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. 2022 Nov 12;11(22):3579. doi: 10.3390/cells11223579

Figure 5.

Figure 5

TLR4/NF-κB signaling cascade and ROS abundance are responsible for S100A8-induced NLRP3 inflammasome-dependent pyroptotic death in macrophages. (A) Western blot analysis of p65, p-p65, IKKα, and p-IKKα expression in THP-1 macrophages treated with GST-rhS100A8 or GST for 0, 30, 60 or 120 min. The protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes relative to the 0 min group (right panel). (B–E) THP-1 macrophages were exposed to 5 µg/mL rhS100A8 with or without 1 h of BAY 11-7082, TAK-242 or FPS-ZM1 pretreatment. The qRT-PCR analysis was performed to detect the mRNA levels of NLRP3 (B), pro-IL-1β (C), and pro-IL-18 (D). Western blot analysis was used to determine the protein expression of NLRP3, GSDMD, GSDMD P30, pro-IL-1β, mature IL-1β, and cleaved caspase-1 (E). The protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes relative to the GST group (right panel). (F) THP-1 macrophages were pretreated with TAK-242 or FPS-ZM1 for 1 h and then exposed to 5 µg/mL of rhS100A8. Western blot analysis was used to determine the expression of p-p65 and p-IKKα. The protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes relative to the GST group (right panel). (G) Flow cytometry analysis of ROS levels in THP-1 macrophages treated with rhS100A8 for 6 h. (H) THP-1 macrophages were exposed to 5 µg/mL of rhS100A8 with or without 1 h of DPI pretreatment. Protein expression levels of NLRP3, GSDMD, GSDMD P30, pro-IL-1β, and mature IL-1β were determined by Western blot. The protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes relative to the GST group (right panel); ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.