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. 2022 Nov 7;20(11):e3001351. doi: 10.1371/journal.pbio.3001351

Fig 7. The B30.2 domain regulates Pyrin activation in human macrophages and is disrupted by FMF mutations.

Fig 7

(A) LPS-primed (10 ng/ml, 3 h or 18 h) hMDM were treated with TcdB (20 ng/ml, 1 h), then lysed and assessed for phosphorylation of Pyrin (S242), Pyrin, or actin by immunoblot. Representative of 3 independent experiments. (B) FACS profile of mCherry induction following doxycycline incubation, percentage positive cells shown. Representative of 2 independent experiments. (C) LDH release from Pyrin-deficient THP1 cells reconstituted with doxycycline-inducible hPyrin, mPyrin, or the vector control and treated with TcdA (1 μg/ml), BAA-473, (10 μM), or nigericin (8 μM) for 2 h. Mean and SEM from 3 independent experiments shown. (D) Table of the genotypes of the different FMF donors. (E) IL-1β release from monocytes from HDs or FMF donors (FMF, donors 2–6 used for the experiment) pretreated with Pam3CSK4 (25 ng/ml, 3 h), incubated with colchicine (2.5 μM) for 20 min, and stimulated with TcdA (1 μg/ml) for 3 h. IL-1β release from hMDM from HDs or FMF donors (FMF) pretreated with Pam3CSK4 (25 ng/ml, 3 h); incubated with CP-456,773 (2.5 μM), VX765 (40 μM), colchicine (2.5 μM), or CP-456,773 and colchicine together (TcdB only) for 20 min; and stimulated with (F) TcdA (1 μg/ml, FMF donors 1–6), (G) TcdB (20 ng/ml, FMF donors 3–6), or nigericin (8 μM) for 2 h. (H) Immunoblot for Pyrin expression in Pam3CSK4 (25 ng/ml, 3 h) primed hMDM from HD or FMF (donors 2 and 3). For experiment in (C) mean and SEM from 3 independent experiments. For (E-G), mean and SEM shown for 3–6 independent donors. * p < 0.05, n.s. not significant. The underlying data can be found in the summary data file in the tab Fig 7C and 7E–7G. FMF, familial Mediterranean fever; HD, healthy donor; hPyrin, human Pyrin-flag; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; mPyrin, mouse Pyrin-flag.