Figure 5.

Effects of ANA on the expressions of cell cycle and cell death regulating genes. (A) Immunoblotting analyses of effects of ANA on cyclinD1 and P21 protein expression in the three types of cells. The cells were treated with ANA for the indicated time periods and the cell lysates were processed for immunoblotting with indicated antibodies. α-tubulin antibody was used as a control for equal protein loading. (B) Effects of ANA on the expressions of Bcl-XL and Bcl-2 proteins (left) and the effects of SLFN12 siRNA on the ANA-induced protein level changes of Bcl-XL and Bcl-2 (right). H4 cells were treated with or without ANA (100 nM), or with or without SLFN12 siRNA as indicate for 12 or 24 hours, and the cell lysates were processed for immunoblotting analyses using antibodies as indicated. (C and D) Effects of ANA on the expressions of TNF-α, DR4, and DR5 mRNAs and the effects of SLFN12 siRNA on the ANA-induced mRNA level changes of TNF-α, DR4, and DR5 in H4 cells. The H4 cells were treated with or without ANA (100nM), or with or without the SLFN12 siRNA as indicated for 18 h, and the cell lysates were processed and the mRNA levels of TNF-α (C), DR4, DR5 (D) were measured by quantitative RT-PCR analyses. (E) Effects of ANA on DR4 and DR5 protein expressions in the three types of cells. The cells were treated with ANA (100 nM) for indicated time periods and the cell lysates were processed for immunoblotting analyses using the indicated antibodies. Anti-tubulin antibody was used as a control for equal protein loading. (F) Effects of ANA or ANA plus SLFN12 siRNA on the activation of caspase 8 and caspase 9, protein expressions of DR4 and DR5, and cleavage of PARP. The H4 cells were treated with ANA, a combination of ANA and SLFN12 siRNA, for the indicated time periods, and the cells were processed for immunoblotting analyses using antibodies as indicated. Anti-tubulin antibody was used as a control for equal protein loading.