Fig. 4

Activation of microglia induced by GRP78 via TLR4. A Quantitative results of cell viability detected by CCK-8 assay for primary microglia incubated with different concentrations of GRP78 (n = 3). Data are mean ± SEM. B–D Comparisons of IL-6 (B), TNF-α (C) and IL-1β (D) in the supernatant of primary microglia incubated with different concentrations of GRP78 (n = 3). E Changes in the morphology of primary microglia upon GRP78 treatment. F Double immunofluorescence for GRP78 (green) and TLR4 (red) of BV2 cells. G Comparisons of IL-6, TNF-α, and IL-1β in the supernatant of primary microglia upon GRP78 stimulation with or without TAK-242 pretreatment (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Tukey’s multiple comparisons test. H: Representative western blotting images showing levels of TLR4, MyD88, p-p65, p65, and β-actin. I–K Densitometric quantification of TLR4/ actin (B), MyD88/ actin (C), and p-P65/ P65 (D) in different groups (n = 3). *Comparison to control, *p < 0.05, **p < 0.01. ^#comparison to GRP78 (5 μg/ml) group, ^##p < 0.01, ^###p < 0.001. Data are mean ± SEM. n = 3. One-way ANOVA and Tukey’s multiple comparisons test