Figure 1.

MyD88-dependent inflammatory response stimulated by LPS couples to tubular damage without affecting Fn14/SCF^Fbxw7α cascade under hypoxia. (A) MTT assay measuring cell viability of HK-2 cells exposed to LPS at the indicated concentrations with or without hypoxia in the presence of 20 µM zVAD-FMK and siRNA targeting control, Fn14 or caspase-8 transfection, respectively. N: normoxia. H: hypoxia. Ctrl.siRNA: control siRNA. C-8.siRNA: caspase-8 siRNA. Experiments were performed three times and data are expressed as mean ± s.d. *P<0.05 versus H, one-way ANOVA, post hoc comparisons, Tukey's test. (B and C) Representative pictures (B) and quantification (C) from Hoechst33342 and PI double-staining assay of HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence of 20µM zVAD-FMK and siRNA targeting Fn14 or caspase-8 transfection, respectively. D: DMSO, L: LPS. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05, **P<0.01, one-way ANOVA, post hoc comparisons, Tukey's test. (D) Western blotting analyses detecting the abundance of Fn14 (top panel) and caspase-8 (bottom panel) protein in HK-2 cells with Fn14 or caspase-8 siRNA transfection. (E) ELISA assay for IL-6 secretion from HK-2 cell cultures exposed to LPS at the indicated concentrations with hypoxia or 10 µM ST2825 treatment or MyD88 siRNA transfection. Data are expressed as mean ± s.d. of at least three experiments. *P<0.05, **P<0.01, ***P<0.001 versus N, one-way ANOVA, post hoc comparisons, Tukey's test. Insert: Western blotting analyses comparing the levels of MyD88 protein in HK-2 cells with or without MyD88 siRNA transfection. (F) Coimmunoprecipitation assay evaluating the interaction between MyD88 and TLR4, IRAK1 and TRAF6 in LPS-stimulated HK-2 cells with or without hypoxia. IP: immunoprecipitation.