American Society of Hematology

Figure 1.

$Figure 1. Both SAHA and ITF2357 inhibit IL-1β secretion but do not affect IL-1β intracellular accumulation and targeting. (A) Monocytes were activated 4 hours with LPS in the absence (lanes 1, 2, 6, and 7) or presence of 50 nM ITF2357 (lanes 4 and 9) or 50 nM of the negative compound ITF2375 (ITF-nil, lanes 3 and 8) or 0.5 μM SAHA (lanes 5 and 10), and then exposed to 1 mM ATP for 15 minutes (lanes 2-5 and 7-10). Supernatants and cell lysates were analyzed by Western blotting for the presence of IL-1β (top panel), caspase-1 (middle panel), and cathepsin D (cath-D, bottom panel). One representative experiment out of 10 performed is shown. (B) Densitometric analyses of Western blots of mature IL-1β, caspase-1, and cathepsin D secreted following exposure to different amounts of the 3 drugs as indicated. Results are expressed as percent of inhibition of secretion plus or minus standard error (SE) of a single experiment performed in triplicate. One representative experiment of at least 5 performed is shown. Data were validated by ELISA (not shown). In 13 different donors, IL-1β secreted in 15 minutes of ATP stimulation by 0.5 × 106 cells ranged between 5 ng and 15 ng (not shown). (C,D) Monocytes were cultured 4 hours with LPS, in the absence or presence of 50 nM ITF2357 or 0.5 μM SAHA, and a postnuclear supernatant (PNS) and an endolysosome enriched fraction were obtained as described in “Materials and methods.” (C) Densitometric analyses of Western blots showing the percent of pro-IL-1β, procaspase-1, and cathepsin-D in the lysosomal fractions of monocytes activated with LPS in the absence (-) or presence of SAHA or ITF2357. (D) Western blot of a representative experiment showing endolysosomal-enriched fractions (Ly, lanes 1 and 2) and aliquots (1/10) of postnuclear supernatants (PNS, lanes 3 and 4) from monocytes exposed for 4 hours to LPS (lanes 1 and 3) or to LPS and ITF2357 (lanes 2 and 4). Filters were hybridized with anti-IL-1β (top panel), anti-caspase-1 (middle panel), anti-cathepsin D (bottom panel). For each panel, one representative experiment of at least 3 performed is shown.$

Both SAHA and ITF2357 inhibit IL-1β secretion but do not affect IL-1β intracellular accumulation and targeting. (A) Monocytes were activated 4 hours with LPS in the absence (lanes 1, 2, 6, and 7) or presence of 50 nM ITF2357 (lanes 4 and 9) or 50 nM of the negative compound ITF2375 (ITF-nil, lanes 3 and 8) or 0.5 μM SAHA (lanes 5 and 10), and then exposed to 1 mM ATP for 15 minutes (lanes 2-5 and 7-10). Supernatants and cell lysates were analyzed by Western blotting for the presence of IL-1β (top panel), caspase-1 (middle panel), and cathepsin D (cath-D, bottom panel). One representative experiment out of 10 performed is shown. (B) Densitometric analyses of Western blots of mature IL-1β, caspase-1, and cathepsin D secreted following exposure to different amounts of the 3 drugs as indicated. Results are expressed as percent of inhibition of secretion plus or minus standard error (SE) of a single experiment performed in triplicate. One representative experiment of at least 5 performed is shown. Data were validated by ELISA (not shown). In 13 different donors, IL-1β secreted in 15 minutes of ATP stimulation by 0.5 × 10⁶ cells ranged between 5 ng and 15 ng (not shown). (C,D) Monocytes were cultured 4 hours with LPS, in the absence or presence of 50 nM ITF2357 or 0.5 μM SAHA, and a postnuclear supernatant (PNS) and an endolysosome enriched fraction were obtained as described in “Materials and methods.” (C) Densitometric analyses of Western blots showing the percent of pro-IL-1β, procaspase-1, and cathepsin-D in the lysosomal fractions of monocytes activated with LPS in the absence (-) or presence of SAHA or ITF2357. (D) Western blot of a representative experiment showing endolysosomal-enriched fractions (Ly, lanes 1 and 2) and aliquots (1/10) of postnuclear supernatants (PNS, lanes 3 and 4) from monocytes exposed for 4 hours to LPS (lanes 1 and 3) or to LPS and ITF2357 (lanes 2 and 4). Filters were hybridized with anti-IL-1β (top panel), anti-caspase-1 (middle panel), anti-cathepsin D (bottom panel). For each panel, one representative experiment of at least 3 performed is shown.

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