EMBO/soda-nel huggingface dataset:
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 6

Figure 6. Chloroquine-induced endosomal autophagy depends on Rabaptin5 and its ATG16L1 binding motif.

A By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK+Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA).

B The same stable HEK293A-derived cell lines were treated without (-CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. Scale bar, 10 µm.

C WIPI2 and LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, ***P<0.001, ****P<0.0001).

D, E The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01).

F, G HEK+Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5, mCherry-ATG16L1, and WIPI2. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (F) or WIPI2 (G) and M2 showing the inverse (mean ± S.D. of three independent experiments; ANOVA for Rbpt5-KO+wt or +AAA vs. HEK+Rbpt5 cells: *P<0.05, **P<0.01, ***P<0.001).

H Wild-type HEK293A cells, HEK+Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Scale bar, 10 µm.

I The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean ± S.D. of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK+Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures. ANOVA: *P<0.05, ****P<0.0001.
Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1 (2021)
Figure 7

Figure 7. Rabaptin5-mediated autophagy contributes to early killing of Salmonella.

A HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± S.D. of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± S.D. of three independent experiments; ANOVA: *P<0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B as cellular markers. Z-stacks for >5'000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± S.D. of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively).

B Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+wt, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, ****P<0.0001).

C Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment - as illustrated on the top left (scale bar, 2 µm) - during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Mean ± S.D. of three independent experiments, analyzing >50 bacteria for each time point.)
The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. (2010)
Figure 1

DHR3 is a specific dS6K interacter.

Induction of UAS constructs and EPs by the ap promoter directed Gal4 expression within the dorsal compartment of the wing imaginal disc: (A) ap-Gal4 control; (B, E, H, J, K) UAS-dS6K; (C, F, I) UAS-S6K1dE/D3E; (D, E, F) DHR3-EP; (G, H, I, K) UAS-DHR3-RNAi and (J) unidirectional DHR3-EP. The bending down of the wing indicates a slight overgrowth of the dorsal compartment, whereas a bending up reveals a slight growth deficit of this compartment.
The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. (2010)
Figure 2

DHR3–EP specifically counteracts Tsc-dependent growth suppression.

Induction of UAS constructs and DHR3-EP by the eyeless promoter-directed Gal4 expression in the developing eye: (A) eye-Gal4 control; (B, D, E) DHR3-EP; (C, D) UAS-dTsc1 and UAS-dTsc2; (E, F) UAS-dPTEN.
The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. (2010)
Figure 3

DHR3 regulates dS6K activity.

(A) The DHR3 locus is schematized at the top with the 2 EP insertions indicated (arrows). The RA and RB transcripts encode a DBD sequence (indicated by the grey box). RS is a putative novel transcript whose first AUG is located beyond the DBD-coding sequences. (B–G) Adult wings in which the ap-Gal4 induces expression of the following UAS constructs: (B) ap-Gal4 control; (C) UAS-dS6K; (D) UAS-DHR3-RS; (E) both UAS-dS6K and UAS-DHR3-RS; (F) UAS-DHR3-RS in a dS6Kl-1 mutant escaper; (G) UAS-dS6K, UAS-DHR3-RS, and UAS-DHR3-RNAi together. (H) Upper panel, H2B substrate phosphorylation by dS6K from larval protein extracts. Ubiquitous induction of UAS-DHR3 RNAi, using either a daughterless-Gal4 (da>Ri) or an actin-Gal4 (ac>Ri) driver, provoked a drop in dS6K activity, as compared with control drivers alone (da, ac). Middle and lower panels are western blots detecting dS6K and α-tubulin, respectively.
The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. (2010)
Figure 4

Immunodetection of DHR3 proteins.

(A–F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue. (G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2). (H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.
The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. (2010)
Figure 5

Mutations in the LBD of DHR3.

(A, B) The ap-Gal4 driver induces dS6K in combination with either (A) the DHR3-EP transgene or (B) the EP with the EMS DHR3 mutation. (C) Structure of the full-length DHR3-PA protein showing previously described DBD mutants (G60S, R107G) and the EMS mutants that lack the LBD (K243X, W284X); the light and dark grey boxes represent the DBD and the LBD respectively. (D) dS6K activity in larval protein extracts measured by H2B target phosphorylation and (E) corresponding western blot of dS6K in the trans-heterozygote LBD-mutant combination (LBD), as compared to heterozygous larvae (Co). (F, G) The eyeless promoter directs the flipase recombinase during eye development. Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye (F) that is homozygous for the wild-type allele of DHR3. (G) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye. A dotted yellow line surrounding the control eye in (F) has been copied and pasted on the mutant eyes (G); insets are higher magnification images of ommatidia showing misorientation of bristles. (H) At the scutellum, a y-marked homozygous DHR3K243X mutant bristle (arrow), recognizable by its light color, is smaller than the symmetrical neighboring bristles.
The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. (2010)
Figure 6

Growth defect in DHR3 LBD mutant cells.

(A) Normalized eye size (mm2) of control (black bar; n = 20), DHR3W284X (grey bar; n = 18) and DHR3K243X (white bar; n = 18) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction (***P<0.001). (B) Ommatidia size of control (black bar), DHR3W284X (grey bar), and DHR3K243X (white bar) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction (**P<0.01). (C–E) Comparison of y-marked homozygous clonal bristles (black arrows) to their neighboring heterozygous bristles (white arrows) at the wing margin. Note that control clonal bristles are of normal size and orientation (C), whereas those of DHR3W284X (D) and DHR3K243X (E) are reduced in size and are misoriented. (F) Size measurements (µm) of the length of homozygous clones (y−) and their neighboring heterozygous (y+) bristles (n = 51 for each bar). The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X (***P<0.001) and DHR3W284X (***P<0.001) mutants, but not for the control (Co) bristles (P = 0.028).
Multinucleation resets human macrophages for specialized functions at the expense of their identity (2022)
Figure 2

Figure 2. Fusion and multinucleation causes the down-regulation of a shared mononuclear phagocyte gene signature between the LGCs, FBGCs and osteoclasts.

(A) Venn diagram showing the commonly downregulated transcripts (n=191) as a result of multinucleation in LGCs, FBGCs and osteoclasts.

(B) KEGG and BioPlanet 2019 pathway analyses on the 191 commonly downregulated genes. Pathways in red are shared between KEGG and BioPlanet.

(C) MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors.

(D) Representative MRC1 (left panel) and CSF1R (right panel) immunofluorescence in mononuclear and multinucleated osteoclasts stained for DAPI (blue). Note the dim MRC1 and CSF1R in multinucleated osteoclasts (dashed lines) compared to surrounding mononucleated ones.

(E) CSF1R, MRC1, TLR2 and FOS relative expression measured by qRT-PCR for LGCs (upper), FBGCs (middle) and osteoclasts (lower), following the cell membrane fusion regulator DC-STAMP knockdown. si-Ctrl, scrambled siRNA; si-DC-STAMP, DC-STAMP siRNA; n=7 donors.

Data information: Error bars are mean ± SD; significance tested by paired t-test; *, P < 0.05; **, P < 0.01, *** P < 0.001. Scale bars, 20 µm (C) and 100 µm (D).
Multinucleation resets human macrophages for specialized functions at the expense of their identity (2022)
Figure 4

Figure 4. LGCs form in granuloma-like clusters and show increased membrane expression of B7-H3.

(A) Venn diagram showing the uniquely differentially expressed transcripts in LGCs (grey box).

(B) KEGG and BioPlanet 2019 pathway analyses on the differentially expressed transcripts in LGCs only. Pathways in red are in common in KEGG and BioPlanet or relevant for LGC function.

(C) Giemsa stained LGCs (left), FBGCs (middle) and osteoclasts (right) differentiated from PBMCs. Note the cell cluster formation in LGCs only.

(D) CD3 immunofluorescence (green) in PBMC-derived LGCs. Hoechst (grey) and phalloidin (red) staining show the nuclei and cytoskeleton, respectively. Merged (CD3, Hoechst, phalloidin) image shows the existence of CD3+ T cells in the granuloma-like clusters.

(E) B7-H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7-H3 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.

Data information: Scale bars, 100 µm (C and D) and 20 µm (E).
Multinucleation resets human macrophages for specialized functions at the expense of their identity (2022)
Figure 6

Figure 6. Osteoclasts show a distinctively increased mitochondrial activity.

(A) Venn diagram showing uniquely differentially expressed transcripts in osteoclasts (grey box).

(B) KEGG and BioPlanet 2019 pathway analyses on differentially expressed transcripts in osteoclasts only. Pathways in red overlap between KEGG and BioPlanet.

(C) TRAP immunohistochemistry (upper panel) and hydroxyapatite resorption (lower panel) in LGCs, FBGCs and osteoclasts. Note the presence of TRAP+ MGCs in all cell types and hydroxyapatite resorption only in osteoclasts.

(D) Oxygen consumption rate (OCR, upper panel), its related readout (middle panel) and extracellular acidification rate (ECAR, lower panel) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts; n=4 donors.

(E) Extracellular acidification rate (ECAR, left) and glycolysis (right) in mononuclear and multinucleated osteoclasts; n=4 donors.

(F) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts; n=3 donors.

(G) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number; n=6 donors.

(H) Cartoon illustrating that macrophage multinucleation causes a shared down-regulation of mononuclear phagocyte identity while the lysosome-iron pathway is induced.

Data information: Error bars are mean ± SD; significance tested by paired t-test; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, non-significant. Scale bars, 40 µm (C, upper panel) and 1 mm (C, lower panel).

Expanded View Figure legends
The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes (2022)
Figure 1

Figure 1. IQGAP1 promotes LPS+ATP-induced exosome release.

A. Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) treatment as indicated.

B. Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP. **, P<0.01 by unpaired two-tailed t test (n=3 biological repeats in one experiments).

C. Size distributions for vesicles as analyzed in D.

D. Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells.

E. Diagram illustrating the principle of flow cytometry based exosome analysis.

F. Flow cytometry analysis for CD63+ exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants. Quantification was done by mean fluorescence intensity. **, P<0.01 by unpaired two-tailed t test (n=4).

Data information: For panel B and F, data were presented as mean ± SEM. All experiments were biologically repeated for 3 times and yielded consistent results; the representative results are shown.
The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes (2022)
Figure 2

Figure 2. The IQ domain of IQGAP1 interacts with the C-terminal domain of GSDMD.

A. Schematics of domain structure of IQGAP1 and GSDMD.

B. Co-immunoprecipitation of GSDMD with full-length and deletion mutants of IQGAP1 tested in HEK293 cells.

C. Co-immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells.

D. Co-immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant (D276A) restored GSDMD KO YAMC cells. Cells were treated as indicated.

E. Co-immunoprecipitation of C-terminal GSDMD (CT) with IQGAP1 overexpressed in HEK293 cells.

F. Co-immunoprecipitation of pyroptosis mutants full-length (I105N FL) and N-terminal GSDMD defective of pyroptosis (I105N-NT) with IQGAP1.

Data information: All experiments were repeated 3 times and yielded consistent results; the representative results are shown.
The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes (2022)
Figure 3

Figure 3. The IQ domain of IQGAP1 is required for LPS+ATP-induced exosome release

A. Imaging analysis for co-localization of CD63 with IQGAP1 in IQGAP1 (FL) and IQ domain truncation (ΔIQ) restored YAMC cell. Cells were treated with LPS plus ATP. The IQGAP1-CD63 co-localization dots (shown by yellow) were counted, and Pearson's correlation coefficient was calculated for the quantification of co-localization. Data were presented as mean ± SEM. *, P<0.05 by unpaired two-tailed student's t test. Scale bar, 10μm.

B. Imaging analysis for co-localization of CD63 with GSDMD in IQGAP1 full length (FL) and IQ domain truncation restored YAMC cell (ΔIQ) after LPS plus ATP treatment. The GSDMD-CD63 co-localized dots were counted and Pearson's correlation coefficient was calculated for quantification. Data were presented as mean ± SEM. *, P<0.05; **, P<0.01 by unpaired two-tailed student's t test. Scale bar, 5μm.

C. Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells. Data were presented as mean ± SEM. ***, P<0.001 by unpaired two-tailed t test. Scale bar, 5μm.

D. Western blots analysis for supernatants collected from IQGAP1 KO YAMC cells, IQGAP1 full length and IQ domain truncation restored IQGAP1 KO YAMC cells. Cells were treated as indicated and the supernatants were subjected to IP with anti-IL-1β.

E. Western blots analysis for whole cell lysate collected from panel D.

F. ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated. Data were presented as mean ± SEM. *, P<0.05; **, P<0.01 by unpaired two-tailed student's t test.

G. Vesicle size distributions for exosome samples collected from panel E.

Data information: All experiments were biologically repeated for 3 times and yielded consistent results; the representative results are shown.
The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes (2022)
Figure 4

Figure 4. IQGAP1 recruits Tsg101 to GSDMD+ late endosome to drive exosomal IL-1β release.

A. Imaging analysis for early endosome (shown by marker EEA1) change in WT and IQGAP1 KO YAMC cells upon indicated treatments. Quantification was done by counting the EEA1 puncta from each cell. Five field views of cells' puncta were counted. Three independent experiments were repeated and data were shown as mean ± SEM. Ns denotes not significant by unpaired two-tailed t test. Scale bar, 10μm.

B. Co-localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells. Yellow dots indicated the co-localization signal. Data were presented as mean ± SEM. *, P<0.05 by two tailed unpaired t test. Scale bar, 10μm.

C. Co-localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells. Data were presented as mean ± SEM. *, P<0.05 by two tailed unpaired t test. Scale bar, 10μm.

D. Co-localization of CD63 with IQGAP1-GSDMD complex, as measured by PLA, shown by the yellow dots after LPS and ATP treatment.

E. Co-localization of CD63 and pro-IL-1β in YAMC and IQGAP1 KO YAMC cells. Scale bar, 10μm. Data were shown as mean ± SEM. *, P<0.05 by two tailed unpaired t test.

F. Western blot analysis of extracellular vesicle secretion from WT and TSG101 knockdown cells upon indicated treatments. Secreted proteins were pulled down by IL-1β in the supernatant and the indicated proteins were probed for.

G. Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment. Data were presented as mean ± SEM. *, P<0.05 by two tailed unpaired t test.

H. Western blot analysis for interaction of TSG101 with IQGAP1 in WT and GSDMD KO cells by pulling down IQGAP1 under indicated treatments.

I. Co-immunoprecipitation of GSDMD with TSG101 in WT and IQGAP1 KO cells by pulling down TSG101 under indicated treatments.

Data information: All experiments were biologically repeated for 3 times and yielded consistent results; the representative results are shown.
The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes (2022)
Figure 5

Figure 5. Active CDC42 promotes IQGAP1- and GSDMD-mediated exosomal IL-1β release.

A. Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO (IQ1 KO), IQGAP1 full length restored (FL) and GRD domain truncation restored (ΔGRD) YAMC cells upon LPS or LPS plus ATP treatment. Data were presented as mean ± SEM. *, P< 0.05 by two-tailed unpaired t test.

B. PLA assay for IQGAP1 and CDC42 interaction in IQGAP1 FL restored cell and ΔGRD restored cell. The green dots showed the interaction of IQGAP1 and CDC42. Data were presented as mean ± SEM. ****, P<0.0001 by unpaired two-tailed t test. Scale bar, 10μm.

C. Colocalization of CD63 with IQGAP1-CDC42 complex (shown by PLA signal) in WT and GSDMD KO cells under LPS treatment. Quantification was performed by counting PLA green dots. Data were presented as mean ± SEM. Two-tailed unpaired t test was used to calculate the p value. Scale bar, 10μm.

D. Co-immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild-type and GSDMD KO cells upon LPS or LPS plus ATP treatment.

E. Co-immunoprecipitation analysis of GTP-bound CDC42 in indicated cells following LPS treatment.

F. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild-type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment.

G. Western blots for exosome secretion upon CDC42 GTPase inhibition. Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity, followed by LPS and ATP treatment as indicated.

H. Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G. Data were presented as mean ± SEM. *, P<0.05 by unpaired two-tailed t test.

I. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 (10µM for 24 hours) treated cells following LPS or LPS plus ATP treatment.

Data information: All experiments were biologically repeated for 3 times and consistent results were yielded. The representative results are shown.