotic and vital cells in comparison to mock-treated cells (*: P < 0.05; **: P < 0.01). (C) HCT116 wild type and HCT116 p53-/- cells were incubated with 50 mM LiCl for the indicated time. Cell lysates were prepared and 50 μg of protein were separated on a 10% SDS-PAGE gel. Proteins were transferred onto a PVDF membrane and probed with an antibody directed against PARP, and against PCNA for loading control. (D) HCT116 wild type and HCT116 p53-/- cells were incubated with 50 mM LiCl for the indicated time. Cell lysates were prepared and 50 μg of protein were separated on a 15% SDS-PAGE gel. Proteins were transferred onto a PVDF membrane and probed with an antibody directed against Caspase-3, and against PCNA for loading control. (E) HCT116 wild type and HCT116 p53-/- cells were incubated for the indicated times with 50 mM LiCl. Fragmented DNA was isolated and separated on a 1.4% agarose gel. (F) HCT116 wild type and HCT116 p53-/- cells were incubated for the indicated times with 50 mM LiCl. The relative amount of apoptosis was determined using the Cell Death ELISA Plus kit (Roche). Mean values and standard deviation of three independent experiments were calculated and plotted. The readings for untreated cells were set to 1. Statistical analysis was performed for alterations in the number of apoptotic, necrotic and vital cells in comparison to mock-treated cells (*: P < 0.05).