Figure 2.
BAL-0028 specifically inhibits NLRP3 inflammasome formation. (A) Western blot for caspase-1 and IL-1β cleavage and NLRP3 expression from PMA-differentiated THP-1 cells stimulated with LPS and nigericin in the presence of BAL-0028 or MCC950 (both 500 nM). (B) Comparison of BAL-0028 and MCC950 effects on ASC speck formation assessed by fluorescence microscopy in PMA-differentiated THP-1 ASC-GFP cells stimulated with LPS and nigericin. (C) Comparison of BAL-0028 and MCC950 effects on ASC speck formation assessed by flow cytometry in HEK293T ASC-BFP cells transfected with human NLRP3 and stimulated with nigericin. (D and E) Effect of BAL-0028 and MCC950 on ASC speck formation assessed by fluorescence microscopy using an anti-ASC antibody in iMacs. (F and G) Effects of BAL-0028, MCC950, and VX-765 on IL-1β release from PMA-differentiated THP-1 cells stimulated with (F) LPS and transfected with poly(dA:dT) or (G) protective antigen (PA) and Lfn-needle protein. (H) Effects of BAL-0028, MCC950, and VX-765 on IL-18 release from human keratinocytes stimulated with talabostat. (A) Representative blots from N = 2 independent experiments. (B) Average ± SEM % ASC-GFP speck-positive cells from N = 2 independent experiments performed in triplicate. (C) Average ± SEM change in nigericin-induced ASC specks normalized to cells without compound treatment from N = 3–4 independent experiments. (D and F–H) Graph symbols show average values relative to vehicle control from independent experiments performed in triplicate (indicated by different symbols) ± SEM. N = 2 (D, G, and H) and N = 3 (F). (E) Representative images from D, scale bar is 100 μM. Source data are available for this figure: SourceData F2.

BAL-0028 specifically inhibits NLRP3 inflammasome formation. (A) Western blot for caspase-1 and IL-1β cleavage and NLRP3 expression from PMA-differentiated THP-1 cells stimulated with LPS and nigericin in the presence of BAL-0028 or MCC950 (both 500 nM). (B) Comparison of BAL-0028 and MCC950 effects on ASC speck formation assessed by fluorescence microscopy in PMA-differentiated THP-1 ASC-GFP cells stimulated with LPS and nigericin. (C) Comparison of BAL-0028 and MCC950 effects on ASC speck formation assessed by flow cytometry in HEK293T ASC-BFP cells transfected with human NLRP3 and stimulated with nigericin. (D and E) Effect of BAL-0028 and MCC950 on ASC speck formation assessed by fluorescence microscopy using an anti-ASC antibody in iMacs. (F and G) Effects of BAL-0028, MCC950, and VX-765 on IL-1β release from PMA-differentiated THP-1 cells stimulated with (F) LPS and transfected with poly(dA:dT) or (G) protective antigen (PA) and Lfn-needle protein. (H) Effects of BAL-0028, MCC950, and VX-765 on IL-18 release from human keratinocytes stimulated with talabostat. (A) Representative blots from N = 2 independent experiments. (B) Average ± SEM % ASC-GFP speck-positive cells from N = 2 independent experiments performed in triplicate. (C) Average ± SEM change in nigericin-induced ASC specks normalized to cells without compound treatment from N = 3–4 independent experiments. (D and F–H) Graph symbols show average values relative to vehicle control from independent experiments performed in triplicate (indicated by different symbols) ± SEM. N = 2 (D, G, and H) and N = 3 (F). (E) Representative images from D, scale bar is 100 μM. Source data are available for this figure: SourceData F2.

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