FIGURE 10.

Inhibition of calcineurin reduces IL-1β mRNA stability and protein levels in response to lipotoxic stimulation. A–C, pMACs were stimulated with palmitate-LPS ± 5 μm CSA, and mRNA expression of IL-1β (A), TNFα (B), and NLRP3 (C) was assessed at the indicated time points by qRT-PCR. D, the expression of pro-IL-1β protein was determined by Western blotting from macrophages stimulated with palmitate (palm)-LPS for 16 h in the presence of vehicle (veh) or CSA. E, quantification of pro-IL-1β protein expression normalized to tubulin. F, pMACs were activated with palmitate-LPS, and vehicle, CSA, or Z-YVAD (50 μm) was added simultaneously with the stimulation (sim) or delayed by 12 h. IL-1β release was quantified by ELISA, and the levels are reported as % vehicle (where 100% means no inhibition). G, macrophages were treated with palmitate-LPS ± CSA or pretreated with LPS (for 2 h) followed by 5 mm ATP ± CSA for 60 min. IL-1β release was quantified by ELISA. H, pMACs were treated with palmitate-LPS for 4 h after which actinomycin D (ActD, 5 μm) was added to the cells in the presence (black bars) or absence (white bars) of CSA (5 μm). IL-1β and TNFα mRNA levels were quantified at the indicated time points by qRT-PCR. Bar graphs indicate the mean ± S.E. for a minimum of three experiments, each performed in triplicate. *, p < 0.05 for vehicle versus inhibitor or early versus delayed stimulations.