EMBO/soda-nel huggingface dataset:
Allosteric inhibition of SHP2 uncovers aberrant TLR7 trafficking in aggravating psoriasis (2021)
Figure 8

Figure 8. SHP2 enhanced activation of TLR7/NF-κB signaling in a phosphatase-dependent manner.

A Immunoblotting of HEK293T cells co-transfected for 48 h with GFP-TLR7, plus HA-tagged SHP2 or SHP2 mutant vectors, followed by immunoprecipitation with anti-GFP.

B, E Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP-TLR7 or GFP-TLR7 mutant vectors.

C, F Immunoblotting of p-p65 in lysates of HEK293T cells transfected with GFP-TLR7 and TLR7 mutant vectors and then treated with IMQ (10 µg/ml) for 30 min.

D, G HEK293T cells transfected with NF-κB-luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ (10 µg/ml) for 24 h were harvested for luciferase assay.

H Immunofluorescence staining of HEK293T cells transfected with GFP-TLR7 and TLR7 mutant vectors, prior to treatment of R848 (10 µg/ml) for 30 min and labelled with specific antibodies. Scale bar: 50 μm.

I Representative p-TLR7 Y1024 staining of skin sections from human. Scale bar: 100 μm.

Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test (D, G). **P<0.01, ns, not significant.
Allosteric inhibition of SHP2 uncovers aberrant TLR7 trafficking in aggravating psoriasis (2021)
Figure 9

Figure 9. The Tlr7-Y1025D mutant alleviated the psoriasis-like phenotype in the IMQ-induced murine model. Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days.

A Phenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 μm. Left: H&E staining data; right: statistical data (mean ± SEM).

B Clinical scores plotted with mean ± SEM. *denotes statistical significance when compared with the Tlr7wt/wt group.

C Body weight change plotted with mean ± SEM. *denotes statistical significance when compared with the Tlr7wt/wt group.

D Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines and other psoriasis-related cytokines in the dorsal skin. Results were normalized to Gapdh expression.

E Representative p-p65 staining of the dorsal skin. Scale bars: 100 μm.

F The graphic illustration of the mechanism of SHP2 accelerating the development of psoriasis. In macrophages of normal skin, when stimulating by TLR7 agonist ssRNA (e.g., IMQ, R848), TLR7 is phosphorylated and SHP2 is recruited to dephosphorylate TLR7, then TLR7 is ubiquitinated, next activates downstream NF-κB signaling. However, in infiltrated macrophages of psoriatic skin, SHP2 is overexpressed. Hyper-dephosphorylation of TLR7 at Tyr1024 by SHP2 leads to hyper-ubiquitination of TLR7 and overactivation of NF-κB activation, resulting in unrestrained skin inflammation. Created in BioRender.com.

Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test (A, D) or Tukey multiple comparison test (B, C). *P<0.05, **P<0.01.
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 2

Figure 2 Function analyses of candidate miRNAs in controlling ESC self-renewal.

Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.

Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.

The heatmap representation of gene expression pattern in miRNA mimic-transfected Dgcr8-/- ESCs.

Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.

Ranking of the miRNAs mainly based on their ability to silence ESC self-renewal program. The color of the circles represented the fold change of these subjects compared to scramble control; the size of the circles represented the magnitude of P values as determined by Student’s t-test. Full list is shown in Supplementary Fig S4A.

AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 3

Figure 3 The essential role of miR-27a and miR-24 in ESC differentiation.

A. The genomic location of miR-23a~27a~24-2 cluster.

B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.

D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).

F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).

G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.

H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs (I).
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 4

Figure 4 miR-27a and miR-24 directly target critical pluripotency associated factors and are restrained by c-Myc in ESCs.

Computer prediction of the binding of miR-27a and miR-24 on the 3′ UTR or CDS of these pluripotency-associated genes.

Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.

The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.

The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.

The schema of Ago2 immunoprecipitation.

F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.

H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.

I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.

J. The five putative c-Myc binding sites scattered within the promoter region of mouse miR-23a~27a~24-2 cluster.

K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.

L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.

M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 5

Figure 5 Inhibition of miR-27a or miR-24 promotes somatic cell reprogramming to iPSC.

The schema of reprogramming MEFs to iPSCs by three transcription factors Oct4, Sox2 and Klf4 (OSK).

Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).

AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.

Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.

Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.

Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.

Bisulfite genomic sequencing of the promoter regions of Oct4 and Nanog in iPSCs, ESCs and MEFs. Open circles indicated unmethylated CpG dinucleotides, while closed circles indicated methylated CpGs.

Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.

Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).

Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 6

Figure 6 Bi-allelic double knockout of miR-23~27~24 clusters in ESCs by CRISPR/Cas9.

Genomic location and sequence alignment of mouse miR-23~27~24 cluster miRNAs. Black box indicates the seed region.

Summary of CRISPR/Cas9-mediated gene targeting in ESCs.

Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.

Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).

AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.

Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).

Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 7

Figure 7 miR-23~27~24 clusters are required for ESC differentiation.

Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).

Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.

Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).

Actinin, ANP and Troponin I immunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.

Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.

Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01.

Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown.

Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.
Non-canonical functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus (2021)
Figure 1

Fig. 1. Deletion of the WD and linker domains of ATG16L1 leads to selective loss of non-canonical autophagy.

A. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 hours as indicated. Cells were fixed and permeabilized and stained for LC3. Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter.

B. Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy. LC3 puncta were identified using spot function software to locate puncta ranging from 0.5-1.0mm diameter. LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter. Data from 10 cells are shown, bars represent the mean ± SD and were compared by Student's t-test (*P<0.05, ****P<0.0001). C-E. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1, δWD and LC3 as indicated. Control MEFS express α and β isoforms of ATG16L1 at 70 kDa, and MEFs from δWD mice express a truncated ATG16L1 at 30kDa. (D) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean (±SD) of three replicate blots. (E) shows fluorescence images of phagosomes following engulfment of Zymosan (red) by bone marrow-derived macrophages (BMDM) from control and δWD mice. White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center. F shows examples of line profile analysis

G, H. (G) shows the percentage of LC3 positive phagosomes per cell and (H) shows line profile analysis of data from 10 cells, bars represent the mean ± SD and were compared by Student's t-test (****P<0.0001).
Non-canonical functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus (2021)
Figure 3

Fig. 3. Systemic loss of non-canonical autophagy leads to extensive lung inflammation and damage.

A-E. Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (A) At the indicated time points, cytokine mRNA transcripts in lung tissue (n = 5) were evaluated by qPCR. Data for individual animals are shown, bars represent the mean ± SD and were compared by 2-way ANOVA with Bonferroni post-tests (*P<0.05, **P<0.01,***P<0.001). (B) Representative lung sections from animals (n = 6) taken at 3 d.p.i. were stained by IH for neutrophils (Ly6G) or neutrophil extracellular traps (NET; anti-H3). Sections at 7 d.p.i. were stained for macrophages (Iba-1). Further micrographs are shown in Appendix Figs. S1 and S2. (C) BAL (n = 5) was taken at 2 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of neutrophils (CD11b+, Ly6G+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05). (D) Single cell suspensions were prepared from lungs taken at 5 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of macrophage/monocytes (CD11b+, F4/80+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05). (E) δWD mice were treated with either anti-PCDA-1 (to deplete plasmacytoid DC) or an isotype-matched control. Litter-mate control mice were used as comparator. Weight loss was measured at the indicated days p.i. (n = 5). Comparisons were made using a repeated-measures two-way ANOVA (Bonferroni post-test, *P<0.05)
Non-canonical functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus (2021)
Figure 4

Fig. 4. Bone marrow-derived lymphoid tissue cannot reverse sensitivity to IAV infection A-D. Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points. Data represent the mean value ± SEM. Comparisons were made using a repeated-measures two-way ANOVA (Bonferroni post-test, **P<0.01, ***P<0.001). (B) Survival was assessed at indicated time points. Comparisons were made using log-rank (Mantel-Cox) test **P<0.01). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6). Data for individual animals are shown using symbols described in (A) and (B), bars represent the mean ± SD. A one-way ANOVA with Tukey's post-hoc analysis was used to determine significance (*P<0.05, **P<0.01). (D) Lungs taken at 5 d.p.i. were analysed for neutrophils (Ly6G), neutrophil extracellular traps (NET; anti-H3) and macrophages (Iba-1) .
Non-canonical functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus (2021)
Figure 5

Fig. 5. Loss of non-canonical autophagy from phagocytes does not increase sensitivity to IAV infection.

A-C. δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells (for construction see Fig. S6A). Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points. Data represent the mean value ± SEM. Comparisons were made using a repeated-measures two-way ANOVA (Bonferroni post-test). (B) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6). Data for individual animals are shown, bars represent the mean ± SD. Mann-Whitney U test was used to determine significance. (C) IL-1β mRNA transcripts in lung at 5 d.p.i. were determined by qPCR. Mann-Whitney U test was used to determine significance (**P<0.01). (D) Precision-cut lung slices from control and δWD mice were infected with IAV. Virus titres were determined at indicated time points. Comparisons were made using two-way ANOVA with Bonferroni post-tests (**P<0.01).
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 1

Figure 1. Irgm1 and Irgm2 but not Irgm3 limit the production of inflammatory cytokines in response to LPS in vivo

(A) WT, Irgm1-/-, Irgm2-/- and Irgm3-/- mice (n = 4 mice/genotype) were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hours post injection (hpi) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel. Relative concentration (fold change relative mean of WT) is shown for the indicated cytokines (absolute cytokine concentrations of same experiment are shown in Fig EV1).

(B) WT (n = 9), Irgm1-/- (n = 7), Irgm2-/- (n = 9) and Casp1-/-Casp11-/- (n = 7) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, IL-18, and TNFα was measured via ELISA.

Data information: Data shown are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 for indicated comparisons or comparison between WT and indicated genotype by one-way ANOVA with Dunnett's multiple comparison test. N.S, non significant.
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 2

Figure 2. Irgm2 suppresses inflammasome activation in LPS-treated macrophages

(A) qPCR measurement of IL-1β, and TNF-α mRNA levels in WT and Irgm2-/- BMMs following 8-hour stimulation with LPS (1 µg/ml).

(B) WT, Irgm2-/- and Casp1-/-Casp11-/- BMMs were treated for 24 hours with LPS at indicated doses and supernatant TNFα, IL-1β, and IL-18 was measured by ELISA.

(C) IFNγ-primed WT and Irgm2-/- BMMs were treated with LPS, Pam3CSK4, poly(I:C) or a combination of Pam3CSK4 and poly(I:C) (1 µg/ml for all treatments) for 24 hours and cell supernatant IL-1β and IL-18 concentrations were assessed by ELISA.

(D) WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and IL-1β, IL-18, and LDH release were assessed at 24 hours post treatment (hpt).

(E) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 hours and subsequently stained with anti-ASC antibody and Hoechst stain (DNA/nuclei). Representative images are shown with white arrows pointing at ASC specks. Number of ASC specks per nuclei was quantified. Scale bars: 20 µm.

(F) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) for 24 h and cell lysates and supernatants collected. Protein levels in cell lysates (caspase-1, and actin) and supernatants (caspase-1 p20) were visualized via immunoblotting.

Data information: Graphs in (A-E) show means ± SEM from n = 3 independent experiments. ***p < 0.001 for indicated comparisons or comparison between WT and indicated genotype by two-way ANOVA with Sidak's (A, C) or Tukey's (B, D, E) multiple comparisons test. Images in panel E are representative of one of three independent experiments. Panel F is representative of n = 2 independent experiments. N.S, non significant.
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 3

Figure 3. Irgm2 suppresses caspase-11 activation in response to LPS and bacterial infections

(A) IFNγ primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 h. Following treatment, cells were stained with anti-ASC antibody and Hoechst (DNA/nuclei). Representative images of ASC specks (white arrows point at specks) are shown and number of ASC specks per nuclei quantified. Scale bars: 20 µm.

(B) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hours-post treatment (hpt).

(C) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were infected with E. coli K-12 at indicated MOIs and cell viability assessed via CellTiter-Glo. Cell death was calculated as a function of relative viability to uninfected cells.

(D) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were infected with E. coli K-12 (MOI 25) and 24 hpi supernatant IL-1β and IL-18 levels were measured by ELISA.

(E) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with OMVs at indicated concentrations for 24 h. Cell viability was assessed via CellTiter-Glo and cell death was calculated as a function of relative viability to untreated cells.

(F) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with OMVs (1 µg/ml) and 24 hpt cell supernatant IL-1β and IL-18 levels were measured via ELISA.

Data information: Shown are means ± SEM from n = 4 (A) or n = 3 (B-F) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 for indicated comparisons by two-way ANOVA with Tukey's multiple comparisons test. Images in panel A are representative of one of four independent experiments. N.S, non significant.
Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock (2020)
Figure 4

Figure 4. Irgm2 deficiency restores caspase-11 activity in the absence of chromosome-3 encoded GBPs

(A) WT, and Irgm2-/- BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected. Lysates were assessed for Casp11, Gbp2, and actin protein levels via immunoblotting.

(B) IFNγ-primed and unprimed WT, Irgm2-/-, Gbpchr3-/- and Aim2-/- BMMs were infected with Francisella novicida (MOI 10) and LDH release measured at 4 hpi.

(C) WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hpt.

(D) IFNγ-primed WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml) for 24 hours and cell lysates and supernatants collected. Protein levels in cell lysates (Caspase-1, and actin) and supernatants (Caspase-1 p20) were visualized via immunoblotting.

(E) Model depicting regulation of caspase-11 activation by Irgm2 and Gbps.

Data information: Graphs show means ± SEM from n = 3 (B, C) independent experiments. **p < 0.01, ***p < 0.001 for indicated comparisons by two-way ANOVA with Tukey's multiple comparisons test. (A) and (D) represent one of two independent experiments. N.S, non significant.