A ) Transcription levels of all M . florum coding sequences ( CDS ) quantified by RNA - seq . Transcription levels were calculated according to the number of fragments per kilobase per million of mapped reads ( FPKM ) observed over six replicates . The corresponding numbers of mRNA copies per cell , estimated from the measured M . florum RNA mass , are also indicated . CDS were sorted from least to most transcribed . The transcription level of selected genes of importance are presented . LDH , L - lactate dehydrogenase ( peg . 600 / mfl596 ) ; G3PDH , glyceraldehyde - 3 - phosphate dehydrogenase ( peg . 583 / mfl578 ) ; PGK , phosphoglycerate kinase ( peg . 582 / mfl577 ) ; L24p and L7 / L12 , large subunit ribosomal proteins L24p ( peg . 133 / mfl134 ) and L7 / L12 ( peg . 605 / mfl601 ) ; S7p , small subunit ribosomal protein S7p ( peg . 626 / mfl623 ) ; RNAPβ , RNAPβ ' , and RNAPα , RNA polymerase subunits β , β ' , and α ( peg . 601 / mfl597 , peg . 602 / mfl598 , and peg . 149 / mfl150 ) ; HPr , phosphotransferase system phosphocarrier protein HPr ( peg . 570 / mfl565 ) ; σ70 , RNA polymerase sigma factor RpoD ( peg . 269 / mfl270 ) .

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

A) Transcription levels of all M. florum coding sequences (CDS) quantified by RNA-seq. Transcription levels were calculated according to the number of fragments per kilobase per million of mapped reads (FPKM) observed over six replicates. The corresponding numbers of mRNA copies per cell, estimated from the measured M. florum RNA mass, are also indicated. CDS were sorted from least to most transcribed. The transcription level of selected genes of importance are presented. LDH, L-lactate dehydrogenase (peg.600/mfl596); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (peg.583/mfl578); PGK, phosphoglycerate kinase (peg.582/mfl577); L24p and L7/L12, large subunit ribosomal proteins L24p (peg.133/mfl134) and L7/L12 (peg.605/mfl601); S7p, small subunit ribosomal protein S7p (peg.626/mfl623); RNAPβ, RNAPβ', and RNAPα, RNA polymerase subunits β, β', and α (peg.601/mfl597, peg.602/mfl598, and peg.149/mfl150); HPr, phosphotransferase system phosphocarrier protein HPr (peg.570/mfl565); σ70, RNA polymerase sigma factor RpoD (peg.269/mfl270).


C ) Expression levels of all M . florum reference proteins quantified by two - dimensional liquid chromatography - tandem mass spectrometry ( 2D LC - MS / MS ) . Abundance was estimated according to the normalized spectral abundance factor ( NSAF ) calculated for each protein . A NSAF value of 0 was assigned to undetected proteins . The corresponding number of protein molecules per cell ( derived from the biomass data ) are indicated . Proteins were sorted from least to most abundant . The selected genes of importance presented in panel A are also highlighted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression levels of all M. florum reference proteins quantified by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Abundance was estimated according to the normalized spectral abundance factor (NSAF) calculated for each protein. A NSAF value of 0 was assigned to undetected proteins. The corresponding number of protein molecules per cell (derived from the biomass data) are indicated. Proteins were sorted from least to most abundant. The selected genes of importance presented in panel A are also highlighted.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) the TAK1 inhibitor NG25 ( 2 μM ) , the IKKβ inhibitor BI605906 ( 5 μM ) or the IKKβ inhibitor PS1145 ( 10 μM ) . The cells were then co - stimulated for 30 min with 100 ng / ml LPS and 4 mM ATP . Cell lysates ( 10 μg protein ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . ( B ) As in A , except that the cells were co - stimulated for 1 h with 100 ng / ml LPS and 5 μM nigericin . Similar results were obtained in three independent experiments in A and B .

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(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25 (2 μM), the IKKβ inhibitor BI605906 (5 μM) or the IKKβ inhibitor PS1145 (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. (B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B.


( C , D ) As in A , B , except that primary human monocyte - derived macrophages were used instead of mouse BMDM . Similar results were obtained in two independent experiments .

O O O O O O O O O O O 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, D) As in A, B, except that primary human monocyte-derived macrophages were used instead of mouse BMDM. Similar results were obtained in two independent experiments.


( A , B ) The Mouse macrophage J774A . 1 cell line was transfected with control siRNA or siRNA against IKKβ ( A ) or IKKα ( B ) . 72 h post - transfection , the cells were co - stimulated for 60 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 5 μM nigericin . Cell lysates ( 10 μg ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . Similar results were obtained in three ( A ) or two ( B ) independent experiments .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) The Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or IKKα (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.


( C , D ) As in ( A , B ) except that BMDM from IKKβ - LysM - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT control mice were co - stimulated without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( C ) or with 100 ng / ml LPS and / or 5 μM nigericin ( D ) . Similar results were obtained in two independent experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) As in (A, B) except that BMDM from IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (C) or with 100 ng/ml LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.


( A , B ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) , the IKKβ inhibitors BI605906 ( 5 μM ) , TPCA - 1 ( 5 μM ) or PS1145 ( 10 μM ) , the TAK1 inhibitor NG25 ( 2 μM ) or the NLRP3 inhibitor MCC950 . The cells were then co - stimulated for 30 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( A ) and / or 5 μM nigericin ( B ) . The cell culture medium was removed and the cells lysed . Protein in the culture medium was precipitated ( see Methods ) , dissolved in SDS . and subjected to SDS - PAGE , along with cell lysates . After transfer to PVDF membranes , immunoblotting was performed with antibodies recognizing both full length ( FL ) and cleaved ( CL ) gasdermin D ( GSDMD ) , the p20 and p10 fragments of caspase - 1 and IL - 18 . Similar results were obtained in two independent experiments . ( C ) As in A , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) and control WT cells were used . Similar results were obtained in three independent experiments .

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

(A, B) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) 5 μM BI605906 , then stimulated for 1 h with ( + ) 100 ng / ml LPS and / or 5 μM nigericin or left unstimulated . The cells were lysed in buffer containing 1 % ( v / v ) Triton - X - 100 , and the Triton - X - 100 - soluble and Triton - X - 100 - insoluble fractions were prepared as in Methods . The Triton - X - 100 - insoluble fraction was dissolved SDS subjected to SDS - PAGE and immunoblotted with the antibodies indicated . The nuclear protein Histone H3B ( H3B ) was used as a loading control in the Tritin X - 100 insoluble fraction . Similar results were obtained in three independent experiments . ( B ) As in A , except that the Triton - X - 100 - insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2 . 0 mM DSS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT BMDM were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v) Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS subjected to SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments. (B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS.


( C , As in A , B except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) mice ( IKKβ ( fl / fl ) ) were used . The U2 Small Nuclear RNA Auxiliary Factor 1 ( U2AF1 ) was used as a loading control . Similar results were obtained in two independent experiments .

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

(C, As in A, B except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) mice (IKKβ (fl/fl)) were used. The U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) was used as a loading control. Similar results were obtained in two independent experiments.


( E , F ) As in A , B except that BMDM from caspase - 1 KO mice were used . Similar results were obtained in two independent experiments .

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

(E, F) As in A, B except that BMDM from caspase-1 KO mice were used. Similar results were obtained in two independent experiments.


( A - H ) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA - 1 ( 5 μM ) ( C , G ) or BI605906 ( 5 μM ) ( D , H ) . The cells were then stimulated for 1 h with 100 ng / ml LPS ( E ) or 5 μM nigericin ( F , G , H ) , or co - stimulated with 100 ng / ml LPS and 5 μM nigericin ( B , C , D ) or left unstimulated ( control ) ( A ) . The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3 . A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity . ( A - H ) the panel images show the PLA signal ( red ) and DAPI staining for nuclei ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were obtained in three independent experiments and representative images are shown . Scale bar = 50 μm .

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

(A-H) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA-1 (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml LPS (E) or 5 μM nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the PLA signal (red) and DAPI staining for nuclei (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.


( I - L ) As in A - H , except that IKKβ inhibitors were omitted and iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated ( control ) . Scale bar = 50 μm .

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(I-L) As in A-H, except that IKKβ inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.


( A - H ) The experiments were performed as in Figs 5A - 5H , except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody , which was visualized using a secondary antibody ( red ) . Nuclei were counterstained with DAPI ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were observed in many fields and obtained in three independent experiments . Representative images are shown Data information : In all panels , scale bar = 50 μm .

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

(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm.


( I - L ) As in A - H , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated . Similar results were obtained in two independent experiments . Data information : In all panels , scale bar = 50 μm .

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

(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm.


C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice were fasted overnight and then re - fed for 6 ( A - C ) Levels of the indicated proteins detected by IB in ( A ) liver and intestinal extracts from C57BL / 6 mice and in intestinal extracts from ( B ) C57BL / 6 or SHP - KO mice or ( C ) FGF15 - KO mice or littermates . Band quantitation was done using Image J , and the LC3 II / I ratio and p62 / actin and lamp1 / actin ratios are shown at the right . ( A - C ) The mean and standard deviation ( SD ) are plotted . Statistical significance was determined by ( A ) two - tailed Student ' s t - tests or ( B , C ) two - way ANOVA with the Tukey post - test . P - values are indicated ( n = 3 mice ) .

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

C57BL/6, SHP-KO, and FGF15-KO or littermate mice were fasted overnight and then re-fed for 6 (A-C) Levels of the indicated proteins detected by IB in (A) liver and intestinal extracts from C57BL/6 mice and in intestinal extracts from (B) C57BL/6 or SHP-KO mice or (C) FGF15-KO mice or littermates. Band quantitation was done using Image J, and the LC3 II/I ratio and p62/actin and lamp1/actin ratios are shown at the right. (A-C) The mean and standard deviation (SD) are plotted. Statistical significance was determined by (A) two-tailed Student's t-tests or (B, C) two-way ANOVA with the Tukey post-test. P-values are indicated (n=3 mice).


C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice as indicated were fasted for 12 h and then , re - fed for 6 h . levels of the indicated proteins in intestinal extracts from ( B ) C57BL / 6 and SHP - KO mice or ( D ) FGF15 - KO mice and littermates with quantitation of the bands at right . , ( B , D ) n = 3 mice . The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are

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

C57BL/6, SHP-KO, and FGF15-KO or littermate mice as indicated were fasted for 12 h and then, re-fed for 6 h. levels of the indicated proteins in intestinal extracts from (B) C57BL/6 and SHP-KO mice or (D) FGF15-KO mice and littermates with quantitation of the bands at right. , (B, D) n=3 mice. The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated


( G ) HT29 cells were transfected with GFP - LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 , control plasmid DNA , and scrambled siRNA . The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 10 μm . G , The mean and SD are plotted . Statistical significance was determined by ( G ) one - way ANOVA with the Tukey post - test . P - values are

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(G) HT29 cells were transfected with GFP-LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19, control plasmid DNA, and scrambled siRNA. The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 10 μm. G, The mean and SD are plotted. Statistical significance was determined by (G) one-way ANOVA with the Tukey post-test. P-values are indicated


( H ) HT29 cells were treated with 50 ng / ml FGF19 with or without 100 nM bafilomycin A1 for 12 h . LC3 and actin levels in cell extracts were determined by IB . Quantification of bands is shown at the right ( n = 3 culture plates ) . H ) The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are

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(H) HT29 cells were treated with 50 ng/ml FGF19 with or without 100 nM bafilomycin A1 for 12 h. LC3 and actin levels in cell extracts were determined by IB. Quantification of bands is shown at the right (n=3 culture plates). H) The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated


( I ) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for the indicated times . ApoB48 and actin levels were determined by IB ( n = 2 culture plates ) .

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

(I) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for the indicated times. ApoB48 and actin levels were determined by IB (n=2 culture plates).


( A ) HT29 cells were transfected with GFP - LC3 and with Atg7 siRNA or control scrambled RNA ( siCtl ) and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and control siRNA . The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 20 μm . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

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

(A) HT29 cells were transfected with GFP-LC3 and with Atg7 siRNA or control scrambled RNA (siCtl) and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and control siRNA. The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 20 μm. The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


( B ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for 12 h . LC3 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

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(B) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for 12 h. LC3 and actin levels in cell extracts determined by IB (n=3 culture plates).


D ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA ( siCtl ) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 12 h . ( D ) ApoB48 and actin levels in cell extracts determined by IB . Bands were quantified and the LC3 - II / LC3 - I ratio is shown at the right ( n = 3 culture plates ) . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

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

D) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA (siCtl) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 12 h. (D) ApoB48 and actin levels in cell extracts determined by IB. Bands were quantified and the LC3-II/LC3-I ratio is shown at the right (n=3 culture plates). The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


Mice were re - fed for 2 h ( B ) after fasting overnight . ( B ) CoIP of TFEB with SHP . Upper panel : Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples . Lower panel : levels of TFEB and SHP in input samples detected by IB . Similar results were obtained in a second experiment .

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Mice were re-fed for 2 h (B) after fasting overnight. (B) CoIP of TFEB with SHP. Upper panel: Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples. Lower panel: levels of TFEB and SHP in input samples detected by IB. Similar results were obtained in a second experiment.


( F , G ) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from ( F ) C57BL / 6 and SHP - KO mice or ( G ) FGF15 - KO mice or littermates re - fed for 6 h after fasting overnight ( n = 3 mice ) .

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

(F, G) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from (F) C57BL/6 and SHP-KO mice or (G) FGF15-KO mice or littermates re-fed for 6 h after fasting overnight (n=3 mice).


( A ) HT29 cells were transfected with GFP - LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA . The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 cells ) . Scale bar , 10 μm . ( A , The mean and SD are plotted . Statistical significance was determined by ( A ) one - way ANOVA with the Tukey post - test , P - values are indicated .

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

(A) HT29 cells were transfected with GFP-LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA. The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20 cells). Scale bar, 10 μm. (A, The mean and SD are plotted. Statistical significance was determined by (A) one-way ANOVA with the Tukey post-test, P-values are indicated.


( B ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or control scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 ( BAF ) for 12 h . Levels of the indicated proteins determined by IB . LC3 II / I and p62 / actin ratios are shown at the right ( n = 3 - 4 culture plates ) . B , The mean and SD are plotted . Statistical significance was determined by ( B ) two - way ANOVA with the Tukey post - test , P - values are indicated .

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

(B) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or control scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 (BAF) for 12 h. Levels of the indicated proteins determined by IB. LC3 II/I and p62/actin ratios are shown at the right (n=3-4 culture plates). B, The mean and SD are plotted. Statistical significance was determined by (B) two-way ANOVA with the Tukey post-test, P-values are indicated.


( C ) HT29 cells were transfected with siRNA for TFEB ( siTFEB ) or control RNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . Protein levels detected by IB of ULK1 and ATGL ( n = 3 culture plates ) .

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(C) HT29 cells were transfected with siRNA for TFEB (siTFEB) or control RNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. Protein levels detected by IB of ULK1 and ATGL (n=3 culture plates).


( E ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . ApoB48 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

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

(E) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. ApoB48 and actin levels in cell extracts determined by IB (n=3 culture plates).


( A , C57BL / 6 ( A ) were re - fed for 6 h after fasting overnight . The cytosolic and nuclear fractions were isolated from intestine or liver , and the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

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(A, C57BL/6 (A) were re-fed for 6 h after fasting overnight. The cytosolic and nuclear fractions were isolated from intestine or liver, and the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.


B ) FGF15 - KO mice or littermates ( B ) were re - fed for 6 h after fasting overnight . the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

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

B) FGF15-KO mice or littermates (B) were re-fed for 6 h after fasting overnight. the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.


FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( C ) The nuclear fraction was isolated and the nuclear levels of TFEB , SHP , and LAMIN were determined by IB .

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FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (C) The nuclear fraction was isolated and the nuclear levels of TFEB, SHP, and LAMIN were determined by IB.


FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( D ) Levels of LC3 , p62 , and actin in cell extracts were determined by IB .

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FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (D) Levels of LC3, p62, and actin in cell extracts were determined by IB.


( E ) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng / ml FGF19 for 30 min . The indicated proteins were detected by IB ( n = 3 culture plates ) .

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(E) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng/ml FGF19 for 30 min. The indicated proteins were detected by IB (n=3 culture plates).


( F ) FGF15 - KO mice or littermates were re - fed for 0 , 2 , or 3 h after fasting overnight . The indicated proteins in intestinal extracts were detected by IB ( n = 2 mice ) .

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

(F) FGF15-KO mice or littermates were re-fed for 0, 2, or 3 h after fasting overnight. The indicated proteins in intestinal extracts were detected by IB (n=2 mice).


( G ) HT29 cells were transfected with expression plasmids for flag - tagged TFEB or its mutants as indicated and 24 h later , cells were treated with FGF19 ( 50 ng / ml ) for 12 h . ∆ CT indicates the TFEB mutant with a C - terminal deletion of 15 amino acids . The indicated proteins were detected by IB ( n = 3 culture plates ) .

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

(G) HT29 cells were transfected with expression plasmids for flag-tagged TFEB or its mutants as indicated and 24 h later, cells were treated with FGF19 (50 ng/ml) for 12 h. ∆CT indicates the TFEB mutant with a C-terminal deletion of 15 amino acids. The indicated proteins were detected by IB (n=3 culture plates).


Substrate was synthesized in reticulocyte lysate in the presence of 35S - methionine and mixed with SecA and SecYEG proteoliposomes . After incubation with ATP , the samples were treated with proteinase K to degrade any non - translocated material . Where indicated , the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X - 100 ( TX - 100 ) . Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT , generating protease - protected full - length ( FL ) substrate or a translocation intermediate ( IM ) , respectively . All samples were analyzed by SDS PAGE followed by autoradiography . Lane 1 shows 10 % of the input sample . The gel is representative of three replicates . Molecular weight markers are estimated from Coomassie - stained gels run in parallel .

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

Substrate was synthesized in reticulocyte lysate in the presence of 35S-methionine and mixed with SecA and SecYEG proteoliposomes. After incubation with ATP, the samples were treated with proteinase K to degrade any non-translocated material. Where indicated, the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X-100 (TX-100). Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT, generating protease-protected full-length (FL) substrate or a translocation intermediate (IM), respectively. All samples were analyzed by SDS PAGE followed by autoradiography. Lane 1 shows 10% of the input sample. The gel is representative of three replicates. Molecular weight markers are estimated from Coomassie-stained gels run in parallel.


Translocation intermediates ( IM ) were incubated in the presence of different nucleotides ( Nuc . ) for different time periods and then treated with proteinase K ( Prot . K ) . One sample received hexokinase and glucose ( HKG ) to generate ADP , other samples ADP • BeFx or ATPγS . The gel is representative of two replicates . Quantification of ( C ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show the average of replicates . Colors correspond to different nucleotides as shown in the inset box . As in ( C ) , but DTT was added concurrently with HKG or nucleotides . The gel is representative of two replicates . Quantification of ( E ) as in ( D ) , except intensities were calculated for the FL bands .

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

Translocation intermediates (IM) were incubated in the presence of different nucleotides (Nuc.) for different time periods and then treated with proteinase K (Prot. K). One sample received hexokinase and glucose (HKG) to generate ADP, other samples ADP•BeFx or ATPγS. The gel is representative of two replicates.   Quantification of (C). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show the average of replicates. Colors correspond to different nucleotides as shown in the inset box.   As in (C), but DTT was added concurrently with HKG or nucleotides. The gel is representative of two replicates.   Quantification of (E) as in (D), except intensities were calculated for the FL bands.


WB SecA with additional mutations ( WT , Y794G , R342E ) was added to preformed intermediates ( IM ) and incubated for the indicated times before proteolysis . The gel is representative of two replicates . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to WB SecA carrying additional mutations , as shown in the inset box .

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WB SecA with additional mutations (WT, Y794G, R342E) was added to preformed intermediates (IM) and incubated for the indicated times before proteolysis. The gel is representative of two replicates.   Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to WB SecA carrying additional mutations, as shown in the inset box.


Imidazole ( imi ) and either WT or mutant SecA were added to translocation intermediates ( IM ) pre - formed with SecA dN20 : : H6 . The samples were incubated for the indicated times before proteolysis with proteinase K ( Prot . K ) . The gel is representative of two replicates , except for the experiment with the R792G / K797G mutant and the 30 - min time points . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to different SecA mutants as shown in the inset box . As in ( B ) , but ATPγS was added together with imidazole . The gel is representative of two replicates . Quantification of ( E ) as in ( C ) . As in ( D ) , but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA . The gel is representative of two replicates . Quantification of ( G ) as in ( F ) , except that both the intensities of the IM ( darker colors ) and FL ( lighter colors ) bands were measured .

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

Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points. Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box. As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates. Quantification of (E) as in (C). As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.   Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.


( a ) Survival rate of wild - type mice ( WT ) and indicated knockout mice after intranasal infection with 750 plaque forming units ( PFU ) of the PR8 virus .

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(a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus.


( b ) Hematoxylin and eosin ( H & amp ; amp ; E ) - stained lung sections from mice infected with PR8 virus on day 2 . Arrows indicate cellular infiltrates and necrotic debris occluding the airways . Original magnification , × 40 . ( c ) Disease severity ( ' lung score ' ) and extent of cell death ( necrosis score ) on day 2 after infection based on analysis of H & amp ; amp ; E - stained lung sections . ( d ) Necrosis score on day 7 , estimated visually from H & amp ; amp ; E - stained lung sections , and flow cytometry analysis for annexin V - stained lung cell populations .

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(b) Hematoxylin and eosin (H&amp;E)-stained lung sections from mice infected with PR8 virus on day 2. Arrows indicate cellular infiltrates and necrotic debris occluding the airways. Original magnification, ×40. (c) Disease severity ('lung score') and extent of cell death (necrosis score) on day 2 after infection based on analysis of H&amp;E-stained lung sections. (d) Necrosis score on day 7, estimated visually from H&amp;E-stained lung sections, and flow cytometry analysis for annexin V-stained lung cell populations.


( e ) Immunohistochemistry staining of neutrophils occluding the airways of PR8 - infected mice . Original magnification , × 20 .

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(e) Immunohistochemistry staining of neutrophils occluding the airways of PR8-infected mice. Original magnification, ×20.


( f , g ) Flow cytometry analysis of neutrophil , dendritic cell ( DC ) and macrophage cell numbers on days 2 and 7 .

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(f,g) Flow cytometry analysis of neutrophil, dendritic cell (DC) and macrophage cell numbers on days 2 and 7.


( h ) Pulmonary viral titers on days 2 and 7 after infection in Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( log - rank test ( a ) and unpaired two - sided Student ' s t - test ( c , d , f , g ) . Data are pooled from three experiments ( a , f - h ; mean ± s . e . m . in f - h ; n = 10 - 12 mice total ) or are representative of three experiments ( b - e ; mean ± s . e . m . ; n = 3 - 5 mice per experiment ) .

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

(h) Pulmonary viral titers on days 2 and 7 after infection in Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (log-rank test (a) and unpaired two-sided Student's t-test (c,d,f,g). Data are pooled from three experiments (a,f-h; mean ± s.e.m. in f-h; n = 10-12 mice total) or are representative of three experiments (b-e; mean ± s.e.m.; n = 3-5 mice per experiment).


( a - c ) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild - type mice ( WT ) and Ripk2 − / − mice with the PR8 virus were analyzed for IL - 6 and TNF ( day 2 ; a ) , IL - 18 and IFN - γ ( b ) , and indicated chemokines ( c ) . * P 0 . 05 , * * P 0 . 01 ( unpaired two - sided Student ' s t - test ) . Data were normalized for total protein ( t . p . ) from lung homogenates . Data are pooled from three experiments ( mean ± s . e . m . ; n = 10 - 12 mice total ) .

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

(a-c) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild-type mice (WT) and Ripk2 −/− mice with the PR8 virus were analyzed for IL-6 and TNF (day 2; a), IL-18 and IFN-γ (b), and indicated chemokines (c). *P 0.05, **P 0.01 (unpaired two-sided Student's t-test). Data were normalized for total protein (t.p.) from lung homogenates. Data are pooled from three experiments (mean ± s.e.m.; n = 10-12 mice total).


( a - c ) Analysis on day 2 after infection with PR8 virus of wild - type mice ( WT ) or Ripk2 − / − mice injected intraperitoneally with control rabbit serum and Ripk2 − / − mice injected intraperitoneally with anti - IL - 18 neutralizing antiserum ( αIL - 18Ab ) 3 h before infection , for IL - 18 , IFN - γ and CCL2 amounts in whole lung homogenates ( a ; t . p . , total protein ) , and for neutrophil numbers by immunohistochemistry ( b ) and by flow cytometry ( c ) . Original magnification , × 20 .

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(a-c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti-IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20.


( d ) Analysis of survival of indicated mice .

(d) Analysis of survival of indicated mice.


( e ) Neutrophil numbers in WT , Ripk2 − / − and Ripk2 − / − Il18 − / − mice on day 2 after PR8 infection .

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(e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection.


( f ) Survival of Ripk2 − / − Il18 − / − , Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ( a , c , e ) and log - rank test ( d , f ) ) . Data are representative of two independent experiments ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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(f) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6-8 mice per experiment).


( a , b ) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL - 18 production by using IL - 18 immunohistochemistry ( a ) and IL - 18 ELISA ( b ) of whole lung homogenates . WT , wild type ; t . p . , total protein . In a , blue arrows denote IL - 18 + macrophages , and red arrows denote IL - 18 + bronchial epithelium . Original magnification , × 200 .

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(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200.


( c ) Flow cytometry analysis of NK cell counts in Ripk2 − / − and wild - type mice .

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(c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice.


( d ) Analysis of wild - type and Ripk2 − / − NK cells for IFN - γ production on a per cell basis .

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(d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis.


( e ) Accumulation of T and B cells in lungs .

(e) Accumulation of T and B cells in lungs.


( f ) Analysis of CD8 + T cells for IFN - γ and CD44 expression in wild - type and Ripk2 − / − mice . * P 0 . 05 , * * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( b ) and unpaired two - sided Student ' s t - test ( d , f ) ) . Data are representative of two experiments ( a , b , f ) or three experiments ( c - e ) ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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(f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c-e) (mean ± s.e.m.; n = 6-8 mice per experiment).


( a ) Immunoblots showing active caspase - 1 ( casp - 1 ) p20 compared to GAPDH loading controls in wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs after PR8 infection .

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(a) Immunoblots showing active caspase-1 (casp-1) p20 compared to GAPDH loading controls in wild-type (WT), Ripk2−/− and Nod2−/− BMDCs after PR8 infection.


( b ) IL - 18 release in PR8 - infected WT , Ripk2 − / − and Nod2 − / − BMDCs .

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(b) IL-18 release in PR8-infected WT, Ripk2−/− and Nod2−/− BMDCs.


( c , d ) Immunoblot analysis showing active casp - 1 p20 ( c ) and IL - 18 production ( d ) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10 .

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(c,d) Immunoblot analysis showing active casp-1 p20 (c) and IL-18 production (d) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10.


( e , f ) IL - 18 and casp - 1 activation in Ripk2 − / − BMDCs treated with the NLRP3 inflammasome - specific inhibitor glyburide ( glyb . ) during PR8 infection .

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(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.


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( g ) Immunoblot analysis of casp - 1 p20 during Listeria infection of WT and Aim2 − / − BMDCs . * P 0 . 01 , * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ) . Data are representative of six experiments ( a , b ) or three experiments ( c - g ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Immunoblot analysis of casp-1 p20 during Listeria infection of WT and Aim2−/− BMDCs. *P 0.01, **P 0.001 (one-way ANOVA with Tukey's post-hoc test). Data are representative of six experiments (a,b) or three experiments (c-g) (mean ± s.d.; n = 2-3 replicates per experiment).


( a ) Immunoblot analysis of wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs for LC3 - II ( marker for autophagy ) after 18 h of PR8 infection .

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(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection.


( b , c ) Numbers of LC3 - II + puncta ( b ) , counted in at least 100 cells from five random fields from confocal images ( c ) of BMDCs after PR8 infection and / or chloroquine treatment . Original magnification , × 400 .

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(b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400.


( d , e ) Immunoblots of LC3 - II and caspase - 1 ( casp - 1 ; e ) and IL - 18 production ( d ) in wild - type and Ripk2 − / − BMDCs after treatment with rapamycin ( Rap ) .

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(d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap).


( f ) Immunoblot analysis for LC3 - II in lung lysates of PR8 - infected wild - type and Ripk2 − / − mice ( 1 - 5 indicate individual mice ) .

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(f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1-5 indicate individual mice).


( g , h ) Lungneutrophil infiltration ( g ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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(g,h) Lungneutrophil infiltration (g) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


( g , h ) IL - 18 and IFN - γ amounts ( h ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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(g,h) IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


( a - c ) Flow cytometry analysis of wild - type ( WT ) and Ripk2 − / − BMDCs stained with the mitochondrial superoxide - specific stain MitoSOX during IAV or Listeria infection ( a , b ) and with the general mitochondrial stain MitoTracker Green during IAV infection ( c ) .

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(a-c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide-specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c).


( d , e ) Analysis of IL - 18 production and immunoblotting to analyze caspase - 1 ( casp - 1 ) p20 activation in IAV - infected BMDCs that had been pretreated with the ROS inhibitor NAC .

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(d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC.


( f ) Immunoblot of casp - 1 p20 in cell lysates of IAV - infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO .

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(f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO.


( g ) Confocal microscopy analysis of BMDCs stained for mitochondria ( red ) and LC3 ( green ) for colocalization as an indicator of mitophagy . Original magnification , × 400 ; inset ( red boxed region ) was digitally enlarged × 10 . * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( d ) and unpaired two - sided Student ' s t - test ( g ) ) . Data are representative of nine experiments ( a ) , three experiments ( b , d - g ) or six experiments ( c ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Confocal microscopy analysis of BMDCs stained for mitochondria (red) and LC3 (green) for colocalization as an indicator of mitophagy. Original magnification, ×400; inset (red boxed region) was digitally enlarged ×10. *P 0.01 (one-way ANOVA with Tukey's post-hoc test (d) and unpaired two-sided Student's t-test (g)). Data are representative of nine experiments (a), three experiments (b,d-g) or six experiments (c) (mean ± s.d.; n = 2-3 replicates per experiment).


( a , b ) IL - 18 amounts ( a ) , and caspase - 1 ( casp - 1 ) p20 and LC3 - II immunoblots ( b ) in PR8 - infected wild - type ( WT ) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580 .

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(a,b) IL-18 amounts (a), and caspase-1 (casp-1) p20 and LC3-II immunoblots (b) in PR8-infected wild-type (WT) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580.


( c ) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2 − / − BMDCs after PR8 infection .

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(c) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2−/− BMDCs after PR8 infection.


( d ) Immunoblots for casp - 1 p20 in untreated or NAC - treated WT and Ulk1 − / − BMDCs after PR8 infection .

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(d) Immunoblots for casp-1 p20 in untreated or NAC-treated WT and Ulk1−/− BMDCs after PR8 infection.


( e ) Flow cytometry analysis of MitoSOX staining in PR8 - infected WT and Ulk1 − / − BMDCs .

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(e) Flow cytometry analysis of MitoSOX staining in PR8-infected WT and Ulk1−/− BMDCs.


( f ) MitoTracker Green staining of PR8 - infected WT and Ulk1 − / − BMDCs .

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(f) MitoTracker Green staining of PR8-infected WT and Ulk1−/− BMDCs.


Effects of basic and bulky hydrophobic amino acids on LT - induced pyroptosis and caspase - 1 activation in RAWRA cells . Cells were pre - treated with 10 mM indicated amino acids prior to LT stimulation . Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates ( A ) . Mock , DMEM medium .

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Effects of basic and bulky hydrophobic amino acids on LT-induced pyroptosis and caspase-1 activation in RAWRA cells. Cells were pre-treated with 10 mM indicated amino acids prior to LT stimulation. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (A). Mock, DMEM medium.


Cell supernatants were subjected to anti - caspase - 1 immunoblotting and the lysates were blotted with the anti - tubulin antibody ( B ) . p10 , mature caspase - 1 ; p45 , caspase - 1 precursor .

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Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin antibody (B). p10, mature caspase-1; p45, caspase-1 precursor.


C , D Effects of basic and bulky hydrophobic amino acids on LT - induced RFP - ASC specks formation in RAWRA cells . Fluorescence images were taken on a confocal microscopy ( C ) . The percentages of cells showing RFP - ASC specks ( mean values ± SD from three replicates ) are in ( D ) .

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C, D Effects of basic and bulky hydrophobic amino acids on LT-induced RFP-ASC specks formation in RAWRA cells. Fluorescence images were taken on a confocal microscopy (C). The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).


E Effects of basic and bulky hydrophobic amino acids on LT - induced caspase - 1 activation in Tlr4 - / - iBMDMs . Cells were treated as that in ( A ) . Cell supernatants were subjected to anti - caspase - 1 immunoblotting and the lysates were blotted with the anti - tubulin or anti - MEK3 antibody .

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E Effects of basic and bulky hydrophobic amino acids on LT-induced caspase-1 activation in Tlr4-/- iBMDMs. Cells were treated as that in (A). Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin or anti-MEK3 antibody.


A RFP - ASC specks formation assay of the effect of Ubr2 knockdown on LT - induced inflammasome activation . RAWRA cells were transfected with a control siRNA or Ubr2 - targeting siRNA mixtures for 60 h followed by LT treatment . The numbers in the merged panel are the percentages of the cells showing RFP - ASC specks .

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A RFP-ASC specks formation assay of the effect of Ubr2 knockdown on LT-induced inflammasome activation. RAWRA cells were transfected with a control siRNA or Ubr2-targeting siRNA mixtures for 60 h followed by LT treatment. The numbers in the merged panel are the percentages of the cells showing RFP-ASC specks.


B Effect of Ubr2 stable knockdown on caspase - 1 activation in RAWRA cells . Cells stably expressing a control or Ubr2 - targeting shRNA were incubated with WT LT ( + ) or its E687C mutant ( - ) for 3 h .

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B Effect of Ubr2 stable knockdown on caspase-1 activation in RAWRA cells. Cells stably expressing a control or Ubr2-targeting shRNA were incubated with WT LT (+) or its E687C mutant (-) for 3 h.


C Effects of Ubr2 knockdown ( siRNA # 05 ) on LT - induced NLRP1B inflammasome activation reconstituted in 293T cells . Cells were treated with WT LT ( + ) or its E687C mutant ( - ) .

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C Effects of Ubr2 knockdown (siRNA #05) on LT-induced NLRP1B inflammasome activation reconstituted in 293T cells. Cells were treated with WT LT (+) or its E687C mutant (-).


D , E Effect of Ubr2 knockout on NLRP1B or NAIP2 - NLRC4 inflammasome activation . WT and Ubr2 - / - iBMDMs were treated with LT or BsaK for 3 h . Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates ( D ) .

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D, E Effect of Ubr2 knockout on NLRP1B or NAIP2-NLRC4 inflammasome activation. WT and Ubr2-/- iBMDMs were treated with LT or BsaK for 3 h. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (D).


A Immunoblotting of caspase - 1 activation in Ubrs - knockdown BMDMs derived from the 129 / Sv mice . Cells were transfected with the indicated siRNA for 60 h , and then treated with WT LT ( + ) or its E687C mutant ( - ) for 3 h .

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A Immunoblotting of caspase-1 activation in Ubrs-knockdown BMDMs derived from the 129/Sv mice. Cells were transfected with the indicated siRNA for 60 h, and then treated with WT LT (+) or its E687C mutant (-) for 3 h.


Immunoblotting of caspase - 1 activation in E2s - knockdown BMDMs derived from the 129 / Sv mice .

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Immunoblotting of caspase-1 activation in E2s-knockdown BMDMs derived from the 129/Sv mice.


Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT - induced caspase - 1 activation ( C ) . Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates .

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Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT-induced caspase-1 activation (C). Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates.


RFP - ASC specks formation assay of the effect of Ube2o knockdown ( siRNA Ube2o - 01 ) on LT - induced inflammasome activation in RAWRA cells . The percentages of cells showing RFP - ASC specks ( mean values ± SD from three replicates ) are in ( D ) .

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RFP-ASC specks formation assay of the effect of Ube2o knockdown (siRNA Ube2o-01) on LT-induced inflammasome activation in RAWRA cells. The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).


F Co - immunoprecipitation interaction of UBE2O and UBR2 . Myc - UBE2O and Flag - UBR2 were co - expressed in 293T cells and cell lysates were subjected to anti - Flag immunoprecipitation followed by immunoblotting analyses as shown .

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F Co-immunoprecipitation interaction of UBE2O and UBR2. Myc-UBE2O and Flag-UBR2 were co-expressed in 293T cells and cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting analyses as shown.


B Effect of LT treatment on NLRP1B protein level . 293T cells were transfected with Flag - NLRP1B expression plasmid for 24 h , and cells were then treated with WT LT ( + ) or its E687C mutant ( - ) for 4 h . Shown is the anti - Flag immunoblot of the total cell lysates .

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B Effect of LT treatment on NLRP1B protein level. 293T cells were transfected with Flag-NLRP1B expression plasmid for 24 h, and cells were then treated with WT LT (+) or its E687C mutant (-) for 4 h. Shown is the anti-Flag immunoblot of the total cell lysates.


C RFP - ASC specks formation in 293T cells expressing an indicated NLRP1B variant . Cells were treated with LT for 3 h . Scale bar , 20 μm . 1 - 983 and 984 - C ( residues 984 to the C terminus ) are the FIIND - mediated autocleaved fragments of NLRP1B .

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C RFP-ASC specks formation in 293T cells expressing an indicated NLRP1B variant. Cells were treated with LT for 3 h. Scale bar, 20 μm. 1-983 and 984-C (residues 984 to the C terminus) are the FIIND-mediated autocleaved fragments of NLRP1B.


Effect of MG132 on LT - induced NLRP1B degradation . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and MG132 for 8 h . Shown are the anti - HA and anti - tubulin immunoblots of the total cell lysates .

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Effect of MG132 on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.


E Effect of MG132 and Leu on LT - induced NLRP1B degradation . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and MG132 for 8 h . 10 mM Leu was added to cells 30 min prior to LT treatment . Shown are the anti - HA and anti - tubulin immunoblots of the total cell lysates .

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E Effect of MG132 and Leu on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. 10 mM Leu was added to cells 30 min prior to LT treatment. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.


F LT - induced ubiquitination of NLRP1B . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and / or MG132 for 6 h . Cells were then harvested for anti - HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies .

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F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( B ) Representative bright - field images of spheroids treated with dmPGE2 . Scale bars , 200 µm .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (B) Representative bright-field images of spheroids treated with dmPGE2. Scale bars, 200 µm.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( C ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with 0 nM dmPGE2 ( average area was 3761 µm2 for 0 nM group ; n = 4 images from two independent experiments ) .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM dmPGE2 (average area was 3761 µm2 for 0 nM group; n = 4 images from two independent experiments).


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( D ) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin . Scale bars , 100 µm .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (D) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin. Scale bars, 100 µm.


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( E ) Quantification of the average expression ± s . e . m . of Cldn4mRNA relative to the 0 nM treatment group ( n = 3 independent experiments ) .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (E) Quantification of the average expression ± s.e.m. of Cldn4mRNA relative to the 0 nM treatment group (n = 3 independent experiments).


B - F Jejunal spheroids were cultured in differentiation medium containing 0 - 1 , 000 nM dmPGE2 or Iloprost . ( F ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with 0 nM Iloprost ( n = 4 images from two independent experiments ) .

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B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments).


A Quantification of the average expression ± s . e . m . of Ptger1 , Ptger2 , Ptger3 , Ptger4 , and Ptgir mRNAs in whole - thickness lung , ileum or colon tissues or in jejunal spheroids cultured in stem cell ( Stem ) , WAE ( dmPGE2 ) or enterocyte ( EP4i ) medium ( n = 3 independent experiments ) . Data are presented as fold change compared to lung tissue . n . d . , not detected .

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A Quantification of the average expression ± s.e.m. of Ptger1, Ptger2, Ptger3, Ptger4, and Ptgir mRNAs in whole-thickness lung, ileum or colon tissues or in jejunal spheroids cultured in stem cell (Stem), WAE (dmPGE2) or enterocyte (EP4i) medium (n = 3 independent experiments). Data are presented as fold change compared to lung tissue. n.d., not detected.


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( B ) Representative bright field images . Scale bars , 200 µm .

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B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (B) Representative bright field images. Scale bars, 200 µm.


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( C ) Quantification of average spheroid area ± s . e . m . relative to spheroids treated with DMSO alone ( n = 4 images from two independent experiments ) .

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B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with DMSO alone (n = 4 images from two independent experiments).


B - D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 ( EP1i , SC 51322 ) , EP2 ( EP2i , PF 04418948 ) , EP3 ( EP3i , L - 798 , 106 ) or EP4 ( EP4i , L - 161 , 982 ) at a concentration of 10 µM or an equivalent volume of DMSO vehicle . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 compared to the DMSO only group by one - way ANOVA and Dunnett ' s post - test . ( D ) Quantification of the average expression ± s . e . m . of Cldn4 mRNA relative to stem cellspheroids ( n = 3 independent experiments ) .

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B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cellspheroids (n = 3 independent experiments).


E , F Tat - Cre mediated recombination of Ptger4flox / flox ( fl / fl ) jejunal spheroids to generate Ptger4flox / Δ ( fl / Δ ) and Ptger4Δ / Δ ( Δ / Δ ) spheroid lines . ( E ) Schematic and representative PCR genotyping results . M , marker lane . bp , base pairs . ( F ) Quantification of the average expression ± s . e . m . of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl / fl genotype ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 by 2 - way ANOVA and Dunnett ' s multiple comparisons post - test with the EP4i - treated fl / fl group set as the control .

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E, F Tat-Cre mediated recombination of Ptger4flox/flox (fl/fl) jejunal spheroids to generate Ptger4flox/Δ (fl/Δ) and Ptger4Δ/Δ (Δ/Δ) spheroid lines. (E) Schematic and representative PCR genotyping results. M, marker lane. bp, base pairs. (F) Quantification of the average expression ± s.e.m. of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl/fl genotype (n = 3 independent experiments). **p<0.01, ****p<0.0001 by 2-way ANOVA and Dunnett's multiple comparisons post-test with the EP4i-treated fl/fl group set as the control.


G - I Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( G ) Representative bright - field images . Scale bars , 200 µm . ( H ) Quantification of average spheroid area ± s . e . m . ( n = 4 images per group from two independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

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G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (G) Representative bright-field images. Scale bars, 200 µm. (H) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from two independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.


G - I Ptger4flox / flox , Ptger4flox / Δ and Ptger4Δ / Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( I ) average expression ± s . e . m . of Cldn4 mRNA ( n = 3 independent experiments ) relative to EP4i - treated Ptger4flox / flox spheroids .

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G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (I) average expression ± s.e.m. of Cldn4 mRNA (n = 3 independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.


J - L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( J ) Representative bright - field images . Scale bars , 200 µm . ( K ) Quantification of average spheroid area ± s . e . m . ( n = 4 images per group from 3 independent donor lines examined over 3 passages each ) relative to stem spheroids .

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J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (J) Representative bright-field images. Scale bars, 200 µm. (K) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from 3 independent donor lines examined over 3 passages each) relative to stem spheroids.


J - L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test . ( L ) the average expression ± s . e . m . of CLDN4 mRNA ( n = 3 independent donor lines ) relative to stem spheroids .

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J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (L) the average expression ± s.e.m. of CLDN4 mRNA (n = 3 independent donor lines) relative to stem spheroids.


B Heatmap showing unsupervised hierarchical clustering of genes that were significantly ( FDR - adjusted p < 0 . 05 ) and differentially expressed between the three groups ( n = 4 samples per treatment from two independent experiments ) . Each column is one sample and each row is one gene . Red and blue indicate genes with enriched or de - enriched expression in a particular group , respectively . The dendrogram for the gene clustering is colored to highlight major gene clusters 1 - 6 . The number of probes in each cluster is in parentheses .

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B Heatmap showing unsupervised hierarchical clustering of genes that were significantly (FDR-adjusted p<0.05) and differentially expressed between the three groups (n = 4 samples per treatment from two independent experiments). Each column is one sample and each row is one gene. Red and blue indicate genes with enriched or de-enriched expression in a particular group, respectively. The dendrogram for the gene clustering is colored to highlight major gene clusters 1-6. The number of probes in each cluster is in parentheses.


E , F Spheroids were cultured as indicated for 24 hours followed by a 1 - hr pulse with EdU to mark the cells undergoing DNA synthesis . ( E ) Representative images of EdU staining ( red ) . Nuclei are visualized with bisbenzimide ( blue ) . Scale bars , 20 µm . ( F ) Quantification of EdU - positive nuclei as a percent of the total nuclei ( n = minimum of 15 spheroids counted per sample from 2 independent experiments ) . * * * * p < 0 . 0001 by 1 - way ANOVA and Tukey ' s post - test .

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E, F Spheroids were cultured as indicated for 24 hours followed by a 1-hr pulse with EdU to mark the cells undergoing DNA synthesis. (E) Representative images of EdU staining (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm. (F) Quantification of EdU-positive nuclei as a percent of the total nuclei (n = minimum of 15 spheroids counted per sample from 2 independent experiments). ****p<0.0001 by 1-way ANOVA and Tukey's post-test.


G Graph of the fold change in background - subtracted luminescence ± s . e . m . ( relative to 0 hr measurement ) of Cdc25A - CBRLuc spheroids ( n = 3 independent experiments with 4 technical replicates ) . * * * * p < 0 . 0001 for dmPGE2 - and EP4i - treated spheroids compared to stem cells by repeated measures 2 - way ANOVA ( variable = treatment ) . p < 0 . 001 at the 16 hr , 20 hr and 24 hr time points by Dunnett ' s post - test comparing dmPGE2 - treated and EP4i - treated spheroids to the stem cell control .

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G Graph of the fold change in background-subtracted luminescence ± s.e.m. (relative to 0 hr measurement) of Cdc25A-CBRLuc spheroids (n = 3 independent experiments with 4 technical replicates). ****p<0.0001 for dmPGE2- and EP4i-treated spheroids compared to stem cells by repeated measures 2-way ANOVA (variable = treatment). p<0.001 at the 16 hr, 20 hr and 24 hr time points by Dunnett's post-test comparing dmPGE2-treated and EP4i-treated spheroids to the stem cell control.


H Quantification of the average expression ± s . e . m . of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell ( Stem ) or in differentiation medium with the indicated supplements relative to the stem cell group ( n = 3 independent experiments ) . * * p < 0 . 01 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - test . n . d . , not detected .

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H Quantification of the average expression ± s.e.m. of Lgr5 and Mki67 mRNAs in jejunal spheroids cultured in stem cell (Stem) or in differentiation medium with the indicated supplements relative to the stem cell group (n = 3 independent experiments). **p<0.01, ****p<0.0001 by one-way ANOVA and Dunnett's post-test. n.d., not detected.


D Representative images of spheroids stained for Cldn4 ( red ) . Nuclei are visualized with bisbenzimide ( blue ) ( n = 2 independent experiments ) . Scale bars , 20 µm .

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D Representative images of spheroids stained for Cldn4 (red). Nuclei are visualized with bisbenzimide (blue) (n = 2 independent experiments). Scale bars, 20 µm.


E , F Jejunal spheroids were cultured as in Figure 2B - D . Quantification of the average expression ± s . e . m . of Dpcr1 ( E ) and Cd55b mRNAs relative to DMSO group ( n = 3 independent experiments ) . * p < 0 . 05 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s post - test .

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E, F Jejunal spheroids were cultured as in Figure 2B-D. Quantification of the average expression ± s.e.m. of Dpcr1 (E) and Cd55b mRNAs relative to DMSO group (n = 3 independent experiments). *p<0.05, ****p<0.0001 by one-way ANOVA and Dunnett's post-test.


G - J Representative transmission electron microscopy ( TEM ) images of spheroids or an in vivo WAE cell from a biopsy - injured mousecolon . ( G ) The basal plasma membranes are outlined in orange solid lines , lateral plasma membranes are indicated with orange arrowheads , and nuclei are outlined with wide yellow dashed lines . Insets show a magnified view of the apical cell surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J Representative transmission electron microscopy (TEM) images of spheroids or an in vivo WAE cell from a biopsy-injured mousecolon. (G) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface.