Fig. 6

Inflammasome and gasdermin D-independent release of HMGB1 in response to Eh. a, b BMDMs from WT and Caspase1^−/− mice were stimulated with Eh for different time points at 20:1 ratio and with LPS + nigericin (LPS 100 ng/ml, nigericin 10 μM), an activator of NLRP3 inflammasome that leads to pyroptosis. c, d WT and ASC-deficient THP-1 macrophages were stimulated with Eh for different time points and with LPS + nigericin (LPS 100 ng/ml, nigericin 10 μM). e, f WT and NLRP3 CRISPR/Cas9-KO THP-1 macrophages were stimulated with Eh for different time points and with LPS + nigericin (LPS 100 ng/ml, nigericin 10 μM). Equal amounts of cell culture supernatants (SNs) were loaded onto the SDS-PAGE gel to detect HMGB1 level. g, h WT and GSDMD CRISPR/Cas9-KO THP-1 macrophages were stimulated with Eh for different time points and with LPS + nigericin (LPS 100 ng/ml, nigericin 10 μM). HMGB1 was detected in the supernatant by western blot and quantified by densitometric analysis. i WT and GSDMD CRISPR/Cas9-KO THP-1 macrophages were stimulated with Eh for 10 min and equal amounts of protein samples were prepared for immunoprecipitation assay. Acetyl lysine antibody was used for immunoprecipitation and immunoblotted with anti-HMGB1 antibody. Data are representative of three independent experiments. Bars represent mean ± SEM.