Figure 1.

AQP3 mRNA and protein expression are decreased following treatment with TNF α and this effect can be prevented by pretreatment with inhibitors of MEK/ERK and NF‐κB signaling pathways. AQP3 mRNA expression was assessed by real‐time RT‐PCR in HT‐29 cells treated with TNF α (25 ng/mL) for the times indicated (A) or for a total of 12 h in a concentration‐dependent fashion as indicated (B). Statistical significance was assessed in comparison to untreated samples using an unpaired t‐test (A) or ANOVA (B) (*P < 0.05, **P < 0.01, ***P < 0.001). n = 3–4. A time course experiment was performed to assess 1,2,3‐³H‐glycerol uptake into untransfected and AQP3 shRNA‐transfected HT‐29 cells and demonstrated significantly decreased uptake at the 20, 30, and 60 min time points (n = 4, *P < 0.05, ***P < 0.001) (C). Uptake of 1,2,3‐³H‐glycerol was assessed in untransfected, scrambled shRNA and AQP3 shRNA‐expressing HT‐29 cells under control and TNF α (25 ng/mL)‐treated conditions (D). Total cellular ³H uptake was assessed using liquid scintillation counting (D, left panel), cell counts were performed by manual hemacytometer on a parallel set of cells (D, middle panel) and data were normalized to determine the total ³H uptake per cell (D, right panel). Statistical significance was assessed by ANOVA and a Tukey's post hoc test (n = 6; *P < 0.05; **P < 0.01; ***P < 0.001). MAPK inhibitor U0126 significantly abrogated the TNF α‐induced downregulation of AQP3 mRNA expression at the 12 h time point as shown by real‐time RT‐PCR (E), with significance assessed by ANOVA and a Newman–Keuls post hoc test (lower right; n = 5; *P < 0.05). TNF α, 25 ng/mL; DMSO ¹, Volume of DMSO equivalent to 10 μmol/L U0126. NF‐κB inhibitor BAY‐11‐7082 abrogated the TNF α‐induced downregulation of AQP3 mRNA expression at the 12 h time point as shown by real‐time RT‐PCR (F), with significance assessed by ANOVA and a Newman–Keuls post hoc test (lower right; n = 4–5). TNF α, 25 ng/mL; DMSO ¹, Volume of DMSO equivalent to 30 μmol/L BAY‐11‐7082.