Fig. 4.

Inhibition of MIF does not prevent transcription or translation of IL-1β. a NF-κB luciferase activity measured in RAW-ELAM macrophages pre-treated with COR123625 (50 µM) for 1 h prior to priming with LPS (100 ng ml⁻¹) for 4 h. b Primary murine WT BMDM or c human undifferentiated THP-1 cells were left untreated, primed with LPS (100 ng ml⁻¹) alone for 4 h, or pre-treated with COR123625 (50 µM) for 1 h before the addition of LPS. Relative expression (RE—relative to 18S) of il1b mRNA was quantified using real-time PCR. Data are mean ± SEM, n = 3 independent experiments. *P < 0.05, ****P < 0.001, one-way ANOVA with a correction provided by the Tukey's multiple comparisons test. d WT BMDMs were left untreated, primed with LPS alone (100 ng ml⁻¹) for 5 h, pre-treated with COR123625 for 1 h prior to the addition of LPS, primed with LPS before inflammasome activation with nigericin (5 µM) for 1 h, or treated with COR123625 before LPS and nigericin stimulation. Western blot analysis of cellular supernatants and lysates to assess levels of NLRP3, ASC, pro-IL-1β, pro-caspase-1, mature IL-1β (p17), caspase-1 (p20), and β-actin was performed. e Densitometry was used to calculate expression of intracellular proteins shown in d. Mean expression was normalized to β-actin and expressed as relative to levels in control (untreated) samples n = 4 separate experiments (four mice)