Fig 5.

The DNMT inhibitor azacytidine and knockdown of DNMT1 by siRNAs render TIR cells sensitive to pyroptosis and enhance MLN64 expression. (A) TIR cells were cultured in the presence or absence of azacytidine (2 μM) for the indicated times. Nontreated RAW264.7 cells (WT), TIR cells, and TIR cells pretreated with azacytidine were exposed to LeTx (500 ng/ml LF and 1 μg/ml PA) for 5 h. Cell death was measured by MTT assay. (B) TIR cells were pretreated with azacytidine (2 μM) for 24 h, and MLN64 mRNA levels were analyzed by quantitative real-time PCR. (A and B) Data are expressed as means and SD (n = 3). *, P < 0.05 (Student's t test). (C to E) TIR cells were transfected with scrambled siRNA or mouse DNMT1-specific siRNA (si-DNMT1) for 48 h. (C) Cells were then treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 5 h, and cell death was measured by MTT assay. Data are expressed as means and SD (n = 3). *, P < 0.05 (Student's t test). (D) MLN64 mRNA expression levels were analyzed by quantitative real-time PCR. Data are expressed as means and SD (n = 3). *, P < 0.05 (Student's t test). (E) MLN64 expression was analyzed by Western blotting (left), and MLN64 immunoreactivity was analyzed using the NIH Image program (right). Data are expressed as means and SD (n = 3). *, P < 0.05 (Student's t test).