Fig. 3

Knockdown of MEKK3 suppresses the expression of p-p65 and the secretion of pro-inflammatory cytokines in BV2 cells. a BV2 cells were treated with an increasing concentration of LPS (0.1, 0.5, and 1 μg/mL). After 12 h, cells were harvested for RNA isolation. Then, RT-qPCR analysis detected changes in the transcript level of MEKK3. b, c BV2 cells were transfected with negative control (NC) or MEKK3 siRNA (MEKK3-si). After 48 h, cells were harvested, and the MEKK3 mRNA (b) and protein expression (c) levels were evaluated using RT-qPCR and western blot analysis. d, e BV2 cells were transfected with NC or MEKK3-si for 48 h. Cells were washed with PBS and then stimulated with LPS (1 μg/mL) for 12 h. Cells were harvested. NF-κB activity was analysed by luciferase assay (d). Western blotting confirmed the protein expression of p-p65 (e). f–h BV2 cells were transfected with NC or MEKK3-si. After 48 h, cells were washed with PBS and then treated with LPS (1 μg/mL), After 12 h, the cells were harvested. The mRNA levels of pro-inflammatory cytokines TNF-α (f), iNOS (g), and IL-6 (h) were examined by RT-qPCR. GAPDH was used as a loading control for normalising the image density. The data are shown as the mean ± SE from three independent experiments. The fold change is statistically significant where *P < 0.05, **P < 0.01, and ***P < 0.001