Figure 10

Suppression of Toll-like receptor 4 (TLR4) with TLR4 antibody or siRNA inhibited Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation induced by hypoxic stress in primary microglia and BV-2 cells. Immunofluorescence images showing NF-κB/p65 expression in primary microglia (A) and BV-2 cells (B). NF-κB/p65 is mainly localized in the cytoplasm of control microglia colocalized with lectin (Aa, Ad, Ag) and control siRNA-transfected BV-2 cells (Ba, Bd). The expression is intensely augmented both in the cytoplasm and nucleus after hypoxic treatment in both cell groups (Ae, Ah; Bd, Be). NF-κB/p65 immunoreactivity in the cytoplasm and nucleus after hypoxic exposure in primary microglia with TLR4 Ab (Af, Ai) is noticeably suppressed. A similar decrease is observed in TLR4 siRNA-transfected BV-2 cells subjected to hypoxic exposure (Bc, Bf). (C) Western blot analysis of NF-κB/p65 protein expression in BV-2 cells of different groups. The upper panel shows specific bands of NF-κB/p65 (65 kDa) and β-actin (43 kDa) and the lower panel bar graph shows significant changes in the optical density of different groups (given as fold change of control BV-2 cells). Note the NF-κB/p65 protein expression, which is increased after hypoxic exposure in control siRNA-transfected BV-2 cells, is significantly decreased after hypoxic exposure in TLR4-transfected groups. (D) ELISA analysis of phospho-NF-kB/p65 in different groups of BV-2 cells showing a similar trend as in C. *P <0.05 and **P <0.01. The values represent the mean ± SD in triplicate. Scale bars in A and B = 20 μm.