Figure 5. LRRK2 kinase activity is... | Rockefeller University Press

$Figure 5. LRRK2 kinase activity is required for NLRC4 inflammasome activation. (a) LPS-primed peritoneal macrophages were treated with LRRK2 inhibitors 2 µM LRRK2–IN-1 (IN-1) or 2 µM GSK2578215A for 1 h and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were lysed with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Insoluble fractions (Insoluble + DSS) and soluble fractions were detected with antibody to ASC by immunoblot. (b and c) LPS-primed macrophages were treated with LRRK2 inhibitors either by 2 µM LRRK2–IN-1 or 2 µM GSK2578215A for 1 h and then infected with S. Typhimurium at an MOI of 100 for 2 h (b) or stimulated with 1 µg/ml LFn-flagellin and anthrax-protective antigen (PA) for 1 h (c). Cell lysates and culture supernatants (Sup) were collected and blotted with the indicated antibodies. (d and e) Reconstitution of the NLRC4 inflammasome activation in HEK293T cells: HEK293T cells were plated in six-well microplates. The cells were transfected with plasmids expressing Flag-tagged human pro–IL-1β (500 ng/well), pro–caspase-1 (300 ng/well), and NLRC4 (500 ng/well) with or without plasmid encoding Myc-tagged LRRK2 (1,500 ng/well) or its G2019S or D2017A mutants for 24 h. IL-1β cleavage was assessed by immunoblot (d) and ELISA analysis of IL-1 β level (e) in the supernatants. Data are shown as means ± SEM; ***, P < 0.001; determined by one-way ANOVA followed by Bonferroni’s post hoc test. (f) LRRK2-deficient iBMDMs were generated by CRISPR-Cas9–mediated deletion. For rescue experiments, Myc-tagged human LRRK2 WT, G2019S, or D2017A mutants were stably expressed in the LRRK2-deficient iBMDMs. These cells were primed with LPS and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were dissolved with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Insoluble fractions (Insoluble + DSS) and soluble fractions were detected with antibody to ASC by immunoblot. (g) LRRK2-deficient iBMDMs were restored with Myc-tagged human LRRK2 WT, G2019S, or D2017A mutants and then infected with S. Typhimurium at an MOI of 100 for 2 h. Cell lysates and culture supernatants were collected and blotted with the indicated antibodies. All the data are representative of three independent experiments.$

Figure 5.

LRRK2 kinase activity is required for NLRC4 inflammasome activation. (a) LPS-primed peritoneal macrophages were treated with LRRK2 inhibitors 2 µM LRRK2–IN-1 (IN-1) or 2 µM GSK2578215A for 1 h and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were lysed with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Insoluble fractions (Insoluble + DSS) and soluble fractions were detected with antibody to ASC by immunoblot. (b and c) LPS-primed macrophages were treated with LRRK2 inhibitors either by 2 µM LRRK2–IN-1 or 2 µM GSK2578215A for 1 h and then infected with S. Typhimurium at an MOI of 100 for 2 h (b) or stimulated with 1 µg/ml LFn-flagellin and anthrax-protective antigen (PA) for 1 h (c). Cell lysates and culture supernatants (Sup) were collected and blotted with the indicated antibodies. (d and e) Reconstitution of the NLRC4 inflammasome activation in HEK293T cells: HEK293T cells were plated in six-well microplates. The cells were transfected with plasmids expressing Flag-tagged human pro–IL-1β (500 ng/well), pro–caspase-1 (300 ng/well), and NLRC4 (500 ng/well) with or without plasmid encoding Myc-tagged LRRK2 (1,500 ng/well) or its G2019S or D2017A mutants for 24 h. IL-1β cleavage was assessed by immunoblot (d) and ELISA analysis of IL-1 β level (e) in the supernatants. Data are shown as means ± SEM; ***, P < 0.001; determined by one-way ANOVA followed by Bonferroni’s post hoc test. (f) LRRK2-deficient iBMDMs were generated by CRISPR-Cas9–mediated deletion. For rescue experiments, Myc-tagged human LRRK2 WT, G2019S, or D2017A mutants were stably expressed in the LRRK2-deficient iBMDMs. These cells were primed with LPS and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were dissolved with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Insoluble fractions (Insoluble + DSS) and soluble fractions were detected with antibody to ASC by immunoblot. (g) LRRK2-deficient iBMDMs were restored with Myc-tagged human LRRK2 WT, G2019S, or D2017A mutants and then infected with S. Typhimurium at an MOI of 100 for 2 h. Cell lysates and culture supernatants were collected and blotted with the indicated antibodies. All the data are representative of three independent experiments.

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