Figure 5

TIR-1 and NSY-1 are required for responses to arsenite. (A) Western blot analysis of the phosphorylation of PMK-1 in wild-type (N2), tir-1 (tm3036), nsy-1 (ag3) and pmk-1 (km25) mutant animals before and following treatment for the indicated times with 5 mM sodium arsenite. This revealed that TIR-1 and NSY-1 are both required for the phosphorylation of PMK-1, which occurs maximally within 5 min of arsenite treatment. β-tubulin levels are shown as a loading control. (B) The basal and arsenite-induced intestinal expression of gcs-1p::gfp is reduced following tir-1 and nsy-1 RNAi treatment of prdx-2 mutant and in wild-type animals treated with arsenite. *** indicates a statistically significant difference from vector control P < 0.001; ** indicates a statistically significant difference from vector control P < 0.01 (chi² test). (C) tir-1 (tm3036) and nsy-1 (ag3) mutant animals are significantly more sensitive to 10 mM arsenite than wild-type (N2) (log-rank N2 vs tir-1, P < 0.001; N2 vs nsy-1, P = 0.004). All experiments were repeated at least twice with similar results. (D) Assessment of the viability of tir-1 RNAi and vector-control-treated wild-type and pmk-1 mutant animals on 7.5 mM arsenite revealed that tir-1 RNAi increases the arsenite-sensitivity of wild-type (N2) but not pmk-1 (km25) animals. Statistical analysis: wild-type vector vs tir-1 RNAi, P <0.001; pmk-1 vector vs tir-1 RNAi, P = 0.32; wild-type vector vs pmk-1 vector, P < 0.001. GFP, green fluorescent protein; RNAi, RNA interference.