Figure 9

LiCl induces apoptosis in MT450 breast cancer cells. (A) MT450 rat mammary tumour cells were plated in 96-well plates at a density of 1 × 10³ cells per well. 24 hours after plating, LiCl and alsterpaullone were added to the indicated concentrations. 72 hours after drug addition, the relative amount of viable cells was determined using the MTT-assay. Mean values and standard deviations of three independent experiments were calculated and plotted. The relative number of cells in the absence of drug was set to 100% (D: DMSO control). Statistical analysis was performed for alterations in relative cell proliferation in comparison to mock-treated cells (*: P < 0.05; **: P < 0.01). (B) 1 × 10³ MT450 cells were mixed with medium and methylcellulose 4000 containing the indicated doses of LiCl. After two weeks, the number of colonies was determined. The graph shows mean values and standard deviation of three independent experiments. The number of colonies in the absence of LiCl was set to 1. (C) MT450 cells were incubated for 36 hours with the indicated doses of LiCl. 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 a loading control. (D) MT450 cells were incubated with the indicated doses of LiCl for 36 hours. 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, or against PCNA for a loading control. (E) MT450 cells were incubated with 10 or 20 mM LiCl for 24 or 48 hours or left untreated for a control. Fragmented DNA was isolated and separated on a 1.4% agarose gel.