Figure 7

LiCl induces FasL. (A) U2OS cells were treated with the indicated concentrations of LiCl for 24 hours. RNA was prepared and the amount of FasL RNA was determined by qRT-PCR. Mean values and standard deviation of 2^ΔCT values of 3 independent experiments were plotted. The data for untreated cells were set to 1. (B) U2OS and H1299 cells were treated with 50 mM LiCl and harvested at the indicated time points. RNA was prepared and the amount of FasL RNA was determined by qRT-PCR. Mean values of 2^ΔCT values of 2 (H1299) and 3 (U2OS) independent experiments were plotted. The data for untreated cells were set to 1. (C) HCT were transfected with siRNA targeted against TNF-α or with a control siRNA. 20 hours after transfection, Nok-1 antibody was added at a dilution of 1:500 where indicated. Four hours later, 50 mM LiCl were added and the cells were incubated for a further 36 hours. Cells were lysed and 50 μg of protein were separated on a 15% SDS-PAGE gel. Proteins were transferred onto a PVDF membrane and probed with antibodies directed against Caspase-3, or against PCNA for a control. (D) U2OS cells were transfected with siRNA targeted against TNF-α or with a control siRNA, or left untransfected for control. 20 hours after transfection, Nok-1 antibody was added where indicated at a dilution of 1:500. Four hours later, 25 mM LiCl were added and the cells were incubated for a further 36 hours. Relative numbers of viable cells were determined using the MTT-assay.