Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation

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Nucleofection and flow cytometry

THP-1 nucleofection and cell death assays were performed as

described previously (Fujisawa et al., 2007; Honda et al., 2021)

with some modifications. THP-1 cells (1 × 106) were transfected

with 500 ng plasmid encoding pAcGFP-fused CDC42 variants or

hMEFV-pAcGFP1-t2a-CDC42 using 4D-Nucleofector and the SG

Cell Line 4D-Nucleofector X Kit (Lonza). Immediately after nucleofection, 10 ng/ml phorbol 12-myristate 13-acetate (FUJIFILM

Wako) was added and the cells were cultured overnight. The

next day, the cells were stained with LIVE/DEAD Fixable Violet

Dead Cell Stain Kit (Thermo Fisher Scientific) as indicated. Cells

were analyzed using a FACSVerse flow cytometer (Becton and

Dickinson Bioscience [BD]) and FlowJo software (BD). Cell death

was calculated as the percentage of the size-gated cells that were

BV421-high.

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Supplemental material

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Figure S1. Increased production of IL-1β and IL-18 in patients carrying CDC42R186C. (A) Levels of IL-1β, IL-6, IL-18, and IFNγ in sera of Patients 1 and 2.

(B) IL-18 release in response to various inflammasome stimuli from iPS-derived MPs established from Patient 1 and healthy controls. (C and D) IL-1β and IL-6

release in response to various inflammasome stimuli from iPS-derived MPs (C) and MLs (D) established from Patient 2 and healthy controls. Data are representative of two independent experiments using three independent clones. Statistical significance was determined by Mann–Whitney’s U test. *, P < 0.05.

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Figure S2. Immunoblot analysis of CDC42 C-terminal variants, cytokine production from iPS-MLs carrying CDC42R66G, and the effect of CDC42 silencing on cytokine production from CDC42WT and CDC42R186C iPS-MLs. (A) CDC42 C-terminal variants were transiently transfected into HEK293T cells,

and their expression levels were evaluated by Western blot analysis. (B) iPSCs carrying CDC42R66G were generated by manipulating WT-iPSCs by single-base

editing and iPS-MPs were differentiated. Cells were stimulated with LPS + TcdA and the production of IL-1β and IL-6 was evaluated. (C) iPS-MLs from Patient

1 and healthy controls were treated with scramble or CDC42 siRNA and the release of IL-1β and IL-6 was monitored in response to LPS + TcdA or LPS + nigericin

stimulation. Immunoblot images at the bottom show the effects of siRNAs. Representative results of (A) two independent experiments with similar results, and

(B and C) two independent experiments with three clones, are shown. Statistical significance was determined by Mann–Whitney’s U test (B) and by Dunnet

test (C). *, P < 0.05; **, P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS2.

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Figure S3. Analyses of NF-κB signaling in iPS-MLs and cytolytic and degranulation activity of NK cells and CTLs from Patient 1. (A and B) Immunoblot

of JNK, ERK, P65, P38 (A) and pyrin (B) molecules and their phosphorylated forms in iPS-MLs from Patient 1 and a healthy control in response to LPS (A) and

LPS ± TcdA (B) stimuli. (C) Cytolytic activity of activated NK cells derived from healthy controls, Patient 1, and a FHL2 patient. (D and E) Degranulation of

activated NK cells (D) and CTLs (E) derived from healthy controls, Patient 1, and a FHL3 patient. Representative results of three (A and B) and two (C–E)

independent experiments with similar results are shown. FHL, familial hemophagocytic lymphohistiocytosis. Source data are available for this figure: SourceData FS3.

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Figure S4. CDC42R186C and CDC42*192C*24 variants do not affect the transport and the localization of COP-I. (A) mScarletI-T2A-CDC42 and VSV-G

(tsO45)-GFP were transiently expressed in COS-1 cells. Cells were incubated at 40°C for 16 h to allow VSV-G protein to accumulate in the ER, and then at 32°C

for 30 min. (B) mScarlet-T2A-CDC42 and EGFP-STING were stably expressed in Sting−/− MEFs as indicated. (C–E) GFP-T2A-CDC42 was transiently expressed

in COS-1 cells. Cells were then fixed and stained for GM130 and for β-COP (C), β9-COP (D), and γ-COP (E). Nuclei were stained with DAPI (blue). Representative

results of two independent experiments with similar results are shown. Scale bars, 10 µm.

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Figure S5. Addition of the C-terminal sequence of CDC42 (R186C) to Rac1 alters its localization to the Golgi. Rac1 or Rac1-CDC42 (C-terminal) was

transiently expressed in COS-1 cells. Cells were then fixed, permeabilized, and stained for GM130. Nuclei were stained with DAPI (blue). The magnified image of

the perinuclear region is shown in the lower left box. Representative results of two independent experiments with similar results are shown. Scale bars, 10 µm.

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