(A) CD11c^– ATMs purified by FACS from epiWAT SVF of 7- to 9-week-old TSOD mice were plated in a 24-well dish and incubated with or without the recombinant mature form of mouse IL-1β (10 ng/mL) in the absence of FBS. The PI3K inhibitor wortmannin (100 nM) was added 10 minutes before the addition of IL-1β (see third chart). After a 24-hour culture, GDF3 protein concentrations in the supernatants (sups) (first chart, n = 3) and GDF3 mRNA levels in the cell pellets (second chart, n = 5; third chart, n = 3) were measured by real-time ELISA and RT-PCR, respectively. (B) EpiWAT (0.5 g) isolated from 7-week-old TSOD mice were incubated with or without rrhGDF3 (400 ng/mL) in the presence of 20% FBS. The NLRP3 inhibitor, MCC950 (10 μM), was also added into some wells 30 minutes before the addition of GDF3. After a 24-hour culture, the mRNA levels in the SVF isolated from approximately 0.35 g of epiWAT were determined (n = 3). (C) TSOD mice were treated with ALK7 mAb (10 mg/kg) or PBS for 6 weeks, as described in Figure 1. The expression levels of S100A8/A9 heterodimer protein and those of S100A8 and S100A9 mRNA in epiWAT were determined by ELISA and real-time RT-PCR, respectively (n = 5). (D) CD11c^– ATMs (1.0 × 10⁶ cells/24-well dish) isolated from epiWAT SVF of 7- to 8-week-old TSOD mice were cultured with or without S100A8 or S100A8/A9 at 10 μg/mL. After a 24-hour culture, the GDF3 protein and the mRNA levels were measured (n = 3), as described in A and B. *P < 0.05, **P < 0.01 by t test. ^#P < 0.05, ^##P < 0.01 by 1-way ANOVA.