Fig. 7
Activation of ERK1/2 and NF-κB pathways by PMA partially reversed the anti-pyroptotic effect of TGF-β1 in vitro. A The protein expression of Cleaved-GSDMD, NLRP3, and IL-1β in cultured microglia under different treatments was evaluated by Western blot. B Quantitative analysis of Western blot was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. **P < 0.01, ****P < 0.0001 versus CT group; #P < 0.05, ##P < 0.01 versus LPS group; &P < 0.05, &&P < 0.01 versus LPS + TGF-β1 group. N = 6 per group. C Representative confocal images (scale bar = 30 μm) and 3D reconstructed images in high power field (scale bar = 10 μm) of Iba1 and NLRP3 in cultured microglia under different treatments. D Quantitative analysis of the volume of NLRP3+ punctures per cell in immunofluorescent staining. One-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis. ****P < 0.0001 versus CT group; # P < 0.05 versus LPS group, &P < 0.05 versus LPS + TGF-β1 group. N = 9 per group. E The level of IL-1β in microglial CM under different treatments was analyzed by ELISA. Quantitative analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001 versus CT group; ###P < 0.001 versus LPS group; &P < 0.05 versus LPS + TGF-β1 group. N = 6 per group. F The level of TNF-α and IL-6 in microglia-CM exposed to varied stimulations was measured by CBA. Quantitative analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001 versus CT group; ###P < 0.001, ####P < 0.0001 versus LPS group; &P < 0.05, n.s. no significance versus LPS + TGF-β1 group. N = 6 per group. G Representative images of bright field (scale bar = 40 μm) and immunostaining with Iba1 (scale bar = 30 μm) in cultured microglia exposed to varied treatments. H Quantitative analysis of the percent of amoeboid-like microglia in bright-field images. ****P < 0.0001 versus CT group; ##P < 0.01 versus LPS group; &P < 0.05 versus LPS + TGF-β1 group. N = 6 per group