FIGURE 1.

HDAC8 inhibition prevents the inhibitory effects of LeTx on the expression of pro-IL-1β induced by LPS. A–F, RAW264.7 cells were treated with a sub-lethal dose of LeTx (100 ng/ml LF and 100 ng/ml PA) for 4 h and further cultured in fresh media for the time indicated. A, LeTx-exposed (day 1–3) or non-exposed cells were stimulated with LPS (100 ng/ml) for 4 h. B, cells cultured for 16–20 h after LeTx treatments were incubated with or without pan-specific HDAC inhibitor PN (1 nm), DNA methyltransferase 1 (DNMT1), inhibitor azacitidine (AZ; 200 nm), and HDAC1–3- and -11-specific inhibitor mocetinostat (MO; 150 nm) for an additional 20 h. These cells were then treated with LPS (100 ng/ml) for 4 h. C, cells were incubated with or without the HDAC8-specific inhibitor PCI for an additional 20 h and treated with LPS (100 ng/ml) for 4 h. D, cells were treated with LeTx and/or PCI (20 nm) and treated with LPS (100 ng/ml) for 3 h. Pro-IL-1β protein (upper panel) and mRNA (lower panel) levels were measured by qPCR. Data are expressed as means ± S.D. (n = 3; *, p < 0.05, Student's t test). E, RAW264.7 cells were transfected with scramble (si-Scramble) or HDAC8-targeting (si-HDAC8) siRNAs. After 10 h of transfection, cells were treated with LeTx for 20 h and stimulated with LPS for 4 h. F, cells stably transfected with pEGFP or pEGFP-HDAC8 were treated with LPS for 4 h. HDAC8-EGFP expression was measured by immunoblotting using anti-EGFP (middle panel). G–I, primary BMDMs from B57/BL6j (G) and 129SvJ (H and I) mice were treated with PCI, scramble (si-scramble) or HDAC8 targeting (si-HDAC8) siRNAs for 10 h. Cells were then treated with LeTx (100 ng/ml LF and 100 ng/ml PA) for 3 h and further cultured for 20 h in fresh culture media. (A–C and E–I) Expression of pro-IL-1β protein and MEK1 degradation in total cell lysates were analyzed by Western blot. Immunoblotting for β-actin was used as the loading control. Data are representative images of three independent experiments.