Inhibition of actin polymerization prevents caspase-1 activation and IL-18 secretion from Wdr1 mutant monocytes. (a) Western blot analysis of caspase-1 in the supernatant from wild-type (WT), caspase-1^−/− (Casp1^−/−), and Wdr1^rd/rd day 4 BMDM culture. A long exposure (long exp.) is also presented for the cleaved caspase-1 (p20) band. Cells were left untreated, stimulated with 1 µg/ml LPS, or incubated with 10 µM nigericin after LPS priming. Additionally, wild-type and Wdr1^rd/rd cells were pretreated with 1 µM latrunculin-b or 1 µM colchicine for 30 min before LPS stimulation. MW, molecular weight. (b–d) Day 4 FLDMs from Wdr1^+/rd or Wdr1^xn/rd were left untreated or treated with LPS plus an increasing dose of latrunculin-b (Lat-b; b) or colchicine (Col; c) for 16 h, or with LPS in the presence of 100 µM CK-666 or 50 µM Y27632 for 48 h (d). Secreted IL-18 in the culture supernatants was measured by ELISA. (e) Day 3 wild-type or Wdr1^rd/rd BMDMs were stimulated with LPS for 24 h in the presence of the inhibitors used previously and then were fixed and stained with phalloidin to detect filamentous actin. Green, phalloidin-FITC; blue, nuclei-DAPI. Bar, 50 µm. All data are representative of two to four independent experiments; error bars represent means ± SEM (n = 3–4). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 by unpaired t test.