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. Author manuscript; available in PMC: 2022 Jan 5.
Published in final edited form as: Cell Rep. 2020 Mar 17;30(11):3743–3754.e6. doi: 10.1016/j.celrep.2020.02.025

Figure 4. Exogenous ASC Can Replace Endogenous ASC in ASC-Deficient Cells and Induce ASC-Dependent Signaling Pathways.

Figure 4.

(A and B) Immunocytochemical detection (A) and quantification (B) of caspase-1 activity in ASC-deficient macrophages treated with or without exogenous ASC.

(C) The caspase-1 subunit p20 was detected by IB analysis in precipitated supernatants from ASC-deficient microglia.

(D–F) IL-1β levels in conditioned medium of ASC-deficient primary microglia after exposure to different concentrations of exogenous ASC: c1 = 0.22 μM, c2 = 0.66 μM, c3 = 1.75 μM (D); IL-1β levels in conditioned medium of ASC-deficient primary microglia treated with ASC, Aβ, or ASC-Aβ composites for 12 and 24 h (E); and IL-1β levels in conditioned medium of primary WT microglia and ASC-deficient microglia treated with ASC-Aβ composites for 12 h (F).

(G and H) IB analysis and quantification (G) of ASC monomers, dimers, and ASC-mCherry in ASC-deficient microglia cell lysates (H) (n = 4).

(I) Immunostaining of ASC-deficient macrophages treated with exogenous ASC and stained for ASC internalization and ASC speck formation after ASC-Aβ composites treatment (arrowheads).

(J and K) CoIP of ASC and Aβ in ASC-deficient microglia cell lysates. IP: ASC. IB: ASC (AL-177) (J), IB: Aβ (82E1) and input controls (K). Empty wells and wells containing a lysate control negative for both proteins are not displayed. Vertical lines in blots indicate spliced sections.

Data were collected from three independent experiments (n = 3) with three technical replicates perassay (N = 9) (D–F). All graphs are presented as mean ± SEM and were analyzed by two-way ANOVA (D and E), one-way ANOVA in conjunction with Tukey’s test (B, C, and G), or unpaired t test (F). Levels of significance are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Asterisks indicate significance between groups connected by lines; plus symbols indicate significance between ASC-Aβ composites and volume-equal buffer control-treated groups. Images were taken at 40× magnification. Scale bars, 10 μm (A and I).