Figure 3. Clathrin-mediated endocytosis regulates MDP-induced NOD2-dependent NF-κ B activation in HEK293T cells.

(A) HEK293T cells were transiently transfected with a NF-κB luciferase reporter plasmid along with a control β-galactosidase plasmid (control), and NOD2 expression plasmid when indicated (NOD2), in triplicate. The cells were treated with medium alone or with medium containing 100 ng/ml MDP (MDP) for 16 h, in the absence (untreated) or presence of mannans, Poly I or CPZ. Luciferase and β-galactosidase activity was measured in cell lysates and values normalized for transfection efficiency (nRLU). (B) Luciferase reporter gene assays were performed as in (A). Transfected cells were treated with media alone (unst.) or with media containing either 100 ng/ml MDP or 10 ng/ml human TNF-α for 16 h, alone (untreated) or in the presence of CPZ. Results are presented as the mean of triplicate wells ± SD and correspond to one representative experiment of three independent experiments. *, p < 0.01 between untreated and CPZ-treated samples (Nod2+MDP). (C) HEK293T cells were transfected with a control plasmid (shRNA -) or two shRNA plasmids targeting the clathrin heavy chain gene (CHC) (shRNA1 and shRNA2). Cell lysates were prepared and blotted with indicated antibodies. (D, E) HEK293T cells were transiently transfected with a control plasmid (shRNA -) or with shRNA plasmids (shRNA1 and shRNA2). Luciferase reporter gene assays were performed as in A. Results are presented as the mean of triplicate wells ± SD and correspond to one representative experiment of three independent experiments. *, p < 0.05 between control shRNA- plasmid and shRNA1 or shRNA2 constructs in MDP stimulated cultures.