( J and K ) Glutamatergic projection neurons derived from FKRP and corrected ( 5D17 , 5D23 and 3B17 ) progenitor cells . The vast majority of neurons contain vGlut1 + punctae in their neurites ( labelled by Tuj1 ) . Right panels are enlarged images from the insets of left panels . Data information : Scale bars , 50 μm .

O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-SUBCELLULAR O O B-CELL_TYPE O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.


( A ) Molecular weight of glycosylated α - dystroglycan ( IIH6 epitope ) in WT - iPSC derived cortical neurons is similar to that in the mouse brain ( ~ 120 kDa ) and lower than that in the mouse muscle ( ~ 156kDa ) . IIH6 reactivity was almost not detected in cortical neurons derived from FKRP - iPSCs . Note that the β - Actin antibody does not cross - react with the muscle sample .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE O O

(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.


( B ) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC - derived cortical neurons . Intensities of IIH6 reactivity are normalized to β - Actin expression , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * * p < 0 . 01 )

O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O 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) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)


( C ) Representative laminin overlays show that targeted gene correction restores laminin - binding activity in iPSC - derived cortical neurons . Intensities of laminin - binding activity are normalized to β - dystroglycan , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * p < 0 . 05 ) .

O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).


( C ) Dose response curve . H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed , stained and imaged as described above . The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non - linear regression curve was fitted for the 3 compounds showing the highest increase in α - dystroglycan glycosylation ( IIH6 immunostaining ) . Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown . HPFPTzone : ( 5Z ) - 5 - [ ( 3 - ethoxy - 4 - hydroxy - phenyl ) methylene ] - 3 - ( 4 - fluorophenyl ) - 2 - thioxo - thiazolidin - 4 - one . 4BPPNit : 4 - ( 4 - bromophenyl ) - 6 - ethylsulfanyl - 2 - oxo - 3 , 4 - dihydro - 1H - pyridine - 5 - carbonitrile . DPPTzone : ( 5E ) - 5 - [ ( 2 , 5 - dimethyl - 1 - phenyl - pyrrol - 3 - yl ) methylene ] - 3 - phenyl - 2 - thioxo - thiazolidin - 4 - one . Regression curve and EC50 values were calculated using GraphPad Prism 7 software . In images shown , green represents glycosylated α - dystroglycan and blue Hoechst staining . Scale bar , 50 µm .

(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.


( E ) Flow cytometry analysis confirmed that 4BPPNit treatment ( 20 µM ) significantly increased IIH6 - reactive glycan in mouse H2K 2B4 cells , compared with untreated cells . There was no significant difference in the percentage of IIH6 - positive cells . Values indicate mean ± s . e . m . ( n = 6 , triplicates of 2 independent experiments ; t - test ; NS , not significant ; * * p < 0 . 01 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).


( A ) FKRP - NSCs treated with 4BPPNit showed increased IIH6 reactivity , compared with untreated control cells . Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR - corrected NSCs . Under the same exposure time , the signal intensity of IIH6 reactivity is saturated in corrected - NSCs . Data information : Intensities of glycosylation are normalized to GAPDH protein expression .

O O O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O

(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression.


( B ) Representative immunoblots with the IIH6 antibody using FKRP - and corrected - NSCs treated with 4BPPNit for 24 , 48 and 72 h , and DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time .

O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O

(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time.


( C ) Quantification of α - dystroglycan glycosylation ( IIH6 ) , compared with DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( D ) Representative immunoblots of laminin - binding analysis using FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( E ) Quantification of laminin - binding activities , compared with DMSO only controls . Data information : Intensities of laminin - binding activity are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( F ) Representative immunoblots with the AF6868 antibody detecting both α - dystroglycan core protein and β - dystroglycan in FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( G ) Quantification of α - dystroglycan core protein and β - dystroglycan expression , compared with DMSO only controls . Data information : Intensities of protein expression are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( A ) DAG1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01


( B ) FKRP gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( C ) LARGE1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

(C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


B - D . Mice were analyzed by Kaplan - Meier survival plot ( B ) , the percentage of body weight ( C ) , and the clinical scores ( D ) of WT and GPx8 - / - mice with colitis induced by 4 % DSS for 6 days ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-ORGANISM O B-DISEASE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( E ) Images and statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . , 5 - 6 mice were used for each experiment . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections . The infiltrated immune cells at the transmural extensions were indicated by arrowhead . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colons treated with 4% DSS for 5 days were collected on day 14. , 5-6 mice were used for each experiment. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( G ) Colon sections were stained with markers for macrophages ( F4 / 80 ) and anti - GPx8 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colons treated with 4% DSS for 5 days were collected on day 14. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing ( n = 5 per group ) and are presented by a heat map , ( I ) , and principal coordinates analysis , ( J ) .

B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing (n = 5 per group) and are presented by a heat map, (I), and principal coordinates analysis, (J).


Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . ( K ) α - diversity indicated bacteria species in each mouse depicted by richness ( total operational taxonomic units , OTUs ) , the Chao1 richness estimator ( Chao1 ) , and the abundance - based coverage estimator ( ACE ) metrics . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. (K) α-diversity indicated bacteria species in each mouse depicted by richness (total operational taxonomic units, OTUs), the Chao1 richness estimator (Chao1), and the abundance-based coverage estimator (ACE) metrics. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


B . Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages ( F4 / 80 ) .

O O B-TISSUE O B-ORGANISM O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O

B. Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages (F4/80).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . ( C ) The percentage of body weight . ( D ) The clinical scores of mice . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

O O B-CELL_TYPE O O O O O B-ORGANISM O B-CELL_TYPE O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). (C) The percentage of body weight. (D) The clinical scores of mice. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( E ) Statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

O O B-CELL_TYPE O O O O O B-ORGANISM O B-CELL_TYPE O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O B-TISSUE O O B-CELL_TYPE O B-ORGANISM O O O O O O B-DISEASE O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (E) Statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

O O B-CELL_TYPE O O O O O B-ORGANISM O B-CELL_TYPE O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O B-TISSUE O O B-CELL_TYPE O B-ORGANISM O O O O O O B-DISEASE O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (F) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( G ) Colon sections stained with anti - F4 / 80 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

O O B-CELL_TYPE O O O O O B-ORGANISM O B-CELL_TYPE O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O B-TISSUE O O B-CELL_TYPE O B-ORGANISM O O O O O O B-DISEASE O O O O O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (G) Colon sections stained with anti-F4/80. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . D ) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

O O B-CELL_TYPE O O O O O B-ORGANISM O B-CELL_TYPE O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O B-TISSUE O O B-CELL_TYPE O B-ORGANISM O O O O O O B-DISEASE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). D) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


A . Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency . BMDMs were primed for 6 h with 0 . 5 μg / ml LPS and then transfected with LPS ( TF LPS ) for 8 h , stimulated with ATP ( 5 mM ) for 3 h , MSU ( 150 μg / mL ) for 8 h , or nigericin ( 5 μM ) for 3 h . Activation of inflammasome was demonstrated by secreted IL - 1β . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency. BMDMs were primed for 6 h with 0.5 μg/ml LPS and then transfected with LPS (TF LPS) for 8 h, stimulated with ATP (5 mM) for 3 h, MSU (150 μg/mL) for 8 h, or nigericin (5 μM) for 3 h. Activation of inflammasome was demonstrated by secreted IL-1β. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


B . GPx8 deficiency enhanced noncanonical inflammasome activation . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SUBCELLULAR B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GPx8 deficiency enhanced noncanonical inflammasome activation. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


C . Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs . BMDMs were primed for 6 h with 1 μg / ml poly ( I : C ) and then transfected with LPS for 8 h . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs. BMDMs were primed for 6 h with 1 μg/ml poly(I:C) and then transfected with LPS for 8 h. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( D ) Activation of noncanonical inflammasome in BMDMs with empty vector ( EV ) or hGPx8 was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O O O O B-SUBCELLULAR O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (D) Activation of noncanonical inflammasome in BMDMs with empty vector (EV) or hGPx8 was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( E ) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats .

O O O O O B-SUBCELLULAR O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (E) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.


F . GPx8 deficiency enhanced caspase - 11 cleavage and activation . Levels of processed caspase - 1 and - 11 in the supernatants ( Sup ) and full - length caspase - 1 , - 11 and GAPDH in the cell lysates ( Cell ext ) were determined by immunoblotting . Fold changes of protein expression were normalized with internal controls and are indicated below the blots .

O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

F. GPx8 deficiency enhanced caspase-11 cleavage and activation. Levels of processed caspase-1 and -11 in the supernatants (Sup) and full-length caspase-1, -11 and GAPDH in the cell lysates (Cell ext) were determined by immunoblotting. Fold changes of protein expression were normalized with internal controls and are indicated below the blots.


G . RNA expression of caspase - 11 was not altered by GPx8 deficiency . Caspase - 11 RNA expression of BMDMs isolated from GPx8 - / - mice was measured after LPS priming . Data information : ata are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O 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. RNA expression of caspase-11 was not altered by GPx8 deficiency. Caspase-11 RNA expression of BMDMs isolated from GPx8-/- mice was measured after LPS priming. Data information: ata are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.


H . GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo . Colon samples from 5 WT or GPx8 - / - mice treated with DSS for 5 days were collected on day 7 . Levels of full - length and processed caspase - 1 , - 11 and gasdermin D ( GSDMD ) in tissue lysates were individually analyzed by immunoblots .

O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O

H. GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo. Colon samples from 5 WT or GPx8-/- mice treated with DSS for 5 days were collected on day 7. Levels of full-length and processed caspase-1, -11 and gasdermin D (GSDMD) in tissue lysates were individually analyzed by immunoblots.


GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( I ) Survival of mice primed with ultra pure LPS ( E . coli O55 : B5 , 400 μg / kg ) and re - challenged 6 h later with LPS ( 40 mg / kg ) ( n = 8 per group ) .

B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-ORGANISM O O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (I) Survival of mice primed with ultra pure LPS (E. coli O55:B5, 400 μg/kg) and re-challenged 6 h later with LPS (40 mg/kg) (n = 8 per group).


GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( J ) Survival of mice primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 10 mg / kg ) ( n = 9 per group ) .

B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (J) Survival of mice primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (10 mg/kg) (n = 9 per group).


GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( K ) IL - 1β production levels in mice sera were measured 2 h after being primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 1 mg / kg ) ( n = 8 - 9 per group ) . In ( K ) , data are presented as the mean ± SEM . NS , no significance . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (K) IL-1β production levels in mice sera were measured 2 h after being primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (1 mg/kg) (n = 8-9 per group). In (K), data are presented as the mean ± SEM. NS, no significance. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


A . Interaction of GPx8 with human caspase - 4 ( Casp4 ) or mouse caspase - 11 ( Casp11 ) was confirmed by co - IP assays . 293T cells were co - transfected with vectors expressing FLAG tagged Casp4 or Casp11 , in addition to GPx8 expressing vectors . Cell lysate containing Casp4 - or Casp11 - FLAG proteins was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 and anti - FLAG Ab ( left panel ) . GPx8 and its interacting proteins were precipitated by anti - GPx8 Ab and analyzed by the indicated Abs ( right panel ) .

O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O

A. Interaction of GPx8 with human caspase-4 (Casp4) or mouse caspase-11 (Casp11) was confirmed by co-IP assays. 293T cells were co-transfected with vectors expressing FLAG tagged Casp4 or Casp11, in addition to GPx8 expressing vectors. Cell lysate containing Casp4- or Casp11-FLAG proteins was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 and anti-FLAG Ab (left panel). GPx8 and its interacting proteins were precipitated by anti-GPx8 Ab and analyzed by the indicated Abs (right panel).


B . Co - localization of Myc - tagged GPx8 ( red ) and endogenous caspase - 4 ( green ) in THP - 1 cells .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

B. Co-localization of Myc-tagged GPx8 (red) and endogenous caspase-4 (green) in THP-1 cells.


C . Interaction between GPx8 and caspase - 4 in THP - 1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay ( PLA ) ( red dots , labeled by arrowheads ) . GPx8 - Myc stably expressing THP - 1 were used to detect the interaction between GPx8 - Myc and endogenous caspase - 4 . Untreated cells ( Ctrl ) or cells primed with LPS ( primed LPS ) were incubated with anti - Myc and anti - caspase - 4 Abs , according to the manufacturer ' s instructions , and the nucleus was stained with DAPI ( blue ) . Results were quantified by counting at least 5 different fields with an average of 300 cells . Reactions without primary Abs were used as the control for the PLA assay ( PLA Ctrl ) . Data information : All data are representative of at least 3 independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Interaction between GPx8 and caspase-4 in THP-1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay (PLA) (red dots, labeled by arrowheads). GPx8-Myc stably expressing THP-1 were used to detect the interaction between GPx8-Myc and endogenous caspase-4. Untreated cells (Ctrl) or cells primed with LPS (primed LPS) were incubated with anti-Myc and anti-caspase-4 Abs, according to the manufacturer's instructions, and the nucleus was stained with DAPI (blue). Results were quantified by counting at least 5 different fields with an average of 300 cells. Reactions without primary Abs were used as the control for the PLA assay (PLA Ctrl). Data information: All data are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( D ) Complexes of GPx8 and caspase - 4 were demonstrated by co - IP assays . Cysteine - dependent covalent interactions of GPx8 and caspase - 4 ( C258S ) were analyzed under non - reducing and reducing conditions . Disulfide - linked complexes of Gpx8 and caspase - 4 are indicated .

B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O

293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (D) Complexes of GPx8 and caspase-4 were demonstrated by co-IP assays. Cysteine-dependent covalent interactions of GPx8 and caspase-4 (C258S) were analyzed under non-reducing and reducing conditions. Disulfide-linked complexes of Gpx8 and caspase-4 are indicated.


293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( E ) Interactions of GPx8 and caspase - 4 in response to H2O2 treatment were demonstrated by co - IP assays of lysates prepared from 293T cells treated with H2O2 ( 200 μM ) .

B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O

293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (E) Interactions of GPx8 and caspase-4 in response to H2O2 treatment were demonstrated by co-IP assays of lysates prepared from 293T cells treated with H2O2 (200 μM).


293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( F ) C79S , C108S , and C2S2 mutants of GPx8 abolished most of the binding to caspase - 4 by co - IP assays .

B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (F) C79S, C108S, and C2S2 mutants of GPx8 abolished most of the binding to caspase-4 by co-IP assays.


C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . THP - 1 cells stably expressing empty vector ( EV ) , WT , or mutant GPx8 proteins were confirmed by immunoblotting ( G ) .

O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O

C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. THP-1 cells stably expressing empty vector (EV), WT, or mutant GPx8 proteins were confirmed by immunoblotting (G).


C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . ( H and I ) Cells were treated with 12 - O - tetradecanoylphorbol 13 - actate ( PMA ) ( 0 . 1 μM ) and differentiated for 3 days then primed and transfected with LPS . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( H ) and LDH release ( I ) . Data information : All data are representative of at least 3 independent experiments . In H and I , data are presented as the mean ± SD , n = 4 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SUBCELLULAR B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. (H and I) Cells were treated with 12-O-tetradecanoylphorbol 13-actate (PMA) (0.1 μM) and differentiated for 3 days then primed and transfected with LPS. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (H) and LDH release (I). Data information: All data are representative of at least 3 independent experiments. In H and I, data are presented as the mean ± SD, n=4, technical repeats. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


J . E . coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase - 4 ( C258S ) to form a disulfide - linked complex ( oxidized GPx8 : oGPx8 ; reduced GPx8 : rGPx8 ) . Lanes 2 and 3 , GPx8 pretreated with 1 and 5 mM H2O2 , respectively . Lanes 4 and 5 , Gpx8 pretreated with 50 and 200 mM DTT , respectively . Disulfide - linked complexes of recombinant Gpx8 and caspase - 4 ( C258S ) are indicated .

O O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

J. E. coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase-4 (C258S) to form a disulfide-linked complex (oxidized GPx8: oGPx8; reduced GPx8: rGPx8). Lanes 2 and 3, GPx8 pretreated with 1 and 5 mM H2O2, respectively. Lanes 4 and 5, Gpx8 pretreated with 50 and 200 mM DTT, respectively. Disulfide-linked complexes of recombinant Gpx8 and caspase-4 (C258S) are indicated.


K . GPx8 inhibited the activation of caspase - 4 by decreasing the oligomerization of caspase - 4 . Oxidized GPx8 and caspase - 4 ( C258S ) were co - incubated for 30 min . Different amounts of LPS were added to the complex and incubated for another 30 min . Oligomerized caspase - 4 was analyzed by native blue PAGE according to the manufacturer ' s instructions . Mock : without GPx8 ; oxidized GPx8 : oGPx8 ; oxidized GPx8C79S : oC79S . Data information : All data are representative of at least 3 independent experiments . In K , quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers / total proteins . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. GPx8 inhibited the activation of caspase-4 by decreasing the oligomerization of caspase-4. Oxidized GPx8 and caspase-4 (C258S) were co-incubated for 30 min. Different amounts of LPS were added to the complex and incubated for another 30 min. Oligomerized caspase-4 was analyzed by native blue PAGE according to the manufacturer's instructions. Mock: without GPx8; oxidized GPx8: oGPx8; oxidized GPx8C79S: oC79S. Data information: All data are representative of at least 3 independent experiments. In K, quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers/total proteins. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


A . Casp4C118S mutant partially abolished the covalent disulfide - link with GPx8 . Interacting GPx8 were co - immunoprecipitated with FLAG - tagged caspase - 4 ( WT , C118S , C329S mutants ) and analyzed under reducing ( + DTT ) or non - reducing ( - DTT ) conditions . Disulfide - linked complexes of GPx8 and caspase - 4 are indicated . Fold change of complexes was normalized with internal controls and indicated below blots .

O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O

A. Casp4C118S mutant partially abolished the covalent disulfide-link with GPx8. Interacting GPx8 were co-immunoprecipitated with FLAG-tagged caspase-4 (WT, C118S, C329S mutants) and analyzed under reducing (+DTT) or non-reducing (-DTT) conditions. Disulfide-linked complexes of GPx8 and caspase-4 are indicated. Fold change of complexes was normalized with internal controls and indicated below blots.


C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . THP - 1 cells stably expressing empty vector ( EV ) , FLAG - tagged caspase - 4 ( WT , C258S , C118S mutants ) , or GPx8 ( WT and C79S mutants ) were confirmed by immunoblotting ( B ) . Data information : All data shown are representative of at least 3 independent experiments .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. THP-1 cells stably expressing empty vector (EV), FLAG-tagged caspase-4 (WT, C258S, C118S mutants), or GPx8 (WT and C79S mutants) were confirmed by immunoblotting (B). Data information: All data shown are representative of at least 3 independent experiments.


C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . ( C - D ) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS . THP - 1 cells were differentiated , primed , and transfected with LPS . Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL - 1β ( C ) and LDH ( D ) . In C and D , data are presented as the mean ± SD , n = 4 , technical repeats . * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. (C-D) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS. THP-1 cells were differentiated, primed, and transfected with LPS. Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL-1β (C) and LDH (D). In C and D, data are presented as the mean ± SD, n=4, technical repeats. ***P < 0.001 (Student's two‐tailed t‐test).


A . The caspase - 4 / 11 and - 1 inhibitor , VX - 765 , inhibited noncanonical inflammasome activation . BMDMs isolated from mice were primed with LPS for 5 - 6 h , treated with 20 μM VX - 765 , and transfected with LPS for 12 h . Data information data are presented as the mean ± SD , n = 5 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-CELL_TYPE O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The caspase-4/11 and -1 inhibitor, VX-765, inhibited noncanonical inflammasome activation. BMDMs isolated from mice were primed with LPS for 5-6 h, treated with 20 μM VX-765, and transfected with LPS for 12 h. Data information data are presented as the mean ± SD, n=5, technical repeats. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and analyzed by a Kaplan - Meier survival plot ( B ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . Data information : , data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-DISEASE O O O O O O O O B-EXP_ASSAY O O O O O B-ORGANISM O B-DISEASE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and analyzed by a Kaplan-Meier survival plot (B) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). Data information: , data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and the percentage of body weight ( C ) and clinical scores ( D ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-DISEASE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O B-DISEASE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and the percentage of body weight (C) and clinical scores (D) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Full - length and cleavage forms of caspase - 11 , caspase - 1 , and GSDMD ( E ) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-DISEASE O B-TISSUE O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Full-length and cleavage forms of caspase-11, caspase-1, and GSDMD (E) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Production of IL - 1β and IL - 6 ( F ) in colon tissue lysates was assessed on day 14 after treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-DISEASE O B-TISSUE O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Production of IL-1β and IL-6 (F) in colon tissue lysates was assessed on day 14 after treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


A . GPx8 was predominantly expressed in macrophages of UC patients . Colon biopsies of non - inflamed regions from UC patients were stained with the pan - macrophage marker , CD68 , or the monocyte / macrophage activating marker , CD163 , combined with anti - GPx8 . Quantification of double - positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double - positive cells . The total cell numbers are the sum of double - and single - positive cells .

O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O B-DISEASE B-ORGANISM O B-TISSUE O O O O O O O B-DISEASE B-ORGANISM O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

A. GPx8 was predominantly expressed in macrophages of UC patients. Colon biopsies of non-inflamed regions from UC patients were stained with the pan-macrophage marker, CD68, or the monocyte/macrophage activating marker, CD163, combined with anti-GPx8. Quantification of double-positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double-positive cells. The total cell numbers are the sum of double- and single-positive cells.


B . Relative expression levels of GPx8 , GPx7 and caspase - 4 in non - inflamed biopsy specimens from healthy controls ( Ctrl ) ( n = 14 ) and UC patients ( n = 25 ) are demonstrated by immunoblots . C . Data are normalized to the mean of healthy controls and presented as the mean ± SEM . Mann Whitney test for GPx8 and Student ' s two ‐ tailed t ‐ test for GPx7 and caspase - 4 expression analysis . Data information : NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O B-EXP_ASSAY 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-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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. Relative expression levels of GPx8, GPx7 and caspase-4 in non-inflamed biopsy specimens from healthy controls (Ctrl) (n = 14) and UC patients (n = 25) are demonstrated by immunoblots. C. Data are normalized to the mean of healthy controls and presented as the mean ± SEM. Mann Whitney test for GPx8 and Student's two‐tailed t‐test for GPx7 and caspase-4 expression analysis. Data information: NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.


D . Paired GPx8 and caspase - 4 expression from each individual are linked by lines and presented as the mean ± SEM . The p - value was analyzed by the Fisher ' s exact test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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D. Paired GPx8 and caspase-4 expression from each individual are linked by lines and presented as the mean ± SEM. The p-value was analyzed by the Fisher's exact test. *P < 0.05; **P < 0.01; ***P < 0.001.


( D ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells 56 h after transfection with the indicated siRNAs . When indicated , cells were exposed to 100 nM rapamycin for the last 3 h . RPTOR ( raptor ) and BECN1 ( beclin1 ) siRNAs served as positive and negative controls , respectively .

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(D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively.


( E ) Autophagic flux was measured as the ratio between luciferase activities in MCF7 - RLuc - LC3WT and MCF7 - RLuc - LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h . Error bars are SDs for a representative ( n = 5 ) triplicate experiment with a minimum of 4 × 10 randomly chosen areas / sample analyzed ( D ) or three independent experiments ( F ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , as compared with similarly treated control siRNA - transfected samples .

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(E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.


( A ) Representative immunoblots of indicated proteins from whole - cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0 - 40 min of amino acid starvation [ − AA ] ) .

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(A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0-40 min of amino acid starvation [−AA]).


( B ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar ( siRNA - resistant CIP2A ) - encoding lentivirus , transfected with the indicated siRNAs for 53 h , and treated with 100 nM rapamycin ( Rapa ) for 0 - 20 min .

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(B) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar (siRNA-resistant CIP2A)-encoding lentivirus, transfected with the indicated siRNAs for 53 h, and treated with 100 nM rapamycin (Rapa) for 0-20 min.


( C ) MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar - encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP - LC3 puncta formation .

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(C) MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar-encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP-LC3 puncta formation.


( D ) MCF7 - EGFP - LC3 cells transfected with control ( − ) and CIP2A siRNAs together with an empty vector ( − ) or MYC - S62A cDNA as indicated were analyzed 56 h later for EGFP - LC3 puncta ( left ) and expression of indicated proteins ( right ) .

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(D) MCF7-EGFP-LC3 cells transfected with control (−) and CIP2A siRNAs together with an empty vector (−) or MYC-S62A cDNA as indicated were analyzed 56 h later for EGFP-LC3 puncta (left) and expression of indicated proteins (right).


( E ) Representative immunoblots ( left ) of indicated proteins from whole - cell lysates of MCF7 cells treated with the control ( C ) , RPTOR ( RP ) , or CIP2A ( CIP ) siRNAs for 56 h and densitometric quantification of three independent experiments ( right ) . Error bars show SDs for three independent experiments . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , compared with Student ' s t test as indicated ( C and D ) or with control siRNA - transfected samples ( E ) . Black lines indicate that intervening lanes have been spliced out .

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(E) Representative immunoblots (left) of indicated proteins from whole-cell lysates of MCF7 cells treated with the control (C), RPTOR (RP), or CIP2A (CIP) siRNAs for 56 h and densitometric quantification of three independent experiments (right). Error bars show SDs for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with Student's t test as indicated (C and D) or with control siRNA-transfected samples (E). Black lines indicate that intervening lanes have been spliced out.


( A , top left ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells infected with control or CIP2A lentiviral shRNAs . Representative immunofluorescence images ( right ) and immunoblots ( bottom ) are shown . ( B ) Mean cell diameters of MCF7 - EGFP - LC3 cells infected as in A were measured with a cytometer ( NucleoCounter NC - 3000 ; Chemometec AS ) equipped with Via1 - Cassettes ( Chemometec AS ) .

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(A, top left) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells infected with control or CIP2A lentiviral shRNAs. Representative immunofluorescence images (right) and immunoblots (bottom) are shown. (B) Mean cell diameters of MCF7-EGFP-LC3 cells infected as in A were measured with a cytometer (NucleoCounter NC-3000; Chemometec AS) equipped with Via1-Cassettes (Chemometec AS).


( C ) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin ( Rapa ) for 72 h . The viable cells were either visualized after fixation and crystal violet staining ( left ; representative figure , n = 4 ) or trypsinized and counted with NucleoCounter NC - 3000 equipped with Via1 - Cassettes ( right ) .

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(C) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin (Rapa) for 72 h. The viable cells were either visualized after fixation and crystal violet staining (left; representative figure, n = 4) or trypsinized and counted with NucleoCounter NC-3000 equipped with Via1-Cassettes (right).


( D ) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA , 2 µM MG132 , 5 µM siramesine ( Sira ) , or 100 µM etoposide ( Etop ) for 48 h , and the metabolic activity ( cell density ) was measured by the MTT assay ( left ) , and cell death was measured by the LDH assay ( right ) .

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(D) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA, 2 µM MG132, 5 µM siramesine (Sira), or 100 µM etoposide (Etop) for 48 h, and the metabolic activity (cell density) was measured by the MTT assay (left), and cell death was measured by the LDH assay (right).


( E ) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A - encoding lentivirus .

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(E) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A-encoding lentivirus.


( F ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF10A ( left ) and HEK - 293 ( right ) cells infected as in E and treated with 100 nM rapamycin for the indicated times . Black lines indicate that intervening lanes have been spliced out .

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(F) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF10A (left) and HEK-293 (right) cells infected as in E and treated with 100 nM rapamycin for the indicated times. Black lines indicate that intervening lanes have been spliced out.


CIP2A expression correlates with mTORC1 activity in primary human breast tumors . TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores ( arbitrary units [ a . u . ] ) for duplicate samples were averaged and plotted according to mean CIP2A expression ( left ) . The red lines are the regression lines , and the stippled red lines define the 95 % confidence intervals . Statistical analysis of data was performed using NCSS 2007 and Prism version 5 . Examples of the staining of breast cancer ( a ) and normal ( b ) cores indicated in the P - T389 - S6K1 / CIP2A plot are shown on the right . rsp , Spearman ' s correlation coefficient .

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CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman's correlation coefficient.


( A and B ) In vitro translated full - length CIP2A was incubated with purified human PP2A complex and HA - Raptor ( A ) or HA - S6K1 ( B ) purified from transiently transfected MCF7 cells . Protein complexes were immunoprecipitated with anti - HA antibody and analyzed by immunoblotting as indicated . Nonspecific IgG served as a control . Note that human reticulocyte ( RC ) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac .

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(A and B) In vitro translated full-length CIP2A was incubated with purified human PP2A complex and HA-Raptor (A) or HA-S6K1 (B) purified from transiently transfected MCF7 cells. Protein complexes were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting as indicated. Nonspecific IgG served as a control. Note that human reticulocyte (RC) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac.


( C ) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right .

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(C) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.


( D ) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control ( Ctr ) or CIP2A shRNA lentiviruses ( Fig . 3 A ) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right .

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(D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.


( E ) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control ( C ) or PPP2R1A ( R1A ) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies . Immunoblots of corresponding cell lysates are shown on the left .

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(E) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control (C) or PPP2R1A (R1A) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies. Immunoblots of corresponding cell lysates are shown on the left.


( F ) HA - S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses ( Fig . 3 A ) , transiently transfected with HA - RPS6K1 cDNA and starved for amino acids for 20 min when indicated , were analyzed by immunoblotting . Immunoblots of corresponding cell lysates are shown on the right .

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(F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right.


( G ) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full - length CIP2A . Error bars are SDs for four ( D ) independent experiments or a representative triplicate experiment ( C and G ) .

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(G) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full-length CIP2A. Error bars are SDs for four (D) independent experiments or a representative triplicate experiment (C and G).


( H ) Representative confocal microscopy images of untreated and amino acid - starved ( 4 h ) MCF7 cells stained for endogenous CIP2A and raptor . Black lines indicate that intervening lanes have been spliced out . − AA , amino acid starvation ; IP , immunoprecipitation ; a . u . , arbitrary unit . Bar , 10 µm .

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(H) Representative confocal microscopy images of untreated and amino acid-starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.


( A ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0 - 24 h .

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(A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0-24 h.


( B ) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A . AA - starv , amino acid starvation ; a . u . , arbitrary unit . Error bars show SDs for three independent experiments .

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(B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments.


( C ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium ( 0 ) or amino acid starvation ( − AA ) for 12 h . Ctr , control .

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(C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control.


( D ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells treated for 0 - 5 h with the indicated combinations of 100 µM cycloheximide ( CHX ) , 100 nM rapamycin , and 2 nM ConA .

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(D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0-5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA.


( E ) Representative confocal images of MCF7 ( left and middle ) and MCF7 - LC3 - EGFP ( right ) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h , fixed , and stained with antibodies against CIP2A . Nuclei in MCF - LC3 - EGFP cells were visualized with Hoechst . Green arrowheads indicate cells with many LC3 - positive puncta and low CIP2A levels , and yellow arrowheads show cells in which LC3 and CIP2A colocalize . Bars , 10 µm .

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(E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm.


( F ) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin ( Rapa ) and 2 nM ConA as indicated . Mouse IgG served as a negative control .

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(F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control.


( G ) MCF7 cells were treated with 100 nM rapamycin , 2 nM ConA , and 2 µM MG132 for 24 h as indicated . Endogenous CIP2A was immunoprecipitated ( IP ) in stringent conditions ( no coimmunoprecipitation of p62 ) and analyzed by immunoblotting using antibodies against CIP2A , monoubiquitin , and polyubiquitin . Short ( < 100 kD ) , medium ( > 150 kD ) , and long ( 100 - 130 kD ) exposures are shown .

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(G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (<100 kD), medium (>150 kD), and long (100-130 kD) exposures are shown.


( H ) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions ( see G ) and analyzed by immunoblotting for CIP2A and monoubiquitin .

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(H) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions (see G) and analyzed by immunoblotting for CIP2A and monoubiquitin.


( I ) Lysates from MCF7 cells transfected with the indicated ubiquitin - HA constructs were subjected to anti - CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA .

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(I) Lysates from MCF7 cells transfected with the indicated ubiquitin-HA constructs were subjected to anti-CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA.


( J ) Lysates from MCF7 cells transfected with the CIP2A - FLAG construct ( Junttila et al . , 2007 ) and the indicated ubiquitin - HA constructs were subjected to anti - FLAG immunopurification in denaturing conditions with 0 . 1 % SDS followed by immunoblotting with the indicated antibodies . Black lines indicate that intervening lanes have been spliced out . Ub , ubiquitin ; WT , wild type .

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(J) Lysates from MCF7 cells transfected with the CIP2A-FLAG construct (Junttila et al., 2007) and the indicated ubiquitin-HA constructs were subjected to anti-FLAG immunopurification in denaturing conditions with 0.1% SDS followed by immunoblotting with the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Ub, ubiquitin; WT, wild type.


D Bax / BAK1 protein levels were detected by western blotting .

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D Bax/BAK1 protein levels were detected by western blotting.


G , Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from HEK293T cells transfected with different constructs as indicated .

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G, Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.


H Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from HEK293T cells transfected with different constructs as indicated .

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H Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.


I Immublotting results with IP or input ( whole - cell lysates ) from mouse midbrain , SH - SY5Y , HEK293T or HeLa cells as indicated . The α - ctrl represents that the magnetic beads used in IP were not pre - incubated with any antibody .

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I Immublotting results with IP or input (whole-cell lysates) from mouse midbrain, SH-SY5Y, HEK293T or HeLa cells as indicated. The α-ctrl represents that the magnetic beads used in IP were not pre-incubated with any antibody.


B Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from cells transfected with Bcl - 2 and TMEM175 or its mutants TMEM175 - V145A , TMEM175 - R377V , as indicated . C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector , TMEM175 , TMEM175 - V145A or TMEM175 - R377V overexpressing HEK293T cells . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - tests ( C and is indicated with NS for not significant ( P > 0 . 05 ) , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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B Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from cells transfected with Bcl-2 and TMEM175 or its mutants TMEM175-V145A, TMEM175-R377V, as indicated. C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector, TMEM175, TMEM175-V145A or TMEM175-R377V overexpressing HEK293T cells. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests (C and is indicated with NS for not significant (P > 0.05), ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


K ( Upper ) schematic diagram showing the strategy of TMEM175 - KO in HEK293T cells . 92 bp in exon 4 and intron 4 were removed from the tmem175 locus . ( Lower ) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western - blotting .

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K (Upper) schematic diagram showing the strategy of TMEM175-KO in HEK293T cells. 92 bp in exon 4 and intron 4 were removed from the tmem175 locus. (Lower) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western-blotting.