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 .

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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.


C . Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells .

C. Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells.


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 .

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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 .

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F. ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated.


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

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


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

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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


B . Co - localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells . Yellow dots indicated the co - localization signal .

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B. Co-localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells. Yellow dots indicated the co-localization signal.


C . Co - localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells .

C. Co-localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells.


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

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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 .

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E. Co-localization of CD63 and pro-IL-1β in YAMC and IQGAP1 KO YAMC cells.


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 .

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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 .

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G. Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment.


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

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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 .

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


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 .

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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.


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 .

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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.


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 .

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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.


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

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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 .

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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 .

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 .

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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.


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 .

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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.


A . Histology staining for IQGAP1 and GSDMD in naive and inflamed ( 3 days of DSS - challenge ) colon tissues .

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A. Histology staining for IQGAP1 and GSDMD in naive and inflamed (3 days of DSS-challenge) colon tissues.


B . Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls .

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B. Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls.


C . Western blot analysis for supernatant collected from colon explants of DSS - treated wild - type ( WT ) , Gsdmd - / - ( GSDMD KO ) and Iqgap1 - / - ( IQGAP1 KO ) mice . Supernatant was first pulled down by IL - 1β antibody and samples were subjected to Western blot analysis . The indicated proteins were probed .

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C. Western blot analysis for supernatant collected from colon explants of DSS-treated wild-type (WT), Gsdmd-/-(GSDMD KO) and Iqgap1-/- (IQGAP1 KO) mice. Supernatant was first pulled down by IL-1β antibody and samples were subjected to Western blot analysis. The indicated proteins were probed.


D . Immunofluorescent staining with anti - GTP - CDC42 in DSS treated colons from Gsdmd + / + ( WT ) and Gsdmd - / - ( GSDMD KO ) , IQGAP1 + / + and IQGAP1 - / - mice .

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D. Immunofluorescent staining with anti-GTP-CDC42 in DSS treated colons from Gsdmd+/+ (WT) and Gsdmd-/-(GSDMD KO), IQGAP1+/+ and IQGAP1-/- mice.


( A ) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC - labeled zymosan ( 3 mg / ml , 10 µl ) in one hind paw . White dotted lines show the position of zymosan .

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(A) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC-labeled zymosan (3 mg/ml, 10 µl) in one hind paw. White dotted lines show the position of zymosan.


Quantification of the number of macrophages and their subtypes 8 , 24 , 48 and 72 hours after zymosan injection based on the MELC images . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * * p < 0 . 001 . # p < 0 . 05 , # # # p < 0 . 001 as compared to naïve mice .

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Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.


FACS analysis of macrophages and their subtypes 8 , 24 and 48 hours after zymosan injection . Data are shown as mean ( n = 8 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 .

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FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.


( A ) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM ( n . d . , not determinable ) .

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(A) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM (n.d., not determinable).


Representative MELC images showing the IL - 4 and Siglec F expression 4 hours ( panel C ) after zymosan injection . The white dotted lines depict the area where the transition between the neighboring regions occurs .

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Representative MELC images showing the IL-4 and Siglec F expression 4 hours (panel C) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.


Representative MELC images showing the IL - 4 and Siglec F expression 24 hours ( panel D ) after zymosan injection . The white dotted lines depict the area where the transition between the neighboring regions occurs .

Representative MELC images showing the IL-4 and Siglec F expression 24 hours (panel D) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.


( E ) Number of IL - 4 - expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM , two - tailed Students t - test * * * p < 0 . 001 .

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(E) Number of IL-4-expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, two-tailed Students t-test ***p<0.001.


( F ) Number of IL - 4 - expressing eosinophils in the three regions 24 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) Number of IL-4-expressing eosinophils in the three regions 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.


( G ) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(G) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, **p<0.01, ***p<0.001.


FACS analysis of eosinophils in the blood B ; n = 6 mice ) at the indicated time points after zymosan injection . Anti - Siglec F antibody or the IgG - control ( control ) were administered with 0 , 883mg / kg 24 hours prior zymosan injection . Data are shown as mean ± SEM , student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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FACS analysis of eosinophils in the blood B; n= 6 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.


FACS analysis of eosinophils in the paws ( panel C n = 8 mice ) at the indicated time points after zymosan injection . Anti - Siglec F antibody or the IgG - control ( control ) were administered with 0 , 883mg / kg 24 hours prior zymosan injection . Data are shown as mean ± SEM , student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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FACS analysis of eosinophils in the paws (panel C n= 8 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.


Zymosan - induced mechanical ( panel E ) hypersensitivity of control or anti - Siglec F antibody - treated mice . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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Zymosan-induced mechanical (panel E) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.


Zymosan - induced thermal ( panel F ) hypersensitivity of control or anti - Siglec F antibody - treated mice . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

Zymosan-induced thermal (panel F) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.


Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


Quantification of eosinophils , the sum of all macrophages , as well as efferocytosis ( panel H ) in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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Quantification of eosinophils, the sum of all macrophages, as well as efferocytosis (panel H) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


Quantification of eosinophils , the sum of all macrophages , neutrophils ( panel I ) in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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Quantification of eosinophils, the sum of all macrophages, neutrophils (panel I) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


( A ) IL - 4 levels in paws from naive and zymosan - injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil - depleted mice . Data are shown as mean ± SEM ( n = 6 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * * p < 0 . 01 , * * * p < 0 . 001 .

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(A) IL-4 levels in paws from naive and zymosan-injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil-depleted mice. Data are shown as mean ± SEM (n=6 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: **p<0.01, ***p< 0.001.


( B ) FACS analysis of the eosinophil number in paws from control and eosinophil - depleted mice with or without IL - 4c treatment 24h prior zymosan injection . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni * * * p < 0 . 001 .

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(B) FACS analysis of the eosinophil number in paws from control and eosinophil-depleted mice with or without IL-4c treatment 24h prior zymosan injection. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni ***p< 0.001.


( C ) Same as panel B except that resident ( Ly6C - ) and monocyte - derived macrophages ( Ly6C + ) were determined .

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(C) Same as panel B except that resident (Ly6C-) and monocyte-derived macrophages (Ly6C+) were determined.


Same as panel B except that M2 - like ( panel D ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

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Same as panel B except that M2-like (panel D) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


Same as panel B except that M1 - like ( panel E ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

Same as panel B except that M1-like (panel E) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


Same as panel B except that M0 macrophages ( panel F ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

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Same as panel B except that M0 macrophages (panel F) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


( A ) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil - depleted mice . Data are mean ± SEM ( n = 6 mice ) . Student ' s One - way ANOVA , significance between control and anti - Siglec F antibody treatment * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil-depleted mice. Data are mean ± SEM (n=6 mice). Student's One-way ANOVA, significance between control and anti-Siglec F antibody treatment *p<0.05; **p<0.01; ***p<0.001.


FACS analysis of the neutrophil count in paws ( panel B ) from control or eosinophil - depleted mice with or without IL - 4c treatment . Data are shown as the mean ± SEM ( n = 6 - 8 ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 .

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FACS analysis of the neutrophil count in paws (panel B) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.


FACS analysis of the neutrophil count in peripheral blood ( panel C ) from control or eosinophil - depleted mice with or without IL - 4c treatment . Data are shown as the mean ± SEM ( n = 6 - 8 ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 .

FACS analysis of the neutrophil count in peripheral blood (panel C) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.


( D ) Same as panel B except that efferocytosis was determined . * p < 0 . 05 ; * * * p < 0 .

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(D) Same as panel B except that efferocytosis was determined. *p<0.05; ***p<0.001


( E ) FACS analysis of alive and dying neutrophils in paws from control or eosinophil - depleted mice . Data are shown as the mean ± SEM ( n = 5 mice ) .

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(E) FACS analysis of alive and dying neutrophils in paws from control or eosinophil-depleted mice. Data are shown as the mean ± SEM (n=5 mice).


( C ) Zymosan - induced thermal hypersensitivity in control or eosinophil - depleted mice with or without IL - 4c treatment 24h hours prior zymosan injection . The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points . Data are shown as the mean ± SEM ( n = 6 mice ) . Two - way ANOVA / Bonferroni , * p < 0 . 05 , * * p < 0 . 01 ; ns , not significant .

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(C) Zymosan-induced thermal hypersensitivity in control or eosinophil-depleted mice with or without IL-4c treatment 24h hours prior zymosan injection. The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points. Data are shown as the mean ± SEM (n=6 mice). Two-way ANOVA/Bonferroni, *p< 0.05, **p< 0.01; ns, not significant.


E Distribution of endogenous WIPI2 and STING in MEFs . F Statistical analysis of the number of STING - and WIPI2 - positive puncta in cells treated as in ( E ) . G Distribution of endogenous ATG16L1 in HEK293T cells stably expressing STING - HA . H Statistical analysis of the number of STING - and ATG16L1 - positive puncta in cells treated as in ( G ) . All the cells were treated with or without cGAMP for 2 h . All statistical data are presented as mean ± SEM of three independent experiments .

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E Distribution of endogenous WIPI2 and STING in MEFs. F Statistical analysis of the number of STING- and WIPI2-positive puncta in cells treated as in (E). G Distribution of endogenous ATG16L1 in HEK293T cells stably expressing STING-HA. H Statistical analysis of the number of STING- and ATG16L1-positive puncta in cells treated as in (G). All the cells were treated with or without cGAMP for 2 h. All statistical data are presented as mean ± SEM of three independent experiments.


A Co - immunoprecipitation of endogenous STING with WIPI1 / 2 - Myc in MEFs transfected with Myc - tagged WIPI1 or WIPI2 . The cells were treated with or without cGAMP for 2 h and the WIPI1 / 2 - Myc precipitates were analyzed by western blot using anti - STING . B Co - immunoprecipitation of endogenous WIPI2 with HA - tagged STING , STING - R238A or STING - S366A from HEK293T cells stably expressing STING - HA STING - R238A - HA or STING - S366A - HA . The cells were treated with or without cGAMP for 2 h .

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A Co-immunoprecipitation of endogenous STING with WIPI1/2-Myc in MEFs transfected with Myc-tagged WIPI1 or WIPI2. The cells were treated with or without cGAMP for 2 h and the WIPI1/2-Myc precipitates were analyzed by western blot using anti-STING. B Co-immunoprecipitation of endogenous WIPI2 with HA-tagged STING, STING-R238A or STING-S366A from HEK293T cells stably expressing STING-HA STING-R238A-HA or STING-S366A-HA. The cells were treated with or without cGAMP for 2 h.


Co - immunoprecipitation of endogenous STING with Myc - WIPI2 or the indicated WIPI2 mutants from MEFs transfected with the indicated plasmids .

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Co-immunoprecipitation of endogenous STING with Myc- WIPI2 or the indicated WIPI2 mutants from MEFs transfected with the indicated plasmids.


J Purified GST - tagged STING - 153 - 379 or STING - 153 - 379 - 3A - 2 was incubated with recombinant WIPI2 or the indicated WIPI2 mutants , and then precipitated with glutathione sepharose beads . The bound WT or mutant WIPI2 was detected by western blot using anti - WIPI2 . K Statistical analysis of ( J ) .

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J Purified GST-tagged STING-153-379 or STING-153-379-3A-2 was incubated with recombinant WIPI2 or the indicated WIPI2 mutants, and then precipitated with glutathione sepharose beads. The bound WT or mutant WIPI2 was detected by western blot using anti-WIPI2. K Statistical analysis of (J).


A WIPI2 - GFP puncta in MEFs stably expressing GFP - tagged WT or mutant WIPI2 . The cells were treated with cGAMP for 2 h or Torin1 for 1 h . B Statistical analysis of the number of WIPI2 - GFP puncta in cells treated as in ( A ) .

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A WIPI2-GFP puncta in MEFs stably expressing GFP-tagged WT or mutant WIPI2. The cells were treated with cGAMP for 2 h or Torin1 for 1 h. B Statistical analysis of the number of WIPI2-GFP puncta in cells treated as in (A).


F LC3 and p62 level in HEK293T cells stably expressing HA - tagged WT or mutant STING . The cells were treated with or without cGAMP for 4 h .

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F LC3 and p62 level in HEK293T cells stably expressing HA-tagged WT or mutant STING. The cells were treated with or without cGAMP for 4 h.


C Co - immunoprecipitation of endogenous WIPI2 with STING - HA from HEK293 cells stably expressing STING - HA with or without BECN1 deletion . The cells were treated with or without Torin1 for 3 h .

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C Co-immunoprecipitation of endogenous WIPI2 with STING-HA from HEK293 cells stably expressing STING-HA with or without BECN1 deletion. The cells were treated with or without Torin1 for 3 h.


E Co - immunoprecipitation of endogenous STING with Myc - tagged WT or mutant WIPI2 from MEFs treated with or without VPS34 - IN1 for 2 h .

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E Co-immunoprecipitation of endogenous STING with Myc-tagged WT or mutant WIPI2 from MEFs treated with or without VPS34-IN1 for 2 h.


G GFP - LC3 punctum formation in MEFs stably expressing GFP - LC3 and STING - HA . The cells were treated with or without cGAMP or VPS34 - IN1 for 2 h . Scale bars , 10 µm .

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G GFP-LC3 punctum formation in MEFs stably expressing GFP-LC3 and STING-HA. The cells were treated with or without cGAMP or VPS34-IN1 for 2 h. Scale bars, 10 µm.


I Western blot assay of p62 and phospho - TBK1 ( Ser172 ) in MEFs . The cells were pretreated with or without VPS34 - IN1 for 1 h and then co - treated with cGAMP or Torin1 for another 4 h along with or without CQ . CQ , chloroquine . J Statistical analysis of p62 protein level in cells treated as in ( I ) .

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I Western blot assay of p62 and phospho-TBK1 (Ser172) in MEFs. The cells were pretreated with or without VPS34-IN1 for 1 h and then co-treated with cGAMP or Torin1 for another 4 h along with or without CQ. CQ, chloroquine. J Statistical analysis of p62 protein level in cells treated as in (I).


A , B Localization of endogenous WIPI2 , WIPI1 , ATG16L1 ( A ) or Myc - tagged WT WIPI2 or WIPI2 mutants ( B ) in MEFs transfected with Cy3 labeled - ISD ( 1 μg / ml ) . ISD , interferon stimulatory DNA .

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A, B Localization of endogenous WIPI2, WIPI1, ATG16L1 (A) or Myc-tagged WT WIPI2 or WIPI2 mutants (B) in MEFs transfected with Cy3 labeled-ISD (1 μg/ml). ISD, interferon stimulatory DNA.


D Statistical analysis of cytoplasmic DNA puncta in cells treated as in ( C ) .

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D Statistical analysis of cytoplasmic DNA puncta in cells treated as in (C).


F Statistical analysis of cytoplasmic DNA puncta in cells treated as in ( E ) .

F Statistical analysis of cytoplasmic DNA puncta in cells treated as in (E).


A Wipi2 - or Sting - deleted MEFs were treated with Ara - C for 12 h , and then cultured in Ara - C - free medium . After 24 h , the cells were lysed and analyzed by western blot using the indicated antibodies . B Statistical analysis of the protein level of phospho - TBK1 ( Ser172 ) and STING in cells treated as in ( A ) . The relative phospho - TBK1 level was normalized to TBK1 , the relative STING level was normalized to Actin .

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A Wipi2- or Sting-deleted MEFs were treated with Ara-C for 12 h, and then cultured in Ara-C-free medium. After 24 h, the cells were lysed and analyzed by western blot using the indicated antibodies. B Statistical analysis of the protein level of phospho-TBK1 (Ser172) and STING in cells treated as in (A). The relative phospho-TBK1 level was normalized to TBK1, the relative STING level was normalized to Actin.


C - E MEFs with Wipi2 or Sting deletion ( C ) , Wipi2 - KO MEFs with transfection of Myc - tagged WT or mutant WIPI2 ( D ) , or Sting - KO MEFs with transfection of HA - tagged WT or mutant STING ( E ) , were treated with Ara - C for 12 h and then cultured in Ara - C - free medium for another 24 h . Total mRNA was extracted and the Ifnb1 mRNA level was measured by real - time PCR and normalized to Actb mRNA . 2RE , WIPI2 - R108E / R125E .

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C-E MEFs with Wipi2 or Sting deletion (C), Wipi2-KO MEFs with transfection of Myc-tagged WT or mutant WIPI2 (D), or Sting-KO MEFs with transfection of HA-tagged WT or mutant STING (E), were treated with Ara-C for 12 h and then cultured in Ara-C-free medium for another 24 h. Total mRNA was extracted and the Ifnb1 mRNA level was measured by real-time PCR and normalized to Actb mRNA. 2RE, WIPI2-R108E/R125E.


F Sting - KO MEFs with transfection of HA - tagged WT or mutant STING were treated with cGAMP for the indicated time . The cells were lysed and analyzed by western blot using the indicated antibodies .

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F Sting-KO MEFs with transfection of HA-tagged WT or mutant STING were treated with cGAMP for the indicated time. The cells were lysed and analyzed by western blot using the indicated antibodies.


( a ) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression ( 18 h cultures ) . Values are means ± s . e . m . of three independent experiments .

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(a) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures). Values are means ± s.e.m. of three independent experiments.


( b ) The cell death regulated by Atg5 is apoptosis . All data presented are from 5 h cultures . Caspase - 3 - like activity analysed in HeLa cells ( graph ) . Morphology of Jurkat cells is also shown . The numbers in the lower right corner indicate the percentage of cell death , which was determined in parallel experiments using flow

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(b) The cell death regulated by Atg5 is apoptosis. All data presented are from 5 h cultures. Caspase-3-like activity analysed in HeLa cells (graph). Morphology of Jurkat cells is also shown. The numbers in the lower right corner indicate the percentage of cell death, which was determined in parallel experiments using flow cytometry


c ) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z - VAD - fmk . Numbers indicate the results of the quantitative analysis ( percentage ) of each dot blot .

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c) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot.


d ) Reduction of drug - induced cell death by silencing of Atg5 . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) . The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting . The asterisks indicate P 0 . 05 . n . s . , no significant difference .

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d) Reduction of drug-induced cell death by silencing of Atg5. Values are means ± s.e.m. of three independent experiments (18 h cultures). The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting. The asterisks indicate P 0.05. n.s., no significant difference.


a ) Spontaneous neutrophil apoptosis . Apoptosis was associated with the appearance of a 24K anti - Atg5 - reactive protein . 33K Atg5 disappeared during apoptosis in a time - dependent manner . HeLa cells overexpressing Atg5 served as positive control .

a) Spontaneous neutrophil apoptosis. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein. 33K Atg5 disappeared during apoptosis in a time-dependent manner. HeLa cells overexpressing Atg5 served as positive control.


b ) Anti - CD95 - induced apoptosis of Jurkat cells . Apoptosis was associated with the appearance of a 24K anti - Atg5 - reactive protein and a reduction of 33K Atg5 in a time - dependent manner . The 24K anti - Atg5 - reactive protein was also visible in Jurkat cells after treatment with actinomycin D ( Act . D ) or cycloheximide ( CHX ; 5 h treatment in both cases ) . GAPDH expression indicates equal loading in each panel . All immmunoblots are representative of at least three independent experiments .

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b) Anti-CD95-induced apoptosis of Jurkat cells. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein and a reduction of 33K Atg5 in a time-dependent manner. The 24K anti-Atg5-reactive protein was also visible in Jurkat cells after treatment with actinomycin D (Act. D) or cycloheximide (CHX; 5 h treatment in both cases). GAPDH expression indicates equal loading in each panel. All immmunoblots are representative of at least three independent experiments.


e ) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation . Calpain cleaved Atg5 at Thr 193 . Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice . The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank . The position of the six - amino - acid deletion causing calpain resistance is indicated in red .

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e) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation. Calpain cleaved Atg5 at Thr 193. Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice. The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank. The position of the six-amino-acid deletion causing calpain resistance is indicated in red.


( d ) Following calpain - mediated cleavage of recombinant Atg5 in vitro , the sample was fractionated by preparative reversed - phase HPLC and analysed by mass spectrometry . The exact relative molecular masses for full - length and truncated Atg5 are indicated .

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(d) Following calpain-mediated cleavage of recombinant Atg5 in vitro, the sample was fractionated by preparative reversed-phase HPLC and analysed by mass spectrometry. The exact relative molecular masses for full-length and truncated Atg5 are indicated.


( f ) A six - amino - acid deletion surrounding the cleavage site ( 35S - Atg5Δ191 - 196 ) led to a loss of the calpain cleavage fragment .

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(f) A six-amino-acid deletion surrounding the cleavage site (35S-Atg5Δ191-196) led to a loss of the calpain cleavage fragment.


( g ) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor - mediated apoptosis of neutrophils . In contrast , Atg6 did not show evidence for cleavage and its levels remained unchanged in this system . Results of 7 h cultures are shown .

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(g) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor-mediated apoptosis of neutrophils. In contrast, Atg6 did not show evidence for cleavage and its levels remained unchanged in this system. Results of 7 h cultures are shown.


( h ) Atg5 is not cleaved in apoptotic HeLa cells ( 5 h staurosporine treatment ) lacking the small subunit of calpain , which was silenced by using specific siRNA .

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(h) Atg5 is not cleaved in apoptotic HeLa cells (5 h staurosporine treatment) lacking the small subunit of calpain, which was silenced by using specific siRNA.


( i ) Calpain resistant Atg5Δ191 - 196 was unable to sensitize HeLa cells lacking endogeneous Atg5 ( due to gene silencing , see Fig . 1c ) to staurosporine - induced cell death . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) .

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(i) Calpain resistant Atg5Δ191-196 was unable to sensitize HeLa cells lacking endogeneous Atg5 (due to gene silencing, see Fig. 1c) to staurosporine-induced cell death. Values are means ± s.e.m. of three independent experiments (18 h cultures).


( a ) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining . Values are means ± s . e . m . of three independent experiments . Bax transfection was used as a positive control . ( b ) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h . Quantitative analysis of these experiments is shown in a . Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining . Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis ( nuclear condensation , cell shrinkage ; approximately 80 % of the cells ) .

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(a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining. Values are means ± s.e.m. of three independent experiments. Bax transfection was used as a positive control. (b) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h. Quantitative analysis of these experiments is shown in a. Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining. Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis (nuclear condensation, cell shrinkage; approximately 80% of the cells).


( c ) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer . Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days . Morphological analysis ( Diff - Quik ) is also shown and revealed that the type of death was apoptosis ( nuclear condensation , cell shrinkage ) .

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(c) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer. Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days. Morphological analysis (Diff-Quik) is also shown and revealed that the type of death was apoptosis (nuclear condensation, cell shrinkage).


( d ) CEM cells that either lacked Bcl - 2 or expressed high levels of Bcl - 2 received truncated Atg5 or GFP by lentiviral gene transfer . In the absence of Bcl - 2 , induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days . Apoptosis was largely blocked in cells expressing high levels of Bcl - 2 . GFP transfection did not affect viability of the cells in this system .

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(d) CEM cells that either lacked Bcl-2 or expressed high levels of Bcl-2 received truncated Atg5 or GFP by lentiviral gene transfer. In the absence of Bcl-2, induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days. Apoptosis was largely blocked in cells expressing high levels of Bcl-2. GFP transfection did not affect viability of the cells in this system.


( e ) Staurosporine - induced Atg5 cleavage ( 5 h treatment ) in CEM cells independent of Bcl - 2 expression . Blots were stripped and reprobed with anti - GAPDH antibody .

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(e) Staurosporine-induced Atg5 cleavage (5 h treatment) in CEM cells independent of Bcl-2 expression. Blots were stripped and reprobed with anti-GAPDH antibody.


( f ) Detection of autophagic activity by staining of acidic vesicular organelles ( VAOs , red ) followed by flow cytometry analysis in genetically modified HeLa cells . That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy ( data not shown ) .

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(f) Detection of autophagic activity by staining of acidic vesicular organelles (VAOs, red) followed by flow cytometry analysis in genetically modified HeLa cells. That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy (data not shown).


( g ) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities . Values are means ± s . e . m . of three independent experiments ( lentiviral gene transfer for 24 h ) . The asterisks indicate P 0 . 05 . n . s . , no significant difference . The scale bars represent 10 μM .

(g) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities. Values are means ± s.e.m. of three independent experiments (lentiviral gene transfer for 24 h). The asterisks indicate P 0.05. n.s., no significant difference. The scale bars represent 10 μM.


( a ) Confocal microscopy of HeLa cells were treated with anti - CD95 antibody for 5 h or left untreated . Cells were stained with anti - Atg5 and anti - VDAC antibodies . In untreated cells , there was only little evidence for colocalization of Atg5 and VDAC . In anti - CD95 antibody treated cells some of the Atg5 colocalized with VDAC . The images of colocalization were prepared using Imaris software . Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated . The error bars represent the s . e . m .

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(a) Confocal microscopy of HeLa cells were treated with anti-CD95 antibody for 5 h or left untreated. Cells were stained with anti-Atg5 and anti-VDAC antibodies. In untreated cells, there was only little evidence for colocalization of Atg5 and VDAC. In anti-CD95 antibody treated cells some of the Atg5 colocalized with VDAC. The images of colocalization were prepared using Imaris software. Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated. The error bars represent the s.e.m.


( b ) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis . After 4 h incubation , cleavage of Atg5 was evident and both full - length and truncated Atg5 were present in the cytosol . After 8 h incubation , some 24K Atg5 but not full - length Atg5 was found in the mitochondrial fraction . Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol . The same data were observed in staurosporine - induced apoptosis of Jurkat cells ( data not shown ) . C , cytosol ; M , mitochondria .

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(b) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis. After 4 h incubation, cleavage of Atg5 was evident and both full-length and truncated Atg5 were present in the cytosol. After 8 h incubation, some 24K Atg5 but not full-length Atg5 was found in the mitochondrial fraction. Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol. The same data were observed in staurosporine-induced apoptosis of Jurkat cells (data not shown). C, cytosol; M, mitochondria.


( c ) Cytochrome c release assay . Truncated and full - length recombinant Atg5 were added to isolated mitochondria and incubated at 37 ° C for 1 h . Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting . Truncated Bid ( tBid ) was used as a positive control for mitochondrial permeabilization . Truncated Atg5 released cytochrome c in a concentration - dependent manner , indicating mitochondrial permeabilization . Full - length Atg5 had only little effect in this system .

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(c) Cytochrome c release assay. Truncated and full-length recombinant Atg5 were added to isolated mitochondria and incubated at 37 °C for 1 h. Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting. Truncated Bid (tBid) was used as a positive control for mitochondrial permeabilization. Truncated Atg5 released cytochrome c in a concentration-dependent manner, indicating mitochondrial permeabilization. Full-length Atg5 had only little effect in this system.


( d ) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase - 3 cleavage . GAPDH expression demonstrates equal loading .

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(d) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase-3 cleavage. GAPDH expression demonstrates equal loading.


( e ) Jurkat cells were treated with staurosporine for 5 h or left untreated . Staurosporine induced Atg5 cleavage and both full - length and truncated Atg5 were present in total lysates . Truncated but not full - length Atg5 was detected in Bcl - xL immunoprecipitates obtained from apoptotic Jurkat cells . No evidence for physical interaction between Atg5 and Bax was obtained . The lower panels demonstrate that both Bax and Bcl - xL were enriched following immunoprecipitation . The asterisk indicates P 0 . 05 . The scale bars represent 10 μM .

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(e) Jurkat cells were treated with staurosporine for 5 h or left untreated. Staurosporine induced Atg5 cleavage and both full-length and truncated Atg5 were present in total lysates. Truncated but not full-length Atg5 was detected in Bcl-xL immunoprecipitates obtained from apoptotic Jurkat cells. No evidence for physical interaction between Atg5 and Bax was obtained. The lower panels demonstrate that both Bax and Bcl-xL were enriched following immunoprecipitation. The asterisk indicates P 0.05. The scale bars represent 10 μM.


A , B Representative images of OG immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 .

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A, B Representative images of OG immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125


A , B Representative images o O - GlcNAc ( B ) immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 .

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A, B Representative images o O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125


C Levels of intestinal epithelial OGT and O - GlcNAcylation in a second cohort of control , UC , and CD patients . ( one - way ANOVA , OGT : Control n = 10 , UC n = 9 , CD n = 6 ; Control vs UC P = 0 . 035 , Control vs CD P = 0 . 003 ; O - GlcNAc : Control n = 10 , UC n = 8 , CD n = 8 ; Control vs UC P = 0 . 0027 , Control vs CD P = 0 . 008

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C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008


D , E Regression plots of average immune - staining scores of OGT and O - GlcNAcylation against histological scores in UC ( D ) and CD ( E ) ( D : OGT n = 19 , O - GlcNAc n = 18 ; E : OGT n = 15 , O - GlcNAc n = 16 )

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D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16)


A Body weight of male wildtype and Vil - Ogt KO mice at 6 weeks of age ( n = 6 , P = 0 . 000015 )

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A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015)


B Incidence of rectal prolapse in male wildtype and Vil - Ogt KO mice ( WT n = 17 , KO n = 9 , P < 0 . 0001 )

B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001)


C Representative colonoscopy images of male wildtype and Vil - Ogt KO mice

C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice


D Body weight of female wildtype and Vil - Ogt KO mice at 9 weeks of age ( WT n = 11 , heterozygous KO n = 4 , homozygous KO n = 5 , P < 0 . 0001 )

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D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001)


E Incidence of rectal prolapse in female wildtype and Vil - Ogt KO mice ( WT n = 9 , heterozygous KO n = 8 , homozygous KO n = 6 , P = 0 . 0439 )

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E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439)


F H & E staining of duodenum , jejunum , ileum , colon , and rectum of 10 - week - old male wildtype and Vil - Ogt KO mice . Scale bars = 100 μm

F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm


A Numbers of Paneth cells in the ileum of wildtype and Vil - Ogt KO mice ( n = 6 , P = 0 . 0048 )

A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048)


B Representative electron microscopic images of Paneth cells . Scale bars = 1 μm

B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm


C Expression of AMP genes in ileum tissues of wildtype and Vil - Ogt KO mice ( n = 6 , Pan Defesin P = 0 . 031 , Defa3 P = 0 . 049 , Defa4 P = 0 . 013 , Defa5 P = 0 . 023 , Defa6 P = 0 . 034 , Ang4 P = 0 . 031 )

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C Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031)


D Immunoblotting of LC3 in IEC cells isolated from WT and Vil - Ogt KO mice

D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice


E Representative images of O - GlcNAc staining in Paneth cells of male wildtype and Defa6 - Ogt KO mice . Stars indicate lysozyme - positive Paneth cells

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E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells


F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 , P = 0 . 024 ) . Arrow indicate Paneth cell . Scale bars = 10 μm

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F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm


G Expression of AMP genes in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 Pan Defesin P = 0 . 0291 , Defa3 P = 0 . 0462 )

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G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462)


H Representative images of cultured organoid on Day 3


I Expression of inflammatory markers in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 )

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I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5)


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes in body weight ( J ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes i colitis scores ( K are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis RT - qPCR of inflammatory genes in the ileum ( L ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049


A Expression of phosphorylated and total STAT3 and NF - κB p65 in the colon of wildtype and Vil - Ogt KO mice

A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice


B Ileum and colon IECs from 10 - week - old wildtype and Vil - Ogt KO mice were isolated for RNA - sequencing analyses . The heatmaps of the sample - to - sample distances are shown

B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown


C Volcano plots of gene expression changes . The x - axis specifies the fold - changes ( FC ) and the y - axis specifies the negative logarithm to the base 10 of the p - values adjusted for multiple testing with the Benjamini - Hochberg procedure . FC > 2 and p < 0 . 05 genes are shown in solid dots

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C Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots


F Levels of STAT1 protein in IECs determined by immunoblotting and O - GlcNAc modification of STAT1 determined by immunoprecipitation using the O - GlcNAc antibody followed by Immunoblotting

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F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting


G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 and global O - GlcNAcylatio were

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G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined


G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 an STAT1 O - GlcNAcylation ( H ) were

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G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined


I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later

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J The pGAS - luc Cis - Reporter plasmid was co - transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 ( n = 4 , P = 0 . 0003 )

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J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003)


A , B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS . Daily changes in body weight ( A ) and colitis score ( B ) ( n = 5 ) . ( B : day6 P = 0 . 0171 , day7 P = 0 . 0445

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A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445


C Intestinal barrier functional assays measuring serum FITC - dextran before DSS treatment ( n = 5 , P = 0 . 0117 )

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C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117)


D - G Albumin levels in feces ( D on day 4 of recovery ( n = 5 )

D-G Albumin levels in feces (D on day 4 of recovery (n = 5)


colon length ( E ) on day 4 of recovery ( n = 5 )

colon length (E) on day 4 of recovery (n = 5)


H & E staining of colon tissues ( F on day 4 of recovery ( n = 5 ) . Scale bars = 100

H&E staining of colon tissues (F on day 4 of recovery (n = 5). Scale bars = 100 μm


RT - qPCR of inflammatory genes in the colon ( G ) on day 4 of recovery ( n = 5 ) . Scale bars = 100 μm . ( E : P = 0 . 0474 ; G : Tnfa P = 0 . 0190 , Il1b P = 0 .

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RT-qPCR of inflammatory genes in the colon (G) on day 4 of recovery (n = 5). Scale bars = 100 μm. (E: P = 0.0474; G: Tnfa P = 0.0190, Il1b P = 0.0301


Dose - dependent increase of C . albicans ( SC5314 ) aggregation after 4 - h exposure ( a ) and filamentation after 24 - h exposure ( b ) to different concentrations of IL - 17A in RPMI - 1640 liquid medium . Cells aliquots were imaged by microscopy and magnified in the insets . Also note the increased turbidity of cell suspension at 24 h . Scale bars , 100 μm .

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Dose-dependent increase of C. albicans (SC5314) aggregation after 4-h exposure (a) and filamentation after 24-h exposure (b) to different concentrations of IL-17A in RPMI-1640 liquid medium. Cells aliquots were imaged by microscopy and magnified in the insets. Also note the increased turbidity of cell suspension at 24 h. Scale bars, 100 μm.


( c ) Percentages of germination of C . albicans untreated ( vehicle - treated , open circle ) or treated with IL - 17A at 1 ( black triangle ) or 10 ng ml − 1 ( black square ) for 4 or 24 h . Data are mean ± s . d . P 0 . 05 , IL - 17A - treated versus vehicle - treated cells , at 4 h ; P 0 . 01 ( 1 ng ) and P 0 . 001 ( 10 ng ) at 24 h ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

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(c) Percentages of germination of C. albicans untreated (vehicle-treated, open circle) or treated with IL-17A at 1 (black triangle) or 10 ng ml−1 (black square) for 4 or 24 h. Data are mean±s.d. P 0.05, IL-17A-treated versus vehicle-treated cells, at 4 h; P 0.01 (1 ng) and P 0.001 (10 ng) at 24 h (one-way analysis of variance Bonferroni post-test) (n=3).


( d , e ) IL - 17A promotes biofilm formation . SEM images of C . albicans SC5314 untreated ( d ) or exposed to 10 ng ml − 1 IL - 17A ( e ) for 4 h and subsequently cultured onto plastic discs for 24 h . Scale bars , 10 μm .

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(d,e) IL-17A promotes biofilm formation. SEM images of C. albicans SC5314 untreated (d) or exposed to 10 ng ml−1 IL-17A (e) for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 10 μm.


( f ) IL - 17A increases resistance to nitrogen starvation . Cells were first grown in YPD medium overnight and then switched to nitrogen - free SLAD medium in the presence of 10 ng ml − 1 IL - 17A . Viability , expressed as a percentage of colony - forming units at zero time , was measured over a 15 - day period . Cells were exposed to 0 . 1 ( black circle ) , 1 ( black triangle ) or 10 ng ml − 1 ( black square ) IL - 17A or drug vehicle ( open circle ) at the beginning of the cultures or , in the case of 1 ng ml − 1 ( open triangle ) , also added every two other days . Data are mean ± s . d . P - values ranged from 0 . 05 to 0 . 001 for all samples treated with 10 ng IL - 17A ; P0 . 05 , on days 3 and 5 for samples treated 1 ng IL - 17A every two other days ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

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(f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3).


( g , l ) C . albicans cells are metabolically active . Untreated ( g - i ) or IL - 17A - treated ( 10 ng ml − 1 in RPMI - 1640 liquid media for 24 h ) ( j - l ) cells were red after staining with FUN - 1 ( g , j ) , green with Con A ( h , k ) and yellow in the merged images ( i , l ) . Metabolically active cells are shown in red , and cell wall polysaccharides are shown in green . Scale bars , 5 μm . Images from one representative experiment out of two are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(g,l) C. albicans cells are metabolically active. Untreated (g-i) or IL-17A-treated (10 ng ml−1 in RPMI-1640 liquid media for 24 h) (j-l) cells were red after staining with FUN-1 (g,j), green with Con A (h,k) and yellow in the merged images (i,l). Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Scale bars, 5 μm. Images from one representative experiment out of two are shown.


( g , h ) TOR inhibition in cells treated with 10 ng ml − 1 IL - 17A , 15 nM rapamycin or vehicle ( none ) by western blotting after 4 - h ( g ) or 24 - h ( h ) exposure .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(g,h) TOR inhibition in cells treated with 10 ng ml−1 IL-17A, 15 nM rapamycin or vehicle (none) by western blotting after 4-h (g) or 24-h (h) exposure.


i - n ) Immunofluorescence of ATG8 in untreated ( i - k ) or IL - 17A - treated ( 10 ng ml − 1 for 4 h ) ( l - n ) cells . Cells were red after TRITC - ATG8 staining ( i , l ) , green after ConA staining ( j , m ) , merged images ( k , n ) . Mean values ( % ATG8 + cells ( merge ) ; mean ± s . d . ) : ATG8 / none , 8 . 2 ± 1 . 6 ; ATG8 / IL - 17A , 58 . 0 ± 11 . 3 ( P = 0 . 002 ) ( n = 3 ) . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

i-n) Immunofluorescence of ATG8 in untreated (i-k) or IL-17A-treated (10 ng ml−1 for 4 h) (l-n) cells. Cells were red after TRITC-ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm.


( o - r ) Monodansilcadaverine ( MDC ) labelling performed on cells unexposed ( o ) or exposed to 1 ( p ) or 10 ng ml − 1 ( q ) IL - 17A for 4 h . Cells were magnified in the inset ( r ) . Representative results from three independent experiments . The mean values ( % hyphal cells with MDC - stained organelles ( merge ) ; mean ± s . d . ) were as follows : none , 9 . 5 ± 3 . 2 ; 1 ng ml − 1 IL - 17A , 14 . 0 ± 4 . 7 ; 10 ng ml − 1 IL - 17A , 56 . 2 ± 18 . 71 ( P = 0 . 004 , IL - 17A 10 ng ml − 1 versus none ) . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(o-r) Monodansilcadaverine (MDC) labelling performed on cells unexposed (o) or exposed to 1 (p) or 10 ng ml−1 (q) IL-17A for 4 h. Cells were magnified in the inset (r). Representative results from three independent experiments. The mean values (% hyphal cells with MDC-stained organelles (merge); mean±s.d.) were as follows: none, 9.5±3.2; 1 ng ml−1 IL-17A, 14.0±4.7; 10 ng ml−1 IL-17A, 56.2±18.71 (P=0.004, IL-17A 10 ng ml−1 versus none). Scale bars, 10 μm.


( s , t ) Quantitative real - time PCR of orfs . 19 . 4325 , 19 . 130 , RIC1 on cDNA from Candida SC5314 ( s ) or the TOR1 - 1 / TOR1 mutant ( t ) strains exposed to vehicle ( open column ) , 10 ng ml − 1 IL - 17A ( black column ) or 15 nM rapamycin ( grey column ) for 4 h . The actual P - values are indicated ( two - tailed t - test ) ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(s,t) Quantitative real-time PCR of orfs. 19.4325, 19.130, RIC1 on cDNA from Candida SC5314 (s) or the TOR1-1/TOR1 mutant (t) strains exposed to vehicle (open column), 10 ng ml−1 IL-17A (black column) or 15 nM rapamycin (grey column) for 4 h. The actual P-values are indicated (two-tailed t-test) (n=3).


( u , v ) Intracellular ( u ) or extracellular ( v ) cAMP levels , vehicle ( open circle ) , 10 ng ml − 1 IL - 17A ( black square ) or rapamycin ( grey square ) . P0 . 05 , IL - 17A - treated versus untreated at 1 h and P0 . 01 , IL - 17A - treated versus untreated at 24 h ( u ) , and P0 . 05 , rapamycin - treated versus untreated at 6 h ( v ) ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(u,v) Intracellular (u) or extracellular (v) cAMP levels, vehicle (open circle), 10 ng ml−1 IL-17A (black square) or rapamycin (grey square). P0.05, IL-17A-treated versus untreated at 1 h and P0.01, IL-17A-treated versus untreated at 24 h (u), and P0.05, rapamycin-treated versus untreated at 6 h (v) (one-way analysis of variance Bonferroni post-test) (n=3).


( w ) orf . 19 . 5258 expression after 24 - h Candida SC5314 exposure as in ( n , o ) . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(w) orf. 19.5258 expression after 24-h Candida SC5314 exposure as in (n,o). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


( a - g ) GFP C . albicans cells were untreated ( a - c ) or treated ( d - g ) for 4 h with 10 ng ml − 1 IL - 17A followed by anti - IL17A - PE and examined by fluorescence microscopy ( scale bars , 5 μm ) . Cells were green in ( a , d ) , red upon staining with anti - IL17A - PE ( b , e ) and double stained in the merged images ( c , f ) . Note the binding of IL - 17A to hyphal tips ( arrows and magnified in g ) ( n = 3 ) . Images are from one representative experiment out of three .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-g) GFP C. albicans cells were untreated (a-c) or treated (d-g) for 4 h with 10 ng ml−1 IL-17A followed by anti-IL17A-PE and examined by fluorescence microscopy (scale bars, 5 μm). Cells were green in (a,d), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Note the binding of IL-17A to hyphal tips (arrows and magnified in g) (n=3). Images are from one representative experiment out of three.


( h , i ) Binding of murine ( h ) or human ( i ) IL - 17A to clinical isolates of C . albicans from human urine ( yellow triangle ) , blood ( green triangle ) , vagina ( red triangle ) and gut ( blue triangle ) . IL - 17A was added to paraformaldehyde - fixed fungal cells followed by biotinylated secondary anti - IL - 17A antibody and streptavidin - horse radish peroxidase . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . P - values of samples exposed to 0 . 1 and 1 ng IL - 17A ranged from 0 . 05 to 0 . 001 ( one - way analysis of variance Bonferroni post - test ) . All samples exposed to 10 ng IL - 17A displayed significant ( P0 . 001 , one - way analysis of variance Bonferroni post - test ) OD values relative to unexposed samples ( 0 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h,i) Binding of murine (h) or human (i) IL-17A to clinical isolates of C. albicans from human urine (yellow triangle), blood (green triangle), vagina (red triangle) and gut (blue triangle). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0).


( j - o ) Direct visualization of IL - 17A on Candida freshly harvested from human vagina . Same experiment as in ( a - g ) on cells previously stained with Con A ( j , m , green ) and incubated with PE - anti - human IL - 17A ( k , n , red ) and double stained in the merged images ( l , o ) . Scale bars , 10 μm . Shown is one representative experiment out of three .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(j-o) Direct visualization of IL-17A on Candida freshly harvested from human vagina. Same experiment as in (a-g) on cells previously stained with Con A (j,m, green) and incubated with PE-anti-human IL-17A (k,n, red) and double stained in the merged images (l,o). Scale bars, 10 μm. Shown is one representative experiment out of three.


( p - r ) Cytospins of vaginal fluids from IL - 17F - deficient ( p ) , IL - 17A - deficient ( q ) and C57BL / 6 ( r ) mice intravaginally infected two days before with C . albicans SC5314 blastospores . Note the presence of hyphae in the vagina of IL - 17F - deficient mice after May - Grünwald - Giemsa staining ( scale bars , 20 μm ) . Shown is one representative experiment out of three .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(p-r) Cytospins of vaginal fluids from IL-17F-deficient (p), IL-17A-deficient (q) and C57BL/6 (r) mice intravaginally infected two days before with C.albicans SC5314 blastospores. Note the presence of hyphae in the vagina of IL-17F-deficient mice after May-Grünwald-Giemsa staining (scale bars, 20 μm). Shown is one representative experiment out of three.


( s ) Binding of IL - 17A to clinical isolates of C . krusei ( red square ) , C . glabrata ( green square ) and S . cerevisiae ( yellow square ) done as in ( h , i ) . IL - 17A was added to paraformaldehyde - fixed fungal cells followed by biotinylated secondary anti - IL - 17A antibody and streptavidin - horse radish peroxidase . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . P - values of samples exposed to 0 . 1 and 1 ng IL - 17A ranged from 0 . 05 to 0 . 001 ( one - way analysis of variance Bonferroni post - test ) . All samples exposed to 10 ng IL - 17A displayed significant ( P0 . 001 , one - way analysis of variance Bonferroni post - test ) OD values relative to unexposed samples ( 0 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(s) Binding of IL-17A to clinical isolates of C. krusei (red square), C. glabrata (green square) and S. cerevisiae (yellow square) done as in (h,i). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0).


( a ) Dose - dependent binding of IL - 17A to fungal Crh11p ( red circle ) and Gas1p ( light blue circle ) proteins , but not to the fungal polysaccharides β - 1 , 3 - D - glucan ( open circle ) , mannan ( blue circle ) and zymosan ( grey circle ) . The lack of binding of IL - 17E to Crh11p ( orange circle ) serves as a negative control . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . Data are mean ± s . d . , n = 3 . P0 . 001 , Crh11p and Gas1p without versus with IL - 17A ( one - way analysis of variance Bonferroni post - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test).


( b ) Co - immunoprecipitation assay of fungal Crh11p and murine IL - 17A proteins . Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP - conjugated antibody . C , irrelevant antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Co-immunoprecipitation assay of fungal Crh11p and murine IL-17A proteins. Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP-conjugated antibody. C, irrelevant antibody.


( c ) Failure of IL - 17A to bind to wild - type ( red square ) or mutant C . albicans strains in which the three genes of the CRH family ( UTR2 , open light blue , CRH1 , open black and CRH12 , open blue square ) had been deleted , and restoration of binding by CRH1 ( black square ) and CRH12 ( blue square ) , but not UTR2 ( light blue square ) , reconstitution . Data are mean ± s . d . ( n = 3 ) . P0 . 001 for red square , open light blue , black square and blue square ( one - way analysis of variance Bonferroni post - test ) .

(c) Failure of IL-17A to bind to wild-type (red square) or mutant C. albicans strains in which the three genes of the CRH family (UTR2, open light blue, CRH1, open black and CRH12, open blue square) had been deleted, and restoration of binding by CRH1 (black square) and CRH12 (blue square), but not UTR2 (light blue square), reconstitution. Data are mean±s.d. (n=3). P0.001 for red square, open light blue, black square and blue square (one-way analysis of variance Bonferroni post-test).


( d , e ) Cellular localization of bound IL - 17A . Electron micrographs of ultrafine sections of C . albicans MKY 378 wild - type ( d ) or the crh11Δ mutant ( e ) incubated with biotinylated IL - 17A and streptavidin gold - conjugated particles . Arrows indicate immunogold particles . Scale bars , 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) Cellular localization of bound IL-17A. Electron micrographs of ultrafine sections of C. albicans MKY 378 wild-type (d) or the crh11Δ mutant (e) incubated with biotinylated IL-17A and streptavidin gold-conjugated particles. Arrows indicate immunogold particles. Scale bars, 200 nm.


Fungi were harvested from the stomachs of C57BL / 6 ( a - c ) , IL - 17F - deficient ( d - f ) or IL - 17A - deficient ( g - i ) mice infected with GFP - Candida for 2 days , and stained using PE - labeled antibody to IL - 17A . Cells were green in ( a , d and g ) , red upon staining with anti - IL17A - PE ( b , e and h ) and double stained in the merged images ( c , f and i ) . Scale bars , 10 μm . Images are from one representative experiment out of two . ( j - l ) SEM images showing fungal germination in the stomachs of C57BL / 6 mice ( j ) , filamentation in IL - 17F - deficient mice ( k ) and neither one in IL - 17A - deficient ( l ) mice intragastrically infected 2 days before . Scale bars , 10 μm . ( a - l ) Images are from one representative experiment out of two .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fungi were harvested from the stomachs of C57BL/6 (a-c), IL-17F-deficient (d-f) or IL-17A-deficient (g-i) mice infected with GFP-Candida for 2 days, and stained using PE-labeled antibody to IL-17A. Cells were green in (a,d and g), red upon staining with anti-IL17A-PE (b,e and h) and double stained in the merged images (c,f and i). Scale bars, 10 μm. Images are from one representative experiment out of two. (j-l) SEM images showing fungal germination in the stomachs of C57BL/6 mice (j), filamentation in IL-17F-deficient mice (k) and neither one in IL-17A-deficient (l) mice intragastrically infected 2 days before. Scale bars, 10 μm. (a-l) Images are from one representative experiment out of two.


( m - o ) C57BL / 6 mice were infected intragastrically with C . albicans MKY 378 strain ( m ) or the crh11Δ ( n ) and crh12Δ ( o ) mutants , and were monitored for fungal growth ( log10 colony - forming units ( CFU ) at 2 ( open circle ) or 8 ( black circle ) days post infection ) . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) for 2 and 8 days ( m ) and for 2 days ( o ) . Fungal cells were untreated ( none ) or exposed to 10 ng ml − 1 IL - 17A for 4 h before injection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(m-o) C57BL/6 mice were infected intragastrically with C. albicans MKY 378 strain (m) or the crh11Δ (n) and crh12Δ (o) mutants, and were monitored for fungal growth (log10 colony-forming units (CFU) at 2 (open circle) or 8 (black circle) days post infection). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test) for 2 and 8 days (m) and for 2 days (o). Fungal cells were untreated (none) or exposed to 10 ng ml−1 IL-17A for 4 h before injection.


Candida SC5314 ( a ) , TOR1 - 1 / TOR1 ( b ) and atg9Δ / atg9Δ ( c ) mutant strains were untreated ( open column ) or pre - incubated with IL - 17A ( filled column ) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion , phagocytosis and killing . Error bars , mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Candida SC5314 (a), TOR1-1/TOR1(b) and atg9Δ/atg9Δ (c) mutant strains were untreated (open column) or pre-incubated with IL-17A (filled column) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion, phagocytosis and killing. Error bars, mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


i - n ) Immunofluorescence of ATG8 in untreated ( i - k ) or IL - 17A - treated ( 10 ng ml − 1 for 4 h ) ( l - n ) cells . Cells were red after TRITC - ATG8 staining ( i , l ) , green after ConA staining ( j , m ) , merged images ( k , n ) . Mean values ( % ATG8 + cells ( merge ) ; mean ± s . d . ) : ATG8 / none , 8 . 2 ± 1 . 6 ; ATG8 / IL - 17A , 58 . 0 ± 11 . 3 ( P = 0 . 002 ) ( n = 3 ) . Scale bars , 10 μm .

i-n) Immunofluorescence of ATG8 in untreated (i-k) or IL-17A-treated (10 ng ml−1 for 4 h) (l-n) cells. Cells were red after TRITC-ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm.


( g ) Fungi purified from the stomachs of mice infected as in ( d - f ) were subjected to RT - PCR for autophagy genes expression . C + , fungal cells grown on SPIDER solid medium . C - , no cells .

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(g) Fungi purified from the stomachs of mice infected as in (d-f) were subjected to RT-PCR for autophagy genes expression. C+, fungal cells grown on SPIDER solid medium. C-, no cells.


( h - j ) Log10 colony - forming units ( CFU ) at 2 days post infection with C . albicans SC5314 ( wild - type ) or different mutant strains in C57BL / 6 ( h ) , IL - 17F - deficient ( i ) or IL - 17A - deficient ( j ) mice . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h-j) Log10 colony-forming units (CFU) at 2 days post infection with C. albicans SC5314 (wild-type) or different mutant strains in C57BL/6 (h), IL-17F-deficient (i) or IL-17A-deficient (j) mice. Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).