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. 2022 Mar 2;165(4):460–480. doi: 10.1111/imm.13454

FIGURE 7.

FIGURE 7

Itaconate and fumarate derivatives inhibit pro‐IL‐1α cleavage in response to ionomycin stimulation. (a–b) WT BMDMs were primed with LPS (1 µg ml−1, 4 h) before treatment with (a) vehicle (DMSO), DMI, 4OI, DMF (125 µM) or MMF (500 µM) for 15 min or (b) vehicle, DMI, 4OI (125 µM), DMF (30 µM), MMF (500 µM) or calpeptin (40 µM) for 3 h. Vehicle (ethanol) or ionomycin (10 µM, 1 h) was then added to the well (n = 4). (ai, bi) Supernatants were assessed for cell death (LDH release). (aii, bii) Supernatants were probed by Western blotting for IL‐1α protein, and (aiii, biii) the ratio of mature IL‐1α:pro‐IL‐1α is shown. For densitometry, see Figure S12bi, bii, ci, cii. (c) WT BMDMs were primed with LPS (1 µg ml−1, 4 h) before treatment with vehicle (DMSO) or DMF (125 µM) for 1 h. Vehicle or ionomycin (10 µM, 5 min) was then added to the well (n = 4). (ci) Supernatants were assessed for cell death. (cii) Cell lysates were assessed for calpain activity using a fluorescent calpain substrate. (ciii) Supernatants were probed by Western blotting for IL‐1α protein, and (civ) the ratio of mature IL‐1α:pro‐IL‐1α is shown. For densitometry, see Figure S12D. (d) WT BMDMs were primed with LPS (1 µg ml−1, 4 h) before treatment with vehicle, DMI, 4OI (125 µM), DMF (30 µM), MMF (500 µM), necrostatin‐1 (50 µM) or calpeptin (40 µM) for 15 min. Vehicle or ZVAD (50 µM, 5 h) was then added to the well (n = 4). (di) Supernatants were assessed for cell death. (dii) Supernatants were probed by Western blotting for IL‐1α protein, and (diii) the ratio of mature IL‐1α:pro‐IL‐1α is shown. For densitometry, see Figure S13. Data are presented as mean ± SEM. Data were analysed using repeated‐measures one‐way (aiii, biii, diii) or two‐way (ai, bi, ci, cii, di) ANOVA with Dunnett's (vs. Veh treatment within each group) or Sidak's post hoc test, or using unpaired t‐test (civ). *p < 0.05; **p < 0.01; and ***p < 0.001. BMDMs, bone marrow‐derived macrophages; DMF, dimethyl fumarate; DMI, dimethyl itaconate; LPS, lipopolysaccharide; MMF, monomethyl fumarate; WT, wild‐type