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. 2008 Sep 8;28(21):6557–6567. doi: 10.1128/MCB.01202-08

FIG. 1.

FIG. 1.

p53 positively regulates the expression of TLR3 in epithelial cells. (A) HCT116 p53+/+ and p53−/− cells were transfected with IL-8 promoter plasmid and treated with 10 μg/ml each of the indicated TLR agonists for 6 h. IL-8 promoter activity was measured 48 h after transfection of IL-8 promoter. The data shown are means ± standard errors from triplicate platings and represent three independent experiments. **, P < 0.001 versus control (Con; phosphate-buffered saline [HCT116 p53+/+]) assessed by ANOVA with Dunnett's procedure. (B) TLR3 mRNA in HCT116 p53+/+ and p53−/− cells was determined by real-time quantitative PCR. The TLR3 mRNA level was normalized to GAPDH (internal control). The data shown are means ± standard deviations from triplicate determinations from three independent experiments. *, P < 0.05 assessed by Student's t test. (C) TLR3 mRNA was examined in HCT116 p53+/+ and in HCT116 p53−/− cells untransfected or transfected with increasing amounts of p53 expression plasmid. (D) TLR3 mRNA was determined in HCT116 p53+/+ and p53−/− cells treated for 24 h with 4 or 8 μM 5-FU. For panels C and D, p21 served as a positive control. GAPDH was used as an internal control for RT-PCR analyses. (E) HCT116 p53+/+ cells were transfected with p53 siRNA (si-p53) or control siRNA duplex (si-GL2), and total RNA from these cells was isolated for analysis of TLR3 and p53 mRNA by RT-PCR. (F) The TLR3 protein level was determined in lysates of HCT116 p53+/+ and p53−/− cells by immunoprecipitation (IP) and Western blotting using anti-TLR3 antibody. Hsc70 was used as internal control. con-IgG, control IgG. (G) A549 cells were treated with 4 or 8 μM 5-FU for 24 h. (H) si-GL2 or the indicated amount of p53 siRNA duplex was transfected in A549 cells. Twenty-four hours later, RNA was extracted. (I to K) HepG2 (I), Caco2 (J), and Calu-3 (K) cells were treated with 400 or 800 μM 5-FU for 8 h. For panels G to K, total RNA was extracted and analyzed by RT-PCR for the expression of TLR3, p21 (positive control), or p53. GAPDH served as the internal control.