Journal: Science Advances

Article Title: USP13 stabilizes NLRP3 to facilitate inflammasome activation by preventing TRIM31-mediated NLRP3 ubiquitination and degradation

doi: 10.1126/sciadv.adx3827

Figure Lengend Snippet: ( A to C ) Cytometric bead array (CBA) analysis of IL-1β secretion from Usp13 +/+ and Usp13 −/− BMDMs primed with LPS for indicated times before stimulation with ATP (A), nigericin (B), or MSU (C). ( D and E ) ELISA analysis of IL-1β from human THP-1 macrophages (D) or HMDMs (E) transduced with lentiviruses expressing shCtrl or shUSP13 before stimulation with LPS and ATP, nigericin, or MSU. ( F ) Immunoblot analysis of indicated proteins in culture supernatants and cell lysates from LPS-primed Usp13 +/+ and Usp13 −/− BMDMs treated with nigericin. ( G ) CBA analysis of IL-1β secretion from LPS-primed Usp13 +/+ and Usp13 −/− BMDMs before stimulation with ATP, nigericin, MSU, poly(dA:dT), or flagellin. ( H ) Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants from LPS-primed Usp13 +/+ and Usp13 −/− BMDMs treated with ATP, nigericin, MSU, poly(dA:dT), or flagellin. ( I and J ) Immunostaining of intracellular ASC specks in LPS-primed Usp13 +/+ and Usp13 −/− BMDMs treated with nigericin (I). Scale bars, 10 μm. Quantification of ASC specks from (I) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei (J). At least 100 cells from each treatment condition were quantified. ( K ) Analysis of LDH in supernatants of untreated or LPS-primed BMDMs stimulated with nigericin. ( L ) Usp13 +/+ and Usp13 −/− BMDMs transduced with control or mouse NLRP3 (K190R and K492R) mutant overexpression lentiviruses were primed with LPS and stimulated with nigericin. CBA analysis of IL-1β in the culture supernatants. Data are representative of three independent experiments [(F), (H), and (I)] or presented as the means ± SEM from three independent experiments [(A) to (E), (G), and (J) to (L)]. Statistical significance was assessed by two-way ANOVA with Bonferroni’s multiple comparisons test [(A) to (C)] or two-tailed unpaired t test [(D), (E), (G), and (J) to (L)].

Article Snippet: Ultrapure LPS (tlrl-pb5lps), Pam3CSK4 (tlrl-pms), ATP (tlrl-atp), nigericin (tlrl-nig), MSU (tlrl-msu), flagellin (tlrl-epstfla), and poly(dA:dT) (tlrl-patn-1) were purchased from Invivogen.

Techniques: Enzyme-linked Immunosorbent Assay, Transduction, Expressing, Western Blot, Immunostaining, Control, Mutagenesis, Over Expression, Two Tailed Test