Association of cytosolic PML with caspase-1 activation and IL-1β production. (A) The level of cytosolic PML in macrophages was increased by LPS stimulation. BMDM cells were primed with LPS for 4 hours, followed by treatment with alum crystal (400 μg/mL) for 1 hour. The amounts of PML in cytosolic extracts (40 μg) and nuclear extracts (25 μg) were determined. The exposure time during development was 4 seconds for cytosolic extracts and 1 second for nuclear extracts. Lamin B and GAPDH were markers for nuclear and cytoplasmic extracts, respectively. (B) Leptomycin B (LMB) decreased the cytosolic presence of PML. LMB (10 ng/mL) was included in the LPS priming stage, and the contents of cytoplasmic and nuclear PML in BMDM determined as in (A). (C-D) LMB treatment suppressed caspase-1 activation and IL-1β production. BMDMs were treated with LPS for 1 to 2 hours, followed by addition of LMB for 2 to 3 hours (4 hours total LPS priming time). Next, alum crystals were added and incubated for an additional 4 hours. Culture supernatants were isolated and cell lysates were prepared as indicated. The levels of IL-1β in supernatants were measured by ELISA (C) and immunoblotting (D). The contents of NLRP3, pro–IL-1β, procaspase-1, ASC, and caspase-1 in cell lysates or supernatants were assessed by Western blotting (D). Representative results of 3 independent experiments are shown. **P < .01 for paired t-test.