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. 2020 Sep 15;11:561948. doi: 10.3389/fimmu.2020.561948

Figure 2.

Figure 2

Enhanced caspase-11-mediated signaling pathways in Gate-16−/−Gabarap−/− macrophages. (A) Western blot analysis of cleaved caspase-1 (p20) and caspase-11 (p26) in cell supernatants, and of pro-caspase-1 and pro-caspase-11 and Actin (loading control) in cell extracts of wild-type (WT) and Gate-16−/−Gabarap−/− BMDMs primed with Pam3CSK4 followed by LPS transfection for 16 h. (B) Fluorescence confocal microscopy of wild-type (WT), Casp11−/−, and Gate-16−/−Gabarap−/− BMDMs primed with Pam3CSK4 followed by transfection of LPS for 6 h and immunostained for ASC (green). The nucleus was stained with DAPI (blue). Squares show cells with ASC speck. Arrow indicates ASC speck itself. Scale bars correspond to 20 μm. (C) Percentage of ASC speck-positive wild-type (WT), Casp11−/− and Gate-16−/−Gabarap−/− BMDMs, as quantified by counting at least 100 cells per coverslip by confocal microscopy. (D) Oligomerization assay of ASC in cell extracts of wild-type (WT) and Gate-16−/−Gabarap−/− BMDMs primed with Pam3CSK4 followed by LPS transfection for 6 h was analyzed by western blot. (E,F) Caspase-1 Pam3CSK4 primed wild-type (WT), Casp11−/− and Gate-16−/−Gabarap−/− BMDMs BMDMs were transfected with LPS for 4 h and added with FAM-FLICA to detect activated caspase-1 by flow cytometry (E) or by analysis for the quantification (F). The data are representative of three independent experiments (A,C,E) and are combined data of more than three independent experiments (C,F). *P < 0.05, ***P < 0.001, two-tailed t-test.