Figure 6

Downregulation of Toll-like receptor 4 (TLR4) after TLR4 siRNA transfection in BV-2 cells. The cells appear more ramified with long extending processes when transfected with either control siRNA or TLR4 siRNA, when compared with the non-transfected control cells under the phase-contrast microscope (A). The viability of BV-2 cells transfected with control siRNA and TLR4 siRNA is 96% and 85%, respectively, of the non-transfected control value (B). Silencing efficiencies were analyzed by reverse transcriptase (RT)-PCR (C), western blots (D) and immunofluorescence staining (E) at 24, 48 and 48 h after transfection respectively. RT-PCR analysis shows that the efficiency of siRNA-mediated suppression of TLR4 is about 88.9% compared to negative control (normalized with β-actin) (C). (D) The upper panel shows the specific band of TLR4 (95 kDa) and β-actin (43 kDa). The lower panel shows bar graphs depicting significant changes in the optical density following hypoxic exposure (given as fold change of the control siRNA transfected group). Note the remaining TLR4 protein expression in TLR4 siRNA-transfected BV-2 cells is about 38% compared to the control siRNA-transfected BV-2 cells. Immunofluorescence images show that TLR4 immunoreactivity is markedly reduced in TLR4 siRNA transfected BV-2 cells compared to negative control (E). *P <0.05 and **P <0.01. The values represent the mean ± SD in triplicate. Scale bars = 100 μm (A) and 20 μm (E).