Figure 5 ( A ) Co - expressing myc - Tau and eGFP - β - catenin increased nuclear translocation of β - catenin , while co - expressing myc - Tau and eGFP - β - cat K49R abolished the nuclear translocation of β - catenin in HEK293 cells . The nuclei were stained using DAPI ( n = 3 from three independent experiments , scale bar , 5 μm ) . Expressing β - cat K49Q increased its nuclear translocation . HEK293 cells were transfected with eGFP - β - cat WT or K49Q for 48 h , and then β - cat protein level in different fractions was measured by Western blottingExpressing β - cat K49Q increased its nuclear translocation . HEK293 cells were transfected with eGFP - β - cat WT or K49Q for 48 h , and then β - cat protein level in different fractions was measured by Western blotting Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs β - cat WT ( C ) Data were analyzed by Student ' s t testExpressing β - cat K49Q increased its nuclear translocation . HEK293 cells were transfected with eGFP - β - cat WT or K49Q for 48 h and immunofluorescent labeling using a laser scanning confocal microscope ( D ) . The nuclei ( blue ) was stained by DAPI ( n = 3 from three independent experiments , scale bar , 2 μm ) . ( E ) Expressing β - cat K49Q further increased its transcription activity measured by dual Topflash / Fopflash reporter luciferase assay after transient transfection of β - cat WT , or K49Q , or Vec in HEK293 cells for 48 h , and the luciferase activity was normalized by β - cat WT or K49Q protein level ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec ; # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat WT Data were analyzed by one - way ANOVA ( F ) Expressing Tau - k18 ( - ) abolished Tau - induced upregulation of mRNA levels of survival genes ( bcl2 and survivin ) and dkk1 ( downstream of Wnt signaling ) in N2a cells measured by Q - PCR ( n = 6 ~ 9 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau ( F Data were analyzed by one - way ANOVA ( G ) Expressing Tau - k18 ( - ) or Tau - 2CA abolished Tau - induced Wnt / β - catenin transcriptional activation measured by dual reporter luciferase activity assay in HEK293 cells ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs vs Tau Data were analyzed by one - way ANOVA ( H ) Expressing phosphor - mimic TauS199E ( TauS199E ) further increased the transcription activity of Wnt / TCF / LEF1 by Tau in HEK293 cells ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec ; # # , P < 0 . 01 , # # # , P < 0 . 001 vs Tau ( H ) . Data were analyzed by , one - way ANOVAThe increased mRNA levels of bcl2 , survivin and dkk1 in the AD brains compared with the age - / sex - matched controls detected by Q - PCR ( n = 7 each group ) Data information : Data are expressed as mean ± SEM , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Ctrl ( I ) Data were analyzed by Student ' s t testThe increased mRNA levels of bcl2 , survivin and dkk1 in the AD brains compared with the age - / sex - matched controls detected by Q - PCR ( n = 7 each group ) , and the positive correlations with the increased K49 - acetylated β - catenin ( see Fig . 1I , J ) analyzed by Pearson analysis .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Co-expressing myc-Tau and eGFP-β-catenin increased nuclear translocation of β-catenin, while co-expressing myc-Tau and eGFP-β-cat K49R abolished the nuclear translocation of β-catenin in HEK293 cells. The nuclei were stained using DAPI (n=3 from three independent experiments, scale bar, 5 μm).Expressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h, and then β-cat protein level in different fractions was measured by Western blottingExpressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h, and then β-cat protein level in different fractions was measured by Western blotting Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs β-cat WT (C) Data were analyzed by Student's t testExpressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h and immunofluorescent labeling using a laser scanning confocal microscope (D). The nuclei (blue) was stained by DAPI (n=3 from three independent experiments, scale bar, 2 μm).(E) Expressing β-cat K49Q further increased its transcription activity measured by dual Topflash/Fopflash reporter luciferase assay after transient transfection of β-cat WT, or K49Q, or Vec in HEK293 cells for 48 h, and the luciferase activity was normalized by β-cat WT or K49Q protein level (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec ; ##, P<0.01, ###, P<0.001 vs β-cat WT Data were analyzed by one-way ANOVA(F) Expressing Tau-k18(-) abolished Tau-induced upregulation of mRNA levels of survival genes (bcl2 and survivin) and dkk1 (downstream of Wnt signaling) in N2a cells measured by Q-PCR (n=6~9 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau (F Data were analyzed by one-way ANOVA(G) Expressing Tau-k18(-) or Tau-2CA abolished Tau-induced Wnt/β-catenin transcriptional activation measured by dual reporter luciferase activity assay in HEK293 cells (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vs Tau Data were analyzed by one-way ANOVA(H) Expressing phosphor-mimic TauS199E (TauS199E) further increased the transcription activity of Wnt/TCF/LEF1 by Tau in HEK293 cells (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec ; ##, P<0.01, ###, P<0.001 vs Tau (H). Data were analyzed by , one-way ANOVAThe increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group) Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Ctrl (I) Data were analyzed by Student's t testThe increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group), and the positive correlations with the increased K49-acetylated β-catenin (see Fig. 1I, J) analyzed by Pearson analysis.


Figure 1Clustered heatmap of the whole chemical genomics screen ; each subsection belongs to an individual strain ' s KO library . Grey cells indicate missing values .

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Figure 1Clustered heatmap of the whole chemical genomics screen; each subsection belongs to an individual strain's KO library. Grey cells indicate missing values.


Figure 2S - score scatterplots for each pairwise strain comparison , highlighting conserved phenotypes ( black points ) , significant changes ( red points ) and gene - condition relationships for which no call can reliably be made ( grey points ) . " r " , pearson ' s r value . Fraction of deletion phenotypes in each strain conserved with other stains in pairwise , three - way and four - way comparisons . Error bars represent standard deviation for all pairwise and three - way comparisons . Only a four - way comparison is possible for each strain so no error bars are represented in these cases . Gene exclusiveness : a measure of each gene ' s propensity to change its chemical genomics profile across strains . The top 10 genes ' names are reported .

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Figure 2S-score scatterplots for each pairwise strain comparison, highlighting conserved phenotypes (black points), significant changes (red points) and gene-condition relationships for which no call can reliably be made (grey points). "r", pearson's r value.Fraction of deletion phenotypes in each strain conserved with other stains in pairwise, three-way and four-way comparisons. Error bars represent standard deviation for all pairwise and three-way comparisons. Only a four-way comparison is possible for each strain so no error bars are represented in these cases.Gene exclusiveness: a measure of each gene's propensity to change its chemical genomics profile across strains. The top 10 genes' names are reported.


Figure 3Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons . Wild type fitness of each strain relative to S288C across the same conditions as in panel A ; each dot represents a specific replicate where colony sizes were measured . Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C ; each dot represents a strain - condition replicate as in panel B . The conditions maltose , glycerol , and NaCl are highlighted . Changes in gene deletion phenotypes for growth on osmotic stress conditions . The top heatmap contains genes belonging to the HOG pathway , while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS . Significant growth phenotypes are marked with " - " . Changes in deletion phenotypes for growth on glycerol and maltose . The top heatmap contains the MAL genes , while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains . Significant growth phenotypes are marked with " - " .

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Figure 3Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons.Wild type fitness of each strain relative to S288C across the same conditions as in panel A; each dot represents a specific replicate where colony sizes were measured.Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted.Changes in gene deletion phenotypes for growth on osmotic stress conditions. The top heatmap contains genes belonging to the HOG pathway, while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS. Significant growth phenotypes are marked with "-".Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".


Figure 4S - score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening . B ) Number of variants significantly associated ( p - value < 1E - 6 ) with phenotypic variation in each growth condition . C ) Number of associated variants that overlap ( i . e . are in a 3kbp window ) with a conditionally essential gene in the same condition , in any of the four KO libraries . Manhattan plots showing examples of overlaps between associated variants and the KO screening . The top plot shows the associations between variants and growth in the natural isolates as a function of the - log10 of the association p - value , while the four bottom plots show the strength of the KO phenotypes across the four yeast strains , as a function of the - log10 of the corrected S - score p - value . Sections shaded in grey indicate the overlap between associations and KO data . Position in the yeast chromosome is reported in kilobase units .

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Figure 4S-score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening. B) Number of variants significantly associated (p-value < 1E-6) with phenotypic variation in each growth condition. C) Number of associated variants that overlap (i.e. are in a 3kbp window) with a conditionally essential gene in the same condition, in any of the four KO libraries.Manhattan plots showing examples of overlaps between associated variants and the KO screening. The top plot shows the associations between variants and growth in the natural isolates as a function of the -log10 of the association p-value, while the four bottom plots show the strength of the KO phenotypes across the four yeast strains, as a function of the -log10 of the corrected S-score p-value. Sections shaded in grey indicate the overlap between associations and KO data. Position in the yeast chromosome is reported in kilobase units.


Figure 1 . ( A and B ) Endogenous FYCO1 binds to GST - LC3B . GST - LC3B bound to glutathione - Sepharose beads was incubated with HeLa cell lysate , and copurified proteins were identified by Coomassie blue staining and mass spectrometry ( A ) or immunoblotting with anti - FYCO1 antibody ( B ) . ( C ) GST - LC3B but not GST - p62 or GST alone binds to FYCO1 in a GST pull - down assay . GST , GST - LC3B , or GST - p62 bound to glutathione - Sepharose beads was incubated with [ 35S ] methionine - labeled myc - FYCO1 . Formed protein complexes were isolated and visualized by autoradiography ( top ) or Coomassie blue staining ( bottom ) . ( D ) GFP - LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate . HeLa cells expressing GFP or GFP - LC3B were lysed and processed as in B . IP , immunoprecipitation ; MM , molecular mass ; WB , Western blot .

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Figure 1.(A and B) Endogenous FYCO1 binds to GST-LC3B. GST-LC3B bound to glutathione-Sepharose beads was incubated with HeLa cell lysate, and copurified proteins were identified by Coomassie blue staining and mass spectrometry (A) or immunoblotting with anti-FYCO1 antibody (B).(C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione-Sepharose beads was incubated with [35S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom).(D) GFP-LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate. HeLa cells expressing GFP or GFP-LC3B were lysed and processed as in B. IP, immunoprecipitation; MM, molecular mass; WB, Western blot.


Figure 2 . ( B ) The region of FYCO1 between aa 1 , 276 and 1 , 294 is essential and sufficient for the interaction with LC3B . GST or GST - LC3B were incubated with [ S35 ] methionine - labeled deletion mutants of FYCO1 and processed as in Fig . 1 C . ( C ) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells . HeLa cells expressing the indicated constructs were imaged by confocal microscopy . Insets show an enlarged field of interest . ( D ) Both N - and C - terminal domains of LC3B are needed for efficient interaction with FYCO1 . GST , GST - LC3B , or its deletion mutants were incubated with [ S35 ] methionine - labeled myc - FYCO1 and processed as in Fig . 1 C . ARG , autoradiography ; CB , Coomassie blue . ( E ) The interaction between FYCO1 and LC3B is direct . GST or GST - LC3B was incubated with recombinant MBP - FYCO1863 - 1 , 478 . Protein complexes were isolated and visualized by immunoblotting with anti - GST or anti - MBP antibodies . WB , Western blot . Bars : ( C ) 10 µm ; ( C , insets ) 1 µm .

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Figure 2.(B) The region of FYCO1 between aa 1,276 and 1,294 is essential and sufficient for the interaction with LC3B. GST or GST-LC3B were incubated with [S35]methionine-labeled deletion mutants of FYCO1 and processed as in Fig. 1 C.(C) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells. HeLa cells expressing the indicated constructs were imaged by confocal microscopy. Insets show an enlarged field of interest.(D) Both N- and C-terminal domains of LC3B are needed for efficient interaction with FYCO1. GST, GST-LC3B, or its deletion mutants were incubated with [S35]methionine-labeled myc-FYCO1 and processed as in Fig. 1 C. ARG, autoradiography; CB, Coomassie blue.(E) The interaction between FYCO1 and LC3B is direct. GST or GST-LC3B was incubated with recombinant MBP-FYCO1863-1,478. Protein complexes were isolated and visualized by immunoblotting with anti-GST or anti-MBP antibodies. WB, Western blot. Bars: (C) 10 µm; (C, insets) 1 µm.


Figure 3 . ( A ) Endogenous FYCO1 localizes to the cytosolic punctuated structures . HeLa cells cultured in complete growth medium ( left ) or in HBSS for 3 h ( right ) were fixed and stained with anti - FYCO1 antibody ( green ) and Draq5 ( blue ) . ( B ) Overexpressed mCherry - FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells . ( C ) FYCO1 partially colocalizes with LC3B , LAMP1 , ATG5 , ATG16L , p62 , and HuntingtinQ64 and does not colocalize with EEA1 . HeLa cells or mouse embryonic fibroblasts ( for ATG16L staining ) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection . For p62 staining , HeLa cells were incubated with 0 . 2 µM BafA1 for 12 h before fixation . ( D ) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures . HeLa cells cotransfected with GFP - FYCO1 , mCherry - LC3B , and myc - p62 were imaged by confocal microscopy 48 h after transfection . ( E and F ) FYCO1 - decorated structures are heterogenous in their luminal content . ( E ) HeLa cells transfected with CFP - FYCO1 and mCherry - YFP - p62 were imaged 48 h after transfection . ( F ) HeLa cells transfected with GFP - FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h in normal growth medium to stain late endocytic compartments ( left ) . Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647 - dextran ( 10 , 000 D ; right ) .

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Figure 3.(A) Endogenous FYCO1 localizes to the cytosolic punctuated structures. HeLa cells cultured in complete growth medium (left) or in HBSS for 3 h (right) were fixed and stained with anti-FYCO1 antibody (green) and Draq5 (blue).(B) Overexpressed mCherry-FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells.(C) FYCO1 partially colocalizes with LC3B, LAMP1, ATG5, ATG16L, p62, and HuntingtinQ64 and does not colocalize with EEA1. HeLa cells or mouse embryonic fibroblasts (for ATG16L staining) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection. For p62 staining, HeLa cells were incubated with 0.2 µM BafA1 for 12 h before fixation.(D) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures. HeLa cells cotransfected with GFP-FYCO1, mCherry-LC3B, and myc-p62 were imaged by confocal microscopy 48 h after transfection. (E and F) FYCO1-decorated structures are heterogenous in their luminal content.(E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection.(F) HeLa cells transfected with GFP-FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h in normal growth medium to stain late endocytic compartments (left). Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647-dextran (10,000 D; right).


Figure 4 . ( A ) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection . Insets show an enlarged field of interest . ( B - D ) Immunoelectron micrographs of HeLa cells transfected with GFP - FYCO1 and stained with anti - GFP polyclonal antibody ( 10 - nm gold particle ) .

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Figure 4.(A) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection. Insets show an enlarged field of interest.(B-D) Immunoelectron micrographs of HeLa cells transfected with GFP-FYCO1 and stained with anti-GFP polyclonal antibody (10-nm gold particle).


Figure 5 . ( E ) HeLa cells transfected with CFP - FYCO1 and mCherry - YFP - p62 were imaged 48 h after transfection . ( B ) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( C ) FYCO1 can self - interact in a CC - dependent manner . Full - length GFP - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc - tagged FYCO1 in rabbit reticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - myc antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation . ( D ) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein - lipid overlay assay . PIP Strips were incubated with 1 - µg / ml solutions of MBP - FYVEFYCO1 , MBP - 2xFYVEFYCO1 , or MBP - FYCO1863 - 1 , 233 for 1 h , and bound proteins were detected by immunostaining with anti - MBP antibody . LPA , lysophosphatic acid ; LPC , lysophosphocholine ; PI , phosphatidylinositol ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine - 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine . ( E ) The construct containing a tandem fusion of two FYVE domains from FYCO1 ( 2xFYVEFYCO1 ) localizes to the nuclear envelope and ER in HeLa cells . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A and E ) Insets show an enlarged field of interest . ( F ) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs ( left ) and PXp40phox ( right ) . HeLa cells were imaged 24 h after transfection with the indicated constructs . Bars , 10 µm .

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Figure 5.(E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection.(B) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles. HeLa cells were imaged 24 h after transfection with the indicated constructs.(C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.(D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein-lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVEFYCO1, MBP-2xFYVEFYCO1, or MBP-FYCO1863-1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.(E) The construct containing a tandem fusion of two FYVE domains from FYCO1 (2xFYVEFYCO1) localizes to the nuclear envelope and ER in HeLa cells. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A and E) Insets show an enlarged field of interest.(F) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs (left) and PXp40phox (right). HeLa cells were imaged 24 h after transfection with the indicated constructs. Bars, 10 µm.


Figure 6 . ( A ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles . HeLa cells transiently transfected with FYCO1990 - 1 , 233 were imaged 24 h after transfection . ( B ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 that can efficiently interact with the full - length FYCO1 . Full - length myc - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP - tagged FYCO1 in rabbitreticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - SDS - PAGE antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation . ( C ) FYCO1990 - 1 , 233 strongly colocalizes with Rab7 , LysoTracker red , and Alexa Fluor 647 - dextran ( 10 , 000 D ) but only weakly with PXp40phox . HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h were imaged 24 h after transfection . ( D ) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 ( FYCO1990 - 1 , 233 + 1 , 156 - 1 , 233 ) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A , C , and D ) Insets show an enlarged field of interest . Bars , 10 µm .

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Figure 6.(A) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles. HeLa cells transiently transfected with FYCO1990-1,233 were imaged 24 h after transfection.(B) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbitreticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-SDS-PAGE antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.(C) FYCO1990-1,233 strongly colocalizes with Rab7, LysoTracker red, and Alexa Fluor 647-dextran (10,000 D) but only weakly with PXp40phox. HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h were imaged 24 h after transfection.(D) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 (FYCO1990-1,233+1,156-1,233) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A, C, and D) Insets show an enlarged field of interest. Bars, 10 µm.


Figure 7 . ( A ) Rab7Q67L recruits FYCO11 , 038 - 1 , 233 and FYCO11 , 091 - 1 , 233 but not FYCO11 , 156 - 1 , 233 to the cytosolic vesicles . HeLa cells transfected with GFP - FYCO11 , 038 - 1 , 233 ( top ) , GFP - FYCO11 , 091 - 1 , 233 ( bottom ) , or GFP - FYCO11 , 156 - 1 , 233 ( middle right ) with or without mCherry - Rab7Q67L were imaged 24 h after transfection . The graph shows the relative number of cells ± SD with GFP - FYCO11 , 038 - 1 , 233 recruited to intracellular membranes without and with cotransfected mCherry - Rab7Q67L . ( B ) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain ( FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 ) but not a 2xFYVE alone to the perinuclear vesicles . HeLa cells transfected with GFP - FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 or GFP - FYCO11 , 156 - 1 , 233 + 1 , 156 - 1 , 233 with or without mCherry - Rab7Q67L were imaged 24 h after transfection . ( C ) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate . HEK293 cells stably transfected with 3xFlag - FYCO1 were cotransfected with GTP - locked mutants of Rab5 , - 7 , - 11 , and - 24 and lysed 24 h after transfection , and protein complexes were immunoprecipitated with anti - Flag M2 agarose affinity gel . Bound proteins were eluted with Flag peptide , resolved by SDS - PAGE , and visualized by immunoblotting with anti - GFP and anti - Flag antibody . IP , immunoprecipitation ; WB , Western blot . ( D ) Leucine - 1151 and tryptophan - 1152 are important for the recruitment of FYCO1 to Rab7 - containing vesicles . HeLa cells transfected with GFP - FYCO1990 - 1 , 233L1151A / W1152A with or without mCherry - Rab7Q67L were imaged 24 h after transfection . ( B and D ) Insets show an enlarged field of interest . ( E ) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles . ( right ) HeLa cells cotransfected with GFP - RILP together with mCherry - FYCO1 and stained with Draq5 were imaged 24 h after transfection . ( left ) Relative number of HeLa cells cotransfected with mCherry - FYCO1 and the indicated constructs , in which FYCO1 is recruited to the intracellular vesicles . Error bars represent SD . Bars , 10 µm .

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Figure 7.(A) Rab7Q67L recruits FYCO11,038-1,233 and FYCO11,091-1,233 but not FYCO11,156-1,233 to the cytosolic vesicles. HeLa cells transfected with GFP-FYCO11,038-1,233 (top), GFP-FYCO11,091-1,233 (bottom), or GFP-FYCO11,156-1,233 (middle right) with or without mCherry-Rab7Q67L were imaged 24 h after transfection. The graph shows the relative number of cells ± SD with GFP-FYCO11,038-1,233 recruited to intracellular membranes without and with cotransfected mCherry-Rab7Q67L.(B) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain (FYCO11,091-1,233+1,156-1,233) but not a 2xFYVE alone to the perinuclear vesicles. HeLa cells transfected with GFP-FYCO11,091-1,233+1,156-1,233 or GFP-FYCO11,156-1,233+1,156-1,233 with or without mCherry-Rab7Q67L were imaged 24 h after transfection.(C) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate. HEK293 cells stably transfected with 3xFlag-FYCO1 were cotransfected with GTP-locked mutants of Rab5, -7, -11, and -24 and lysed 24 h after transfection, and protein complexes were immunoprecipitated with anti-Flag M2 agarose affinity gel. Bound proteins were eluted with Flag peptide, resolved by SDS-PAGE, and visualized by immunoblotting with anti-GFP and anti-Flag antibody. IP, immunoprecipitation; WB, Western blot.(D) Leucine-1151 and tryptophan-1152 are important for the recruitment of FYCO1 to Rab7-containing vesicles. HeLa cells transfected with GFP-FYCO1990-1,233L1151A/W1152A with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (B and D) Insets show an enlarged field of interest.(E) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles. (right) HeLa cells cotransfected with GFP-RILP together with mCherry-FYCO1 and stained with Draq5 were imaged 24 h after transfection. (left) Relative number of HeLa cells cotransfected with mCherry-FYCO1 and the indicated constructs, in which FYCO1 is recruited to the intracellular vesicles. Error bars represent SD. Bars, 10 µm.


Figure 8 . ( A ) Overexpressed FYCO1 is enriched in the cell periphery , close to the MT plus end . HeLa cells were imaged 48 h after transfection with mCherry - FYCO1 and GFP - tubulin . ( B ) Peripheral enrichment of FYCO1 is dependent on intact MTs . HeLa cells were transfected with GFP - FYCO1 and cultured in normal medium ( left ) or treated with 5 µM colcemid ( middle ) or 5 µM latrunculin A ( right ) for 2 h . ( C ) FYCO1 - decorated vesicles move along the MT tracks . HeLa cells transfected with mCherry - FYCO1 and GFP - tubulin were live imaged by confocal microscopy 48 h after transfection . Movements of FYCO1 - decorated structures were tracked with ImageJ MTrackJ plug - in ( National Institutes of Health ) and presented as 8 - min - long tracks in the overlay image . ( D ) Full - length FYCO1 but not the FYCO1 lacking aa 555 - 1 , 136 can redistribute Rab7 and ORP1L to the cell periphery . HeLa cells were transfected with the indicated constructs and stained with DRAQ5 . ( E ) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1 - decorated structures . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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Figure 8.(A) Overexpressed FYCO1 is enriched in the cell periphery, close to the MT plus end. HeLa cells were imaged 48 h after transfection with mCherry-FYCO1 and GFP-tubulin.(B) Peripheral enrichment of FYCO1 is dependent on intact MTs. HeLa cells were transfected with GFP-FYCO1 and cultured in normal medium (left) or treated with 5 µM colcemid (middle) or 5 µM latrunculin A (right) for 2 h.(C) FYCO1-decorated vesicles move along the MT tracks. HeLa cells transfected with mCherry-FYCO1 and GFP-tubulin were live imaged by confocal microscopy 48 h after transfection. Movements of FYCO1-decorated structures were tracked with ImageJ MTrackJ plug-in (National Institutes of Health) and presented as 8-min-long tracks in the overlay image.(D) Full-length FYCO1 but not the FYCO1 lacking aa 555-1,136 can redistribute Rab7 and ORP1L to the cell periphery. HeLa cells were transfected with the indicated constructs and stained with DRAQ5.(E) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1-decorated structures. HeLa cells were imaged 24 h after transfection with the indicated constructs.


Figure 9 . ( A ) Western blot ( WB ) of cell lysates from HeLa cells stably expressing GFP - LC3 48 h after transient transfection with siRNA against FYCO1 ( siRNAs : A , Applied Biosystems ; D , Thermo Fisher Scientific ) . MM , molecular mass . ( B ) Representative images of HeLa cells with or without the perinuclear clusters of GFP - LC3 - positive vesicles ( right ) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 ( left ) . Error bars represent SDs based on three independent experiments . ( C , D , and E ) The perinuclear clusters of GFP - LC3 - positive vesicles colocalize with mCherry - Rab7 but not with Golgi protein marker 58K or LysoTracker red . GFP - LC3 HeLa cells were transiently transfected with siRNA against FYCO1 ( Thermo Fisher Scientific ) 48 h before imaging or cotransfected with mCherry - Rab7 . Cells were either fixed and stained with anti - Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h . Insets show an enlarged field of interest . Bars , 10 µm .

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Figure 9.(A) Western blot (WB) of cell lysates from HeLa cells stably expressing GFP-LC3 48 h after transient transfection with siRNA against FYCO1 (siRNAs: A, Applied Biosystems; D, Thermo Fisher Scientific). MM, molecular mass.(B) Representative images of HeLa cells with or without the perinuclear clusters of GFP-LC3-positive vesicles (right) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 (left). Error bars represent SDs based on three independent experiments.(C, D, and E) The perinuclear clusters of GFP-LC3-positive vesicles colocalize with mCherry-Rab7 but not with Golgi protein marker 58K or LysoTracker red. GFP-LC3 HeLa cells were transiently transfected with siRNA against FYCO1 (Thermo Fisher Scientific) 48 h before imaging or cotransfected with mCherry-Rab7. Cells were either fixed and stained with anti-Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h. Insets show an enlarged field of interest. Bars, 10 µm.


Figure 1A , B . Representative images of H & amp ; amp ; E - ( A ) and PAS - ( B ) stained colon sections from wild - type and Angptl2 - / - mice . Scale bar = 100μm . C , D . Representative images of colon crypts as assessed by Ki - 67 ( C ) and pH3 ( D ) staining . Scale bar = 100 μm . E . mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild - type ( n = 4 ) and Angptl2 - / - ( n = 4 ) mice based on qRT - PCR analysis . F . Western blotting analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 served as an internal control . Numbers below panels represent normalized expression of proteins . G . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice . Scale bar = 50 μm .

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Figure 1A, B. Representative images of H&amp;E- (A) and PAS- (B) stained colon sections from wild-type and Angptl2-/- mice. Scale bar = 100μm.C, D. Representative images of colon crypts as assessed by Ki-67 (C) and pH3 (D) staining. Scale bar = 100 μm.E. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild-type (n=4) and Angptl2-/- (n=4) mice based on qRT-PCR analysis.F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 served as an internal control. Numbers below panels represent normalized expression of proteins.G. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice. Scale bar = 50 μm.


Figure 2A . Survival rates of wild - type ( n = 33 ) and Angptl2 - / - ( n = 33 ) mice supplied with drinking water containing 2 . 5 % DSS for 6 days and then untreated water for 6 more days , from two different experiments . * * * p < 0 . 001 , by log - rank test . B . Representative images of colons from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . C . Colon length of wild - type ( n = 7 ) and Angptl2 - / - ( n = 6 ) mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . D , E , F . Representative images of colon crypts as assessed by H & amp ; amp ; E ( D ) , Ki - 67 ( E ) , and pH3 ( F ) staining in wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . Scale bar = 100 μm . G , H . The number of regenerating crypts as quantified using Ki - 67 ( G ) and pH3 ( H ) staining . Each data point represents the number of Ki - 67 - positive viable crypts or the number of pH3 - positive cells in a single field of view . Multiple areas in at least 6 mice per genotype were quantified . Numbers below panels represent the average . I . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . Scale bar = 50 μm . J . mRNA levels of indicated genes in isolated crypts from wild - type ( n = 4 ) and Angptl2 - / - ( n = 4 ) mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) .

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Figure 2A. Survival rates of wild-type (n=33) and Angptl2-/- (n=33) mice supplied with drinking water containing 2.5 % DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***p < 0.001, by log-rank test.B. Representative images of colons from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).C. Colon length of wild-type (n=7) and Angptl2-/- (n=6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).D, E, F. Representative images of colon crypts as assessed by H&amp;E (D), Ki-67 (E), and pH3 (F) staining in wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 100 μm.G, H. The number of regenerating crypts as quantified using Ki-67 (G) and pH3 (H) staining. Each data point represents the number of Ki-67-positive viable crypts or the number of pH3-positive cells in a single field of view. Multiple areas in at least 6 mice per genotype were quantified. Numbers below panels represent the average.I. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 50 μm.J. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).


Figure 3A . Weight loss of wild - type ( n = 4 ) and Angptl2 - / - ( n = 5 ) mice in days following 12 Gy irradiation . B . Small intestine and colon length from wild - type ( n = 4 ) and Angptl2 - / - ( n = 5 ) mice 5 days after 12 Gy irradiation . C , D . Representative images of crypts of small intestine as assessed by H & amp ; amp ; E ( C ) and Ki - 67 ( D ) staining in wild - type and Angptl2 - / - mice 5 days after 12 Gy irradiation . Scale bar = 100 μm . E . The number of regenerating crypts in small intestine as quantified by Ki - 67 ( D ) staining at days 3 and 5 . Each data point represents the number of Ki - 67 - positive viable crypts in a single field of view . Multiple areas in at least 3 mice per genotype were quantified . Numbers below panels represent average . F , G . Representative images of colon crypts as assessed by H & amp ; amp ; E ( F ) or Ki - 67 ( G ) staining in wild - type and Angptl2 - / - mice 5 days after 12 Gy irradiation . Scale bar = 100 μm . H . The number of regenerating colon crypts as quantified by using Ki - 67 ( G ) staining at days 3 and 5 . Each data point represents the number of Ki - 67 - positive viable crypts in a single field of view . Multiple areas in at least 3 mice per genotype were quantified . Numbers below panels represent average .

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Figure 3A. Weight loss of wild-type (n=4) and Angptl2-/- (n=5) mice in days following 12 Gy irradiation.B. Small intestine and colon length from wild-type (n=4) and Angptl2-/- (n=5) mice 5 days after 12 Gy irradiation.C, D. Representative images of crypts of small intestine as assessed by H&amp;E (C) and Ki-67 (D) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.E. The number of regenerating crypts in small intestine as quantified by Ki-67 (D) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.F, G. Representative images of colon crypts as assessed by H&amp;E (F) or Ki-67 (G) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.H. The number of regenerating colon crypts as quantified by using Ki-67 (G) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.


Figure 4A . Angptl2 mRNA levels in small intestine ( proximal , mid and distal ) , cecum and colon ( proximal and distal ) of wild - type mice based on qRT - PCR analysis . n = 5 . B . ANGPTL2 IHC in colon from wild - type and Angptl2 - / - mice . The Angptl2 - / - colon image serves as a negative control . Scale bar = 50 μm . C . mRNA levels of indicated genes in wild - type ( n = 4 ) mice based on qRT - PCR analysis . D . Representative IF images of distal colon from wild - type mice as assessed with anti - ANGPTL2 ( red ) and anti - α - SMA ( green ) . Nuclei are counterstained with DAPI ( blue ) . Scale bar = 100 μm .

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Figure 4A. Angptl2 mRNA levels in small intestine (proximal, mid and distal), cecum and colon (proximal and distal) of wild-type mice based on qRT-PCR analysis. n=5.B. ANGPTL2 IHC in colon from wild-type and Angptl2-/- mice. The Angptl2-/- colon image serves as a negative control. Scale bar = 50 μm.C. mRNA levels of indicated genes in wild-type (n=4) mice based on qRT-PCR analysis.D. Representative IF images of distal colon from wild-type mice as assessed with anti-ANGPTL2 (red) and anti-α-SMA (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.


Figure 5A , B , C . mRNA levels of indicated genes in ISEMFs from wild - type ( n = 4 ) and Angptl2 - / - ( n = 3 ) mice based qRT - PCR analysis . D . Representative staining for P - Smad1 / 5 in crypts from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( DSS ) or untreated water for 12 days ( Untreated ) . Dashed lines indicate half of the crypt and arrowheads show P - Smad1 / 5 - positive cells in lower halves of crypts . Scale bar = 50 μm . E . The number of P - Smad1 / 5 - positive cells in the crypt base ( n = 60 ) from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( DSS ) or untreated water for 12 days ( Untreated ) . Multiple areas in 3 mice per genotype were quantified . F . Western blotting analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 serves as an internal control . Numbers below panels represent normalized protein expression .

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Figure 5A, B, C. mRNA levels of indicated genes in ISEMFs from wild-type (n=4) and Angptl2-/- (n=3) mice based qRT-PCR analysis.D. Representative staining for P-Smad1/5 in crypts from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Dashed lines indicate half of the crypt and arrowheads show P-Smad1/5-positive cells in lower halves of crypts. Scale bar = 50 μm.E. The number of P-Smad1/5-positive cells in the crypt base (n=60) from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Multiple areas in 3 mice per genotype were quantified.F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 serves as an internal control. Numbers below panels represent normalized protein expression.


Figure 6A . mRNA levels of indicated genes in colon and cultured ISEMFs from wild - type mice ( n = 4 ) based on qRT - PCR analysis . Inta5 , Integrin α5 ; Intb1 , Integrin β1 . B . Representative IHC of integrin α5 and β1 in colon from wild - type mouse . Scale bar = 50 μm . C , D . mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild - type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors . n = 3 . U0126 , ERK inhibitor ; LY294002 , PI3K inhibitor ; BAY11 - 7085 , NF - κB inhibitor .

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Figure 6A. mRNA levels of indicated genes in colon and cultured ISEMFs from wild-type mice (n=4) based on qRT-PCR analysis. Inta5, Integrin α5; Intb1, Integrin β1.B. Representative IHC of integrin α5 and β1 in colon from wild-type mouse. Scale bar = 50 μm.C, D. mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild-type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors. n=3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11-7085, NF-κB inhibitor.


Figure 7A . Colon organoid cultures treated with vehicle or rANGPTL2 protein . Scale bar = 200 μm . B . Organoid growth efficiency and size following treatment with vehicle or rANGPTL2 . rA2 , recombinant ANGPTL2 . n = 4 . Data from vehicle / Wnt ( + ) was set at 1 . D . Colon organoid cultures in presence of ISEMFs with or without Noggin ( Nog ) . Scale bar = 50 μmE . Organoid growth efficiency and size in presence of ISEMFs with or without Noggin ( Nog ) . n = 4 . Data from Wild - type organoids / Wild - type ISEMFs / Nog ( + ) was set at 1 .

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Figure 7A. Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm.B. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n=4. Data from vehicle/Wnt (+) was set at 1.D. Colon organoid cultures in presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μmE. Organoid growth efficiency and size in presence of ISEMFs with or without Noggin (Nog). n=4. Data from Wild-type organoids/Wild-type ISEMFs/Nog (+) was set at 1.


Figure 1 ( A ) ELISA of supernatant IL - 1β for unprimed THP - 1 cells ( UT ) or LPS - primed ( 500 ng / ml , 3 hrs ) THP - 1 cells then infected with ZIKV ( MOI = 1 ) for 36 hrs . Uninfected cells serves as mock control . ( B ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells , left untreated or primed by LPS ( 500 ng / ml , 3 hrs ) , after ZIKV infection ( MOI = 1 ) for 36 hrs . ( C ) ELISA of IL - 1β in the supernatants of wild type ( WT ) , NLRP3 knockout ( KO ) # 1 or AIM2 KO # 1 THP - 1 cells left untreated ( UT ) , or pretreated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) stimulation , or stimulated with poly ( dA : dT ) ( 1mg / ml , 6 hrs ) , or infected with ZIKV ( MOI = 1 , 36 hrs ) . ( D ) ELISA of IL - 1β in the supernatants of two clones of NLRP3 KO THP - 1 cells with or without ZIKV infection ( MOI = 1 ) at the indicated time points . ( E ) ELISA of IL - 1β in the supernatants of PBMCs from two donors transfected with control ( ctrl ) siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for 36 hrs . ( F ) ELISA of IL - 1β in the supernatant of WT , NLRP3 KO or AIM2 KO THP - 1 cells with ZIKV ( MR766 or GZ01 ) ( MOI = 1 ) or DENV - 2 ( MOI = 1 ) infection for 36 hrs . ( G ) ELISA of IL - 1β in the supernatants of THP - 1 cells pre - infected with ZIKV for 24 hrs or mock control . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) treatment for 6 hrs . ( H ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells pre - infected with ZIKV ( MOI = 1 ) or mock control for 24 hrs . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) stimulation for 6 hrs . ( I ) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3 , ASC , pro - caspase - 1 and pro - IL - 1β together with NS1 . ( J ) Immunoblot analysis of protein extracts of Flag - tagged NS1 - inducible THP - 1 cells treated with increasing doses of doxycycline ( Dox ) for 24 hrs . ( K ) ELISA of IL - 1β in the supernatants of Flag - NS1 - inducible THP - 1 cells left unprimed then treated with ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) , or pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation . ( L ) Immunoblot analysis of supernatants ( Sup ) and cell extracts ( Lys ) of Flag - NS1 - inducible THP - 1 cells pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation .

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Figure 1(A) ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 hrs) THP-1 cells then infected with ZIKV (MOI=1) for 36 hrs. Uninfected cells serves as mock control.(B) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated or primed by LPS (500 ng/ml, 3 hrs), after ZIKV infection (MOI=1) for 36 hrs.(C) ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1 or AIM2 KO#1 THP-1 cells left untreated (UT), or pretreated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) stimulation, or stimulated with poly (dA:dT) (1mg/ml, 6 hrs), or infected with ZIKV (MOI=1, 36 hrs).(D) ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI=1) at the indicated time points.(E) ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for 36 hrs.(F) ELISA of IL-1β in the supernatant of WT, NLRP3 KO or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI=1) or DENV-2 (MOI=1) infection for 36 hrs.(G) ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 hrs or mock control. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) treatment for 6 hrs.(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.(I) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1 and pro-IL-1β together with NS1.(J) Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 hrs.(K) ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs), or pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.(L) Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.


Figure 2 ( A ) Relative qRT - PCR analysis of ZIKV RNA in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( B ) Relative qRT - PCR analysis of ZIKV RNA in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( C ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs by the indicated antibodies . ( D ) Relative qRT - PCR analysis of ZIKV RNA in WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points . ( E ) Plaque titration of ZIKV in supernatants of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points . ( F ) Immunoblot analysis of extracts of WT or caspase - 1 KO THP - 1 cells by the indicated antibodies . ( G ) Relative qRT - PCR analysis of ZIKV RNA in WT or caspase - 1 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( H ) Relative qRT - PCR analysis of ZIKV RNA in NS - 1 - inducible THP - 1 cells treated with or without doxycycline ( Dox ) , then left untreated or treated with Ac - YVAD - cmk ( 20 μΜ ) for 3 hrs followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( I ) Relative qRT - PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors . ( J ) Relative qRT - PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs . ( K ) qRT - PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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Figure 2(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.(B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(C) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs by the indicated antibodies.(D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.(E) Plaque titration of ZIKV in supernatants of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.(F) Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies.(G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points.(I) Relative qRT-PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors.(J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


Figure 3 ( A ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - NS1 together with Flag - NLRP3 , Flag - ASC , Flag - caspase - 1 and Flag - IL - 1β . WCL , whole cell lysates . ( B ) Co - immunoprecipitation and immunoblot analysis of extracts of NS1 - inducible THP - 1 cells , which expressed Flag - tagged NS1 , treated with or without doxycycline ( Dox ) ( 100 ng / ml ) . ( C ) Immunoblot analysis of extracts of THP - 1 cells treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) followed by increasing doses of Dox treatment . ( D ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - NS1 . Below , caspase - 1 mRNA RT - PCR ; RPL13A mRNA RT - PCR serves as a loading control . ( E ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - DENV - 2 - NS1 . ( F ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 followed by cycloheximide ( CHX ) treatment ( 100 μg / ml ) for the indicated time points . ( G ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 then treated with MG132 ( 10 mM ) , Lactacystin ( 5 μM ) , Carfilzomib ( 100 mM ) or CQ ( 50 mM ) for 6 hrs . ( H ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) . ( I ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - NS1 , followed by MG132 ( 10 mM ) treatment for 6 hrs .

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Figure 3(A) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1 and Flag-IL-1β. WCL, whole cell lysates.(B) Co-immunoprecipitation and immunoblot analysis of extracts of NS1-inducible THP-1 cells, which expressed Flag-tagged NS1, treated with or without doxycycline (Dox) (100 ng/ml).(C) Immunoblot analysis of extracts of THP-1 cells treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs) followed by increasing doses of Dox treatment.(D) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-NS1. Below, caspase-1 mRNA RT-PCR; RPL13A mRNA RT-PCR serves as a loading control.(E) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-DENV-2-NS1.(F) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 followed by cycloheximide (CHX) treatment (100 μg/ml) for the indicated time points.(G) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), Lactacystin (5 μM), Carfilzomib (100 mM) or CQ (50 mM) for 6 hrs.(H) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml).(I) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-NS1, followed by MG132 (10 mM) treatment for 6 hrs.


Figure 4 ( A ) Immunoblot analysis of extracts of wild type ( WT ) and USP8 knockout ( KO ) 293T cells transfected with Flag - caspase - 1 together with empty vector or Myc - NS1 . ( B ) Immunoblot analysis of extracts of transfected with Flag - caspase - 1 together with empty vector or Myc - USP8 then treated with or without MG132 ( 10 mM ) for 6 hrs . ( C ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) then transfected with or without poly ( I : C ) ( 2 mg / ml , 6 hrs ) . ( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 and Myc - USP8 together with HA - NS1 followed by MG132 treatment ( 10 mM ) for 6 hrs . ( E ) Co - immunoprecipitation and immunoblot analysis of extracts of THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs . ( F ) Co - immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs . ( G ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin ( Ub ) or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - USP8 , followed by MG132 ( 10 mM ) treatment for 6 hrs . ( H ) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag - caspase - 1 , HA - K11 - Ub together with empty vector or Myc - NS1 , followed by two - step immunoprecipitation with anti - Flag beads and immunoblot analysis with anti - HA . ( I ) Co - immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag - caspase - 1 and HA - K11 - Ub together with WT or inactive mutant C786A of USP8 .

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Figure 4(A) Immunoblot analysis of extracts of wild type (WT) and USP8 knockout (KO) 293T cells transfected with Flag-caspase-1 together with empty vector or Myc-NS1.(B) Immunoblot analysis of extracts of transfected with Flag-caspase-1 together with empty vector or Myc-USP8 then treated with or without MG132 (10 mM) for 6 hrs.(C) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml) then transfected with or without poly (I:C) (2 mg/ml, 6 hrs).(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 and Myc-USP8 together with HA-NS1 followed by MG132 treatment (10 mM) for 6 hrs.(E) Co-immunoprecipitation and immunoblot analysis of extracts of THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.(F) Co-immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.(G) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin (Ub) or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-USP8, followed by MG132 (10 mM) treatment for 6 hrs.(H) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag-caspase-1, HA-K11-Ub together with empty vector or Myc-NS1, followed by two-step immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA.(I) Co-immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag-caspase-1 and HA-K11-Ub together with WT or inactive mutant C786A of USP8.


Figure 5 ( A ) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA - NS1 together with Flag - caspae - 1 wild type ( WT ) and mutants ( K37R , K44R or K134R ) . Lower panel shows the intensity analysis of the bands from the three independent experiments . ( B ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - K11 - ubiquitin ( Ub ) together with Flag - caspase - 1 ( WT , K37R , K44R , K134R ) . ( C ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - NS1 . Lower panel shows the intensity analysis of the bands from the three independent experiments . ( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - USP8 . Lower panel shows the intensity analysis of the bands from the three independent experiments .

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Figure 5(A) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA-NS1 together with Flag-caspae-1 wild type (WT) and mutants (K37R, K44R or K134R). Lower panel shows the intensity analysis of the bands from the three independent experiments.(B) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-K11-ubiquitin (Ub) together with Flag-caspase-1 (WT, K37R, K44R, K134R).(C) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-NS1. Lower panel shows the intensity analysis of the bands from the three independent experiments.(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-USP8. Lower panel shows the intensity analysis of the bands from the three independent experiments.


Figure 6 ( A ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in wild type ( WT ) or caspase - 1 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( B ) Relative qRT - PCR analysis of IFNB mRNA levels in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( C ) Relative qRT - PCR analysis of IFNB mRNA levels in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( D ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag - NS1 - inducible THP - 1 cells treated with or without Dox then left untreated or treated with YVAD ( 20 μM ) for 3 hrs , followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( E ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs . ( F ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs . ( G ) ELISA of IFN - β in the supernatants of PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs . ( H ) ELISA of IL - 1β production in brain of WT or Nlrp3 - / - 1 - day - old neonatal mice ( n = 3 per group ) intracerebrally injected with 1 × 105 plaque - forming units ( PFU ) of ZIKV for 5 days . ( I ) Relative qRT - PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV infection ( J ) ELISA of IFN - β production in brains of WT or Nlrp3 - / - neonatal mice ( K ) Relative qRT - PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV infection ( L ) Plaque titration of ZIKV in brain tissues of WT or Nlrp3 - / - neonatal mice ( M ) Body weight gain of WT and Nlrp3 - / - neonatal mice ( n = 4 per group ) inoculated with ZIKV ( 1 × 105 PFUs ) .

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Figure 6(A) Relative qRT-PCR analysis of IFNB, IFIT2 and IFIT1 mRNA levels in wild type (WT) or caspase-1 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.(C) Relative qRT-PCR analysis of IFNB mRNA levels in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points.(E) Relative qRT-PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.(F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.(G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.(H) ELISA of IL-1β production in brain of WT or Nlrp3-/- 1-day-old neonatal mice (n=3 per group) intracerebrally injected with 1×105 plaque-forming units (PFU) of ZIKV for 5 days.(I) Relative qRT-PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection(J) ELISA of IFN-β production in brains of WT or Nlrp3-/- neonatal mice(K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection(L) Plaque titration of ZIKV in brain tissues of WT or Nlrp3-/- neonatal mice(M) Body weight gain of WT and Nlrp3-/- neonatal mice (n=4 per group) inoculated with ZIKV (1×105 PFUs).


Figure 7 ( A ) Relative qRT - PCR analysis of ZIKV RNA levels in wild type ( WT ) or cGAS knockout ( KO ) THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points . ( B ) Relative qRT - PCR analysis of IFNΒ , IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points . ( C ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP , cGAS or NLRP3 and infected with ZIKV ( MOI = 1 ) for 36 hrs . ( D ) Relative qRT - PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP - 1 cells treated with or without YVAD ( 20 μM ) , followed by ZIKV infection ( MOI = 1 ) for indicated times points . ( E ) Immunoblot analysis of extracts of Flag - cGAS - overexpressed THP - 1 cells treated with or without YVAD ( 20 μM ) then infected with ZIKV ( MOI = 5 ) for 24 hrs or infected with HSV - 1 ( MOI = 1 ) for 24 hrs . Immunoblot analysis of WT , NLRP3 KO ( F ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . Immunoblot analysis of WT , caspase - 1 KO ( G ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points . ( H ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points . ( I ) Immunoblot analysis of PBMCs from Donor # 1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for the indicated time points . ( J ) Immunoblot analysis of extracts of 293T cells transfected with Myc - cGAS ( tagged on C - terminal ) , GFP - ASC , Flag - caspase - 1 , together with empty vector or HA - NS1 . ( K ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in WT , cGAS KO THP - 1 cells as well as cGAS KO THP - 1 cells reconstituted with WT or D140A / D157A mutant of cGAS , followed by ZIKV infection ( MOI = 1 ) for 24 hrs .

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Figure 7(A) Relative qRT-PCR analysis of ZIKV RNA levels in wild type (WT) or cGAS knockout (KO) THP-1 cells after ZIKV infection (MOI=1) for indicated time points.(B) Relative qRT-PCR analysis of IFNΒ, IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP-1 cells after ZIKV infection (MOI=1) for indicated time points.(C) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP, cGAS or NLRP3 and infected with ZIKV (MOI=1) for 36 hrs.(D) Relative qRT-PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP-1 cells treated with or without YVAD (20 μM), followed by ZIKV infection (MOI=1) for indicated times points.(E) Immunoblot analysis of extracts of Flag-cGAS-overexpressed THP-1 cells treated with or without YVAD (20 μM) then infected with ZIKV (MOI=5) for 24 hrs or infected with HSV-1 (MOI=1) for 24 hrs.Immunoblot analysis of WT, NLRP3 KO (F) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.Immunoblot analysis of WT, caspase-1 KO (G) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.(H) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.(I) Immunoblot analysis of PBMCs from Donor#1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for the indicated time points.(J) Immunoblot analysis of extracts of 293T cells transfected with Myc-cGAS (tagged on C-terminal), GFP-ASC, Flag-caspase-1, together with empty vector or HA-NS1.(K) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in WT, cGAS KO THP-1 cells as well as cGAS KO THP-1 cells reconstituted with WT or D140A/D157A mutant of cGAS, followed by ZIKV infection (MOI=1) for 24 hrs.


Figure 1Sexing the established ESCs by detecting the Y - linked Zfy gene using genomic DNA ( gDNA ) - qPCR , normalized to Gapdh . n . d . : not detected . Average of presumed males was set to 1 . DNA - FISH detection of the number of X - linked Atrx gene foci . Representative images of the DNA - FISH are shown . Nuclei are surrounded by white dotted lines . Magenta : Atrx ( white arrowheads ) , cyan : DAPI . N > 100 . Heatmap analysis from RNA - seq . Each log10 ( FPKM + 1 ) value was used for clustering . Genes with high or low expression are shown in red and blue , respectively . FPKM : fragments per kilobases per million reads . X and Y chromosome mapping . Genes with high expression ( up ) are shown in orange and low expression ( down ) are shown in blue . The defined ratio of up or down genes is > 1 . 5 and < two - thirds , respectively . The comparison is ( each female vs the average for males ) or ( each male vs the average for females ) . The location controls for the expressed genes ( exp . genes ) on the X - chromosome and Y - chromosome are plotted ( red squares ) for genes whose FPKM averages were above 1 for females or males .

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Figure 1Sexing the established ESCs by detecting the Y-linked Zfy gene using genomic DNA (gDNA)-qPCR, normalized to Gapdh. n.d.: not detected. Average of presumed males was set to 1.DNA-FISH detection of the number of X-linked Atrx gene foci. Representative images of the DNA-FISH are shown. Nuclei are surrounded by white dotted lines. Magenta: Atrx (white arrowheads), cyan: DAPI. N>100.Heatmap analysis from RNA-seq. Each log10(FPKM+1) value was used for clustering. Genes with high or low expression are shown in red and blue, respectively. FPKM: fragments per kilobases per million reads.X and Y chromosome mapping. Genes with high expression (up) are shown in orange and low expression (down) are shown in blue. The defined ratio of up or down genes is > 1.5 and < two-thirds, respectively. The comparison is (each female vs the average for males) or (each male vs the average for females). The location controls for the expressed genes (exp. genes) on the X-chromosome and Y-chromosome are plotted (red squares) for genes whose FPKM averages were above 1 for females or males.


Figure 2Clonogenic assay for ESCs in response to etoposide ( A ) or camptothecin ( B ) . Non - treated cells were set to 1 . Nanog HR assay . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown . The results for each cell line are shown at right . Nanog expression confirmed by RNA - seq . Detection of the number of X - linked Atrx gene foci by DNA - FISH in GFP - positive subclones of the Nanog - targeting assay ( nine independent clones , N > 100 each ) , performed as described in Figure 1C .

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Figure 2Clonogenic assay for ESCs in response to etoposide (A) or camptothecin (B). Non-treated cells were set to 1.Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown. The results for each cell line are shown at right.Nanog expression confirmed by RNA-seq.Detection of the number of X-linked Atrx gene foci by DNA-FISH in GFP-positive subclones of the Nanog-targeting assay (nine independent clones, N> 100 each), performed as described in Figure 1C.


Figure 3Sexing of established ESCs ( gDNA - qPCR , Zfy / Gapdh ) , performed as described in Figure 1B . Detection of the number of X - linked Atrx gene foci by DNA - FISH with or without dox addition for 96 h ( N > 100 ) , performed as described in Figure 1C . Detection of the Xist cloud in # B3 by RNA - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image is shown [ magenta : Xist ( white arrowhead ) , cyan : DAPI . Scale bar : 10 μm ] . Allele - distinguishable RT - PCR for detecting Hprt , G6pdx , and Rnf12 X - linked genes . Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown . Nanog HR assay . The percentage of GFP - positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C . # B2 or # B3 were cultured with or without dox addition for 96 h . The combined number of GFP - positive cells and total colonies from all experiments are listed and summarized at right .

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Figure 3Sexing of established ESCs (gDNA-qPCR, Zfy/Gapdh), performed as described in Figure 1B.Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C.Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm].Allele-distinguishable RT-PCR for detecting Hprt, G6pdx, and Rnf12 X-linked genes. Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown.Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.


Figure 4Detection of the Xist foci by RNA - FISH , with or without dox addition for 96 h ( N > 200 each ) . A representative image with percentage of Xist - positive cells is shown [ magenta : Xist , cyan : DAPI . ] . Detection of the number of X - linked Atrx gene foci by DNA - FISH , with or without dox addition for 96 h ( N > 100 each ) , performed as described in Figure 1C . Estimated repair events . The flow cytometry plots are shown in Figure EV4 . The percentage of GFP - positive cells in TX - DRGFP # 11 and TX - EJ5GFP # 23 was normalized to the transfection efficiency detected by the VENUS expression vector . Dox ( - ) was set to 1 and the ratio of relative GFP - positive cells is shown ( n = 3 ) .

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Figure 4Detection of the Xist foci by RNA-FISH, with or without dox addition for 96 h (N >200 each). A representative image with percentage of Xist-positive cells is shown [magenta: Xist, cyan: DAPI.].Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.Estimated repair events. The flow cytometry plots are shown in Figure EV4. The percentage of GFP-positive cells in TX-DRGFP#11 and TX-EJ5GFP#23 was normalized to the transfection efficiency detected by the VENUS expression vector. Dox (-) was set to 1 and the ratio of relative GFP-positive cells is shown (n=3).