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. 2021 Apr 7;12:649572. doi: 10.3389/fimmu.2021.649572

Figure 5.

Figure 5

Effects of PU.1 and IRF8 knockdown on NLRP3 expression and activation of the NLRP3 inflammasome in human macrophages. (A–D) THP-1 (A, D), U937 (B), or human macrophages (C) were transfected with SPI1 siRNA (siPU.1), IRF8 siRNA (siIRF8), or negative control siRNA (siNega) and incubated for 48 h. (A–C) Relative mRNA expression was determined by qPCR and normalized to expression of Gapdh mRNA. (D) Cellular protein expression was determined by western blotting. (E–G) THP-1 cells were differentiated into macrophages by stimulating with 25 ng/mL PMA for 3 h and incubation for 21 h. Cells were primed with 1 μg/mL LPS for 4 h and then stimulated with 10 μM nigericin for 0.5 h (E, F) or 2 h (G). (E) Cellular protein expression was determined by western blotting. (F) Amount of IL-1β in the culture medium was measured by ELISA. (G) Rate of dead cells was determined by flow cytometry. Data are presented as the mean + S.D. (n = 3). (A–C) *p < 0.05, two-tailed student’s t-test analysis. (F, G) *p < 0.05, Tukey–Kramer test.