Fig 2: Bid is involved in caspase-1-induced apoptosis in macrophages. a BMMs from Gsdmd-/- and Casp1-/- mice were infected with S. Typhimurium as in Fig. 1d. Bid in cell lysates was detected by Western blotting. b–e Gsdmd-/- BMMs were transfected with control siRNA or Bid siRNAs (b, c). Two days after transfection, the cells were again transfected with the same siRNAs and incubated for an additional 2 days (b, c). BMMs were prepared from Gsdmd-/- and Gsdmd-/- Bid-/- mice (d, e). The cells were then infected with S. Typhimurium as in Fig. 1d (b, d), Fig. 1c (c) or Fig. 1 b (e). Bid and cleaved caspase-3 in cell lysates were detected by western blotting (b, d). Microscopic images (c). LDH release (e). f–h RAW264.7 cells of the indicated genotypes were infected with S. Typhimurium as in Fig. 1f (f, g) or Fig. 1e (h). Western blot detection of cleaved caspase-3 and Bid in cell lysates (f, g). Microscopic images (h). In e, the graph depicts the mean ± SD of triplicate cultures. Data are from one representative of three biologically independent experiments (a–e) and three independent experiments (f–h) with similar results. Source data are provided as a Source Data file. (See also Supplementary Figs. 10 and 11.)

Fig 3: Effect of lentinan on expression of cytokine and inflammasome components. (A) BMDMs were treated with the indicated dosage of lentinan (LNT) or LPS (10 ng/mL). Expression of IL-1α, TNFα, IL-6, IL-1rn, IL-10, Pro-IL-1β, NLRP3, NLRC4, AIM2, and Pro-Casp1 mRNAs was measured by RT-PCR and the right bar graph indicates relative band density. (B) BMDMs were treated with LPS (10 ng/mL) or LNT (1 mg/mL) with/without TAK-242, TLR4 inhibitor. Indicated gene expression levels were assessed by RT-PCR. (C) BMDMs were treated with LNT purified by endotoxin removal resin (Endotoxin-removed LNT) or intact LNT as indicated. NLRP3 and Pro-IL-1β proteins were measured by immunoblotting. (D) LPS-primed BMDMs were treated with endotoxin-removed LNT with/without dsDNA. Secretion of IL-1β was measured by ELISA. All RT-PCR data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD.

Fig 4: Effect of lentinan on priming step of inflammasome activation and formation of Asc pyroptosome. (A) BMDMs were treated with lentinan (LNT) or LPS as the 1st signal, after which cells were replaced by media containing nigericin (NG, 2nd signal). Secretion of IL-1β was measured by ELISA, and Casp1 secretion and pro-IL1β expression were analyzed by immunoblotting. (B) LPS-primed BMDMs were transfected with dsDNA in the presence of LNT. Casp1 secretion and Asc pyroptosome formation were analyzed by immunoblotting using cell culture supernatants (Sup), cell lysates (Lys), and cross-linked pellets (Pellet) from whole cell lysates. (C) Recombinant human caspase-1 (rhCasp1) was incubated with its substrate (YVAD-pNA) in the presence of LNT as indicated. Bar graph presents the mean ± SD of relative fluorescence unit (RFU).

Fig 5: Effect of MB on non-canonical inflammasome activation. (A) LPS-primed BMDMs were transfected with LPS for non-canonical inflammasome activation in the presence of MB as indicated. (B) LPS-primed BMDMs were infected with E.coli (MOI = 10) in the presence of MB as indicated. Cspase-1 (Casp1) and IL-1β secretions were analyzed by immunoblotting and ELISA. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD.