Different inhibitory effects of HDAC inhibitors on IL-1β, IL-8, and TNF-α secretion. (A) Monocytes were stimulated for 3 hours with LPS in the absence (lanes 1 and 2) or presence of TSA (1 μM, lane 3), HC-toxin (HC-T; 1 μM, lane 4), SAHA (0.5 μM, lane 5), or tubacin (Tub; 20 μM, lane 6) and then exposed to 1 mM ATP for 15 minutes (lanes 2-6). Supernatants and cell lysates were analyzed by Western blotting for the presence of IL-1β. (B) HDAC inhibitors do not affect ATP-induced K⁺ efflux. Monocytes as in panel A, untreated or exposed to 1 mM ATP for 20 minutes, were analyzed for K⁺ intracellular content. K⁺ depletion is expressed as percent of intracellular K⁺ in LPS-stimulated monocytes (control cells). (C) HDAC inhibitors do not affect ATP-induced [Ca²⁺]i rise. Monocytes as in panel A were loaded with Fura-2-AM and exposed to 1 mM ATP. Results are the mean of fluorescence of at least 50 cells in each experiment monitored for 15 minutes, and are expressed as increase in [Ca²⁺]i above non-ATP stimulated cells (Δ[Ca²⁺]i). (D) Monocytes were cultured for 18 hours with LPS and supernatants were assayed for the presence of IL-1β (□), IL-8 (), or TNF-α (▪) by ELISA. Results are expressed as percent of secretion by LPS-stimulated monocytes (control cells). One experiment of 3 performed is shown. In this experiment, 18 hours' secretion by 0.7 × 10⁶ LPS-stimulated monocytes was: 9.5 ± 0.1 ng/mL for IL-1β; 305 ± 2 ng/mL for IL-8; 6.1 ± 0.3 ng/mL for TNF-α.