Figure 4(A, B) THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times. (C, Sorted human CD14+ monocytes were electroporated with plasmids encoding nucleocapsid or control vector for 36 h and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (C). Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. D) Sorted human CD14+ monocytes were electroporated with plasmids encoding nucleocapsid or control vector for 36 h and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Otherwise, cells were stained with PI and the percentage of PI positive cells was calculated (D). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times. (E, F) GSDMD+/+ and GSDMD-/- THP-1 cells stably expressing nucleocapsid or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an ELISA assay to determine IL-1β levels (E). Cell pellets were subjected to a cell viability assay through checking cellular ATP levels (F). For E-F, Student's t-test was used. Data were shown as means±SD of three technical replicates. NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times. (G) An in vitro cleavage assay was performed by incubating GSDMD and caspase-1 p20/p10 complex with or without the presence of nucleocapsid, followed by immunoblotting with antibodies against the indicated protein tags. FLAG-GSDMD was immunoprecipitated from HEK293T cells using anti-FLAG coupled magnetic beads. His-tagged p20, p10 and nucleocapsid were purified from E. coli strain BL21. (H, I) THP-1 cells stably expressing full-length nucleocapsid or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted with antibodies against the indicated proteins (H). Cells were also stained with PI and the percentage of PI positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.