Figure 1 ( A ) Histogram ( mean , 95 % CI ) of fold induction of PXR activity reporter assay ( luc , luciferase ) in LS180 cells transiently transfected with wild - type PXR and p3A4 - luc reporter plasmids . Chemical structures of FKK5 , FKK6 and FKK999 compounds are overlaid . The bar graph ( s ) depicts one representative experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of cells . * p < 0 . 05 , one - way ANOVA with Dunnett ' s post hoc test . * significant over vehicle ( DMSO ) control . ( B ) , same as ( A ) , in HepG2 cells stably expressing pCYP1A1 luciferase plasmid ( AhR reporter ) . Chemical structures of FKK5 , FKK6 and FKK999 compounds are overlaid . Indole is colored blue . The bar graph ( s ) depicts one representative experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of cells . * p < 0 . 05 , one - way ANOVA with Dunnett ' s post hoc test . * significant over vehicle ( DMSO ) control . C , Histogram ( mean , 95 % CI ) of fold mRNA expression , CYP1A1 ( top panel ) , CYP3A4 ( middle panel ) and MDR1 ( bottom panel ) in LS180 cells with or without ( mock ) transfected PXR plasmid , HepaRG hepatic progenitor cells ( PXR - knockout , PXR - KO ; AhR - knockout , AhR - KO ; parental control 5F clone ) and primary human hepatocytes ( HEP ) from four donors is shown . The bar graph represents one experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of LS180 cells ; n = 2 independent experiments with one well / compound and RT - PCR performed in triplicate for each HepaRG genotype ; for each donor hepatocyte , n = 1 well / compound and RT - PCR performed in triplicate . * , # p < 0 . 05 , two - way ANOVA with Tukey ' s post hoc test . * significant over vehicle control . # significant over the same treatment in corresponding mock transfected or knock - out cells . D , Chromatin Immunoprecipitation ( ChIP ) assay in LS174T cells . Top panel , PCR products cells exposed to vehicle or FKK6 and run on a 2 % agarose gel . DNA 100 base pair marker ; Vehicle , 10 % DMSO ; FKK6 ( 10 μM ) ; 1 / 10 Input - 0 . 2 million cells before IP ; IgG - IP with polyclonal rabbit IgG ; PXR - IP with PXR antibody . Bottom panel , quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH ( fold expression ) . Dash line , 2 - fold expression . The data is one representative experiment of two independent experiments ( each n = 3 biological replicates , n = 4 technical replicates ) . Mean fold expression is expressed above each histogram ( mean + SD ) . E , PXR TR - FRET assay . TR - FRET ratio 520 / 495 nm are plotted against concentration of compound ( s ) . Half - maximal inhibitory concentrations IC50 were obtained from interpolated standard curves ( sigmoidal , 4PL , variable slope ) ; error bars show standard deviation of n = 2 independent experiments each with four technical replicates . F , Microcalorimetric titrations of PXR ligand binding domain ( LBD ) with FKK5 and FKK6 . A single - site binding model was used for fitting the data . The upper panel shows the output signal , dQ / dt , as a function of time . The middle panel shows the integrated heats as a function of the ligand / PXR molar ratio in the cell . The solid line represents the best non - linear least - squares fit of the data . The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain .

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

Figure 1(A) Histogram (mean, 95 %CI) of fold induction of PXR activity reporter assay (luc, luciferase) in LS180 cells transiently transfected with wild-type PXR and p3A4-luc reporter plasmids. Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.(B), same as (A), in HepG2 cells stably expressing pCYP1A1 luciferase plasmid (AhR reporter). Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. Indole is colored blue. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.C, Histogram (mean, 95 %CI) of fold mRNA expression, CYP1A1 (top panel), CYP3A4 (middle panel) and MDR1 (bottom panel) in LS180 cells with or without (mock) transfected PXR plasmid, HepaRG hepatic progenitor cells (PXR-knockout, PXR-KO; AhR-knockout, AhR-KO; parental control 5F clone) and primary human hepatocytes (HEP) from four donors is shown. The bar graph represents one experiment of a series of experiments (n > 3) performed in four consecutive passages of LS180 cells; n = 2 independent experiments with one well/compound and RT-PCR performed in triplicate for each HepaRG genotype; for each donor hepatocyte, n = 1 well/compound and RT-PCR performed in triplicate. *, #p < 0.05, two-way ANOVA with Tukey's post hoc test. *significant over vehicle control. #significant over the same treatment in corresponding mock transfected or knock-out cells.D, Chromatin Immunoprecipitation (ChIP) assay in LS174T cells. Top panel, PCR products cells exposed to vehicle or FKK6 and run on a 2% agarose gel. DNA 100 base pair marker; Vehicle,10% DMSO; FKK6 (10 μM); 1/10 Input - 0.2 million cells before IP; IgG - IP with polyclonal rabbit IgG; PXR - IP with PXR antibody. Bottom panel, quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH (fold expression). Dash line, 2-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biological replicates, n = 4 technical replicates). Mean fold expression is expressed above each histogram (mean + SD).E, PXR TR-FRET assay. TR-FRET ratio 520/495 nm are plotted against concentration of compound(s). Half-maximal inhibitory concentrations IC50 were obtained from interpolated standard curves (sigmoidal, 4PL, variable slope); error bars show standard deviation of n = 2 independent experiments each with four technical replicates.F, Microcalorimetric titrations of PXR ligand binding domain (LBD) with FKK5 and FKK6. A single-site binding model was used for fitting the data. The upper panel shows the output signal, dQ/dt, as a function of time. The middle panel shows the integrated heats as a function of the ligand/PXR molar ratio in the cell. The solid line represents the best non-linear least-squares fit of the data. The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain.


Figure 2A , PXR ( luciferase ) reporter assay in HEK293T cells transiently transfected with PXR plasmids ( wild - type and ligand binding domain mutant C285I / C301A ) . RLU , relative light units are shown normalized to beta - galactosidase ( β - Gal ) expression . The histogram represents one experimental data ( of n > 2 independent experiments each performed in quadruplicate ) mean ( 95 % CI ) ; n . s . not significant ; * p < 0 . 05 , two - way ANOVA . B , Histogram represents fold change in mRNA expression , normalized to GAPDH , by RT - qPCR from Caco - 2 cells exposed to compounds . DMSO , 0 . 1 % DMSO vehicle ; 9 . 3 is 9 . 3 - fold expression . The data is one representative experiment of two independent experiments ( each n = 3 biologic replicates , n = 4 technical replicates ) . Points and error represent mean ( 95 % CI ) values for control ( TNFα 5 ng / ml ) normalized NF - κB ( luciferase ) reporter activity in LS180 cells after C , Passage 13 ( P13 ) and D , Passage 8 ( P8 ) and C , D , Line graph depicts one representative experiment of a series of experiments ( n > 4 ) performed in four consecutive passages of cells . Data was expressed as mean with 95 % CI of four technical replicates . E , LS174T PXR - KO ( knockout ) cells transiently transfected with pNL3 . 2 . NF - κB - RE vector . mean ( + SD ) . * , * * p < 0 . 05 , two way ANOVA with Tukey ' s multiple comparisons test .

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 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 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

Figure 2A, PXR (luciferase) reporter assay in HEK293T cells transiently transfected with PXR plasmids (wild-type and ligand binding domain mutant C285I/C301A). RLU, relative light units are shown normalized to beta-galactosidase (β-Gal) expression. The histogram represents one experimental data (of n > 2 independent experiments each performed in quadruplicate) mean (95 %CI); n.s. not significant; * p < 0.05, two-way ANOVA.B, Histogram represents fold change in mRNA expression, normalized to GAPDH, by RT-qPCR from Caco-2 cells exposed to compounds. DMSO, 0.1% DMSO vehicle; 9.3 is 9.3-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biologic replicates, n = 4 technical replicates).Points and error represent mean (95 %CI) values for control (TNFα 5 ng/ml) normalized NF-κB (luciferase) reporter activity in LS180 cells after C, Passage 13 (P13) and D, Passage 8 (P8) and C, D, Line graph depicts one representative experiment of a series of experiments (n > 4) performed in four consecutive passages of cells. Data was expressed as mean with 95% CI of four technical replicates.E, LS174T PXR-KO (knockout) cells transiently transfected with pNL3.2.NF-κB-RE vector. mean (+ SD). *, ** p < 0.05, two way ANOVA with Tukey's multiple comparisons test.


Figure 3A In Caco - 2 monolayer cells , Log10 relative expression of IL8 mRNA with or without cytokines ( 50 ng / ml IL - 1 , 10 ng / ml IFNγ , 10 ng / ml TNF α ) , and cytokine cocktail plus FKK drugs ( 10 and 25 μM ) with 2 h or 12 h incubations . B , Salmonella invasion in colony forming units per ml ( CFU / ml ) at the different time periods of incubation . C , confocal images of NFĸB ( red ) and the nucleus ( blue ) . Scale bars = 80 µm . Arrows represent localization of NFĸB ( red , cytoplasmic ; purple , nuclear ) . Confocal images shown are representative images from ( n = 3 ) biological replicates . Log10 relative expression of IL8 mRNA with or without cytokines ( 50 ng / ml IL - 1 , 10 ng / ml IFNγ , 10 ng / ml TNF α ) , and cytokine cocktail plus FKK drugs ( 10 and 25 μM ) with 2 h or 12 h incubations . In human intestinal organoids ( HIO ) , Salmonella invasion in colony forming units per ml ( CFU / ml ) at the different time periods of incubation . In human intestinal organoids ( HIO ) , confocal images of NFĸB ( red ) and the nucleus ( blue ) . Scale bars = 80 µm . n human intestinal organoids ( HIO ) ,

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 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 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 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 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 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

Figure 3A In Caco-2 monolayer cells, Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations.B, Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation.C, confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm. Arrows represent localization of NFĸB (red, cytoplasmic; purple, nuclear). Confocal images shown are representative images from (n = 3) biological replicates.Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations. In human intestinal organoids (HIO),Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation. In human intestinal organoids (HIO),confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm. n human intestinal organoids (HIO),


Figure 4Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per individual sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK5 ( n = 9 paired samples ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an individual patient . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc test . Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per individual sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK6 ( n = 3 paired samples ; one removed due to poor inflammatory response ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an individual patient . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc test . Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per individual sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK9 ( n = 5 ; 3 removed due to poor response and tissue integrity ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an individual patient . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc test . Fold induction ( mRNA ) of genes in C57BL / 6 mice gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . The entire experiment was repeated two independent times and one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays and normalized to internal control , GAPDH . The histograms show mean ( 95 % CI ) values for gene expression . The dotted line marks basal fold expressionFold induction ( mRNA ) of genes in pxr - / - mice gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . The entire experiment was repeated two independent times and one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays and normalized to internal control , GAPDH . The histograms show mean ( 95 % CI ) values for gene expression . The dotted line marks basal fold expressionFold induction ( mRNA ) of genes in hPXR mice ( mice expressing the human PXR gene ) gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . The entire experiment was repeated two independent times and one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays and normalized to internal control , GAPDH . The histograms show mean ( 95 % CI ) values for gene expression . The dotted line marks 2 - fold gene induction .

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 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 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 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 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 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

Figure 4Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK5 (n = 9 paired samples). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK6 (n = 3 paired samples; one removed due to poor inflammatory response). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK9 (n = 5; 3 removed due to poor response and tissue integrity). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.Fold induction (mRNA) of genes in C57BL/6 mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expressionFold induction (mRNA) of genes in pxr -/- mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expressionFold induction (mRNA) of genes in hPXR mice (mice expressing the human PXR gene) gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks 2-fold gene induction.


Figure 5After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . A , % weight loss from baseline ( day 1 vs day 10 ) After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . B , colon length ( cm ) After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . C , Inflammation scoreAfter coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . D , Fecal lipocalin 2 ( pg / ml ) After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . E , serum FITC - dextran ( μg / ml ) . After coin - toss randomization , hPXR mice ( mice expressing the human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage and intra - rectal delivery starting day 1 - 10 of DSS administration . F , fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS ( n = 3 / group ; each PCR performed in quadruplicate ) .

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

Figure 5After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. A, % weight loss from baseline (day 1 vs day 10)After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. B, colon length (cm)After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. C, Inflammation scoreAfter coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. D, Fecal lipocalin 2 (pg/ml)After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. E, serum FITC-dextran (μg/ml).After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. F, fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS (n = 3/group; each PCR performed in quadruplicate).


Figure 6As in Fig 5 , pxr - / - mice underwent the identical experimental procedure and analysis . The entire experiment was repeated three independent times and one representative experiment is shown . As in Fig 5 , pxr - / - mice underwent the identical experimental procedure and analysis . The entire experiment was repeated three independent times and one representative experiment is shown . As in Fig 5 , pxr - / - mice underwent the identical experimental procedure and analysis . The entire experiment was repeated three independent times and one representative experiment is shown . As in Fig 5 , pxr - / - mice underwent the identical experimental procedure and analysis . The entire experiment was repeated three independent times and one representative experiment is shown . As in Fig 5 , pxr - / - mice underwent the identical experimental procedure and analysis . The entire experiment was repeated three independent times and one representative experiment is shown .

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Figure 6As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.


Figure 1A Infiltration of GRIp65 - 84 induced cell death similar to GRIp31 - 96 . Bacterially produced 37 nM GST , GST ‐ GRIp31 - 96 or biochemically pure GRI ‐ peptides ( GRIp31 - 96 , GRIp31 - 51 , GRIp47 - 68 , GRIp65 - 84 , GRIp80 - 96 ) were infiltrated into leaves of Col ‐ 0 plants . B Infiltration of increasing concentrations of GRIp65 - 84 into Col ‐ 0leaves , electrolyte leakage was measured after 12 h . Background ( red line ) shows ion leakage from infiltration of leaves with increasing concentrations of ( inactive ) GRIp80 - 96 . C Infiltration of leaves with 37 nM GRIp31 - 96 induced elevated ion leakage in Col ‐ 0 and prk4 , but not in prk5 ‐ 1 or prk5 ‐ 2 . Infiltration with GST caused the same background effect for all lines . D Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31 - 96 . E Enzymatic superoxide production from xanthine / xanthine oxidase ( XXO ) induced more electrolyte leakage in gri compared to Col ‐ 0 or prk5 ‐ 1 , prk5 ‐ 2 and prk4 after infiltration into leaves . Infiltration with xanthine buffer ( X ) was used as a control .

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Figure 1A Infiltration of GRIp65-84 induced cell death similar to GRIp31-96. Bacterially produced 37 nM GST, GST‐GRIp31-96 or biochemically pure GRI‐peptides (GRIp31-96, GRIp31-51, GRIp47-68, GRIp65-84, GRIp80-96) were infiltrated into leaves of Col‐0 plants.B Infiltration of increasing concentrations of GRIp65-84 into Col‐0leaves, electrolyte leakage was measured after 12 h. Background (red line) shows ion leakage from infiltration of leaves with increasing concentrations of (inactive) GRIp80-96.C Infiltration of leaves with 37 nM GRIp31-96 induced elevated ion leakage in Col‐0 and prk4, but not in prk5‐1 or prk5‐2. Infiltration with GST caused the same background effect for all lines.D Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31-96.E Enzymatic superoxide production from xanthine/xanthine oxidase (XXO) induced more electrolyte leakage in gri compared to Col‐0 or prk5‐1, prk5‐2 and prk4 after infiltration into leaves. Infiltration with xanthine buffer (X) was used as a control.


Figure 2B PRK5 ‐ CFP localized to the cell periphery in Col ‐ 0mesophyll protoplasts . C - F Co ‐ localization of PRK5 ‐ CFP and PM localized CAAX ‐ YFP in Col ‐ 0mesophyll protoplasts . ( C ) overlay ; ( D ) PRK5 ‐ CFP ( 465 - 510 nm ) ; ( E ) CAAX ‐ YFP ( 521 - 587 nm ) ; ( F ) chloroplast ( 636 - 711 nm ) . H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P ‐ ATP and myelin ‐ basic protein ( MBP ) as a substrate in the presence of 10 mM MnCl2 . GST ‐ PRK5 and GST ‐ PRK4 did not show kinase activity . Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST ‐ PRK5H500DA520G kinase activity . GST ‐ CRK7 was used as a positive control . Upper panel shows autoradiograph , and lower panel shows the Coomassie ‐ stained 15 % SDS - polyacrylamide gel .

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Figure 2B PRK5‐CFP localized to the cell periphery in Col‐0mesophyll protoplasts.C-F Co‐localization of PRK5‐CFP and PM localized CAAX‐YFP in Col‐0mesophyll protoplasts. (C) overlay; (D) PRK5‐CFP (465-510 nm); (E) CAAX‐YFP (521-587 nm); (F) chloroplast (636-711 nm).H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P‐ATP and myelin‐basic protein (MBP) as a substrate in the presence of 10 mM MnCl2. GST‐PRK5 and GST‐PRK4 did not show kinase activity. Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST‐PRK5H500DA520G kinase activity. GST‐CRK7 was used as a positive control. Upper panel shows autoradiograph, and lower panel shows the Coomassie‐stained 15% SDS-polyacrylamide gel.


Figure 3A In vitro ‐ produced 35S ‐ labeled ectodomains of PRK5 ( PRK540 - 281 ) or PRK4 ( PRK440 - 279 ) were incubated with bacterially produced GST , GST ‐ GRI or GST ‐ GRI31 - 96 and purified . GST ‐ GRI31 - 96 but not GST directly bound to the ectodomain of PRK5 . Binding of GST ‐ GRI to PRK5 ectodomain and binding of GST ‐ GRI31 - 96 and GST ‐ GRI to the ectodomain of PRK4 were strongly reduced . Upper part : autoradiograph , lower part : Coomassie ‐ stained 12 % SDS - polyacrylamide gel . Asterisks in the Coomassie ‐ stained gel indicate GST , GST ‐ GRI and GST ‐ GRI31 - 96 , respectively . Supplementary Fig S12 shows a Western blot of the GST ‐ tagged proteins . B Infiltration of 37 nM GST , GRIp31 - 96 , GRIp65 - 84 or Y ‐ GRIp65 - 84 into Col ‐ 0 or prk5 ‐ 2 leaves . Tyrosine ‐ labeled GRIp65 - 84 still induced cell death in Col ‐ 0 but not in prk5 ‐ 2 plants . C 125I ‐ labeled Y ‐ GRIp65 - 84 ( 0 . 46 nM ) bound specifically to Col ‐ 0membrane fractions ( light gray bars ) , the binding was significantly reduced in prk5 plants . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background levels ( dark gray bars ; all bars show the average of three samples , triangles show individual data points ) . D Immunoprecipitation of PRK5 ‐ c ‐ myc , PRK4 ‐ c ‐ myc or YFP expressed in protoplasts with rabbit polyclonal anti ‐ c ‐ myc antibody , immunoblotted with mouse monoclonal anti ‐ c ‐ myc antibody . E Binding of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) to immunoprecipitates ( using anti ‐ c ‐ myc antibodies ) from prk5 ‐ 2 protoplasts transfected with PRK5 ‐ c ‐ myc , PRK4 ‐ c ‐ myc or YFP , respectively . Binding was competed out with 10 μM unlabeled Y ‐ GRIp65 - 84 . Bars show the average of two samples , and triangles show individual data points . Western blot is shown in panel ( D ) . F Analysis of 125I ‐ Y ‐ GRIp65 - 84 ( 0 . 46 nM ) binding competed out with increasing amounts of unlabeled Y ‐ GRIp65 - 84 to Col ‐ 0membrane extracts . Fifty percent inhibition ( IC50 ) occurred at 25 . 2 nM . Red line shows binding average competition according to a sigmoid curve , dotted lines show 95 % confidence intervals , and circles show data points . G Excess of GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed the binding of 0 . 46 nM 125I ‐ Y ‐ GRIp65 - 84 to membrane extracts from Col ‐ 0 ( all bars show the average of two samples , triangles show individual data points ) .

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Figure 3A In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540-281) or PRK4 (PRK440-279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31-96 and purified. GST‐GRI31-96 but not GST directly bound to the ectodomain of PRK5. Binding of GST‐GRI to PRK5 ectodomain and binding of GST‐GRI31-96 and GST‐GRI to the ectodomain of PRK4 were strongly reduced. Upper part: autoradiograph, lower part: Coomassie‐stained 12% SDS-polyacrylamide gel. Asterisks in the Coomassie‐stained gel indicate GST, GST‐GRI and GST‐GRI31-96, respectively. Supplementary Fig S12 shows a Western blot of the GST‐tagged proteins.B Infiltration of 37 nM GST, GRIp31-96, GRIp65-84 or Y‐GRIp65-84 into Col‐0 or prk5‐2 leaves. Tyrosine‐labeled GRIp65-84 still induced cell death in Col‐0 but not in prk5‐2 plants.C 125I‐labeled Y‐GRIp65-84 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of three samples, triangles show individual data points).D Immunoprecipitation of PRK5‐c‐myc, PRK4‐c‐myc or YFP expressed in protoplasts with rabbit polyclonal anti‐c‐myc antibody, immunoblotted with mouse monoclonal anti‐c‐myc antibody.E Binding of 125I‐Y‐GRIp65-84 (0.46 nM) to immunoprecipitates (using anti‐c‐myc antibodies) from prk5‐2 protoplasts transfected with PRK5‐c‐myc, PRK4‐c‐myc or YFP, respectively. Binding was competed out with 10 μM unlabeled Y‐GRIp65-84. Bars show the average of two samples, and triangles show individual data points. Western blot is shown in panel (D).F Analysis of 125I‐Y‐GRIp65-84 (0.46 nM) binding competed out with increasing amounts of unlabeled Y‐GRIp65-84 to Col‐0membrane extracts. Fifty percent inhibition (IC50) occurred at 25.2 nM. Red line shows binding average competition according to a sigmoid curve, dotted lines show 95% confidence intervals, and circles show data points.G Excess of GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp65-84 to membrane extracts from Col‐0 (all bars show the average of two samples, triangles show individual data points).


Figure 4A Recombinant AtMC9 ( rAtMC9 ) cleaved GRI25 - 168in vitro . Bacterially produced MBP ‐ GRI25 - 168 ( from 0 to 2 pmol left to right ) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut ( rAtMC9C147AC29A ) , and cleavage products were analyzed by Western blot with anti ‐ MBP and anti ‐ AtMC9 antibodies . Arrowheads in the anti ‐ MBP blot ( from top to bottom ) indicate MBP ‐ GRI25 - 168 ( 63 . 5 kDa ) , MBPMCS ( 53 . 5 kDa ) , rAtMC9 ‐ cleaved MBP ‐ GRI25 - 168 ( 46 kDa ) . Arrowheads in the anti ‐ AtMC9 blot indicate : 1 : N ‐ terminal domain + p20 + p10 subunits of AtMC9 , 2 : p20 + p10 subunits of AtMC9 , 3 : N ‐ terminal domain + p20 subunit of AtMC9 , 4 : p20 subunit of AtMC9 . B Infiltration of wild ‐ type , prk5 ‐ 1 and atmc9 ‐ 1 leaves with 37 nM GRIp31 - 96 , GRIp65 - 84 , GRIp68 - 78 or GST . GRIp65 - 84 and GRIp68 - 78 but not GRIp31 - 96 were able to induce elevated ion leakage in the atmc9 ‐ 1 mutant . D Infiltration of Col ‐ 0leaves with 37 nM of GRIp65 - 84 , GRIp68 - 78 , Y ‐ GRIp68 - 78 , GRIp31 - 96 or GST . Y ‐ GRIp68 - 78 showed similar activity in cell death induction compared to the other GRI ‐ derived peptides . E 125I ‐ labeled Y ‐ GRIp68 - 78 ( 0 . 46 nM ) bound specifically to Col ‐ 0membrane fractions ( light gray bars ) , the binding was significantly reduced in prk5 ‐ 1 and prk5 ‐ 2 plants . Excess of non ‐ radioactive Y ‐ GRIp65 - 84 ( 10 μM ) reduced binding to background levels ( dark gray bars ; all bars show the average of four samples , triangles show individual data points ) . F Excess of GRIp68 - 78 and GRIp65 - 84 ( 10 μM ) but not of GRIp31 - 96 or other peptides ( GRIp31 - 51 , GRIp47 - 68 , GRIp80 - 96 ) competed the binding of 0 . 46 nM 125I ‐ Y ‐ GRIp68 - 78 to membrane extracts from Col ‐ 0 ( all bars show average of four samples , triangles show individual data points ) . G Saturation binding curve for 125I ‐ Y ‐ GRIp68 - 78 to Col ‐ 0membrane extracts . Specific binding was calculated by subtracting non ‐ specific binding from the total binding . The affinity of 125I ‐ Y ‐ GRIp68 - 78 to the receptor ( Kd = 1 . 9 nM ) was calculated by non ‐ linear regression analysis . Scatchard plot is shown in Supplementary Fig SI7 .

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Figure 4A Recombinant AtMC9 (rAtMC9) cleaved GRI25-168in vitro. Bacterially produced MBP‐GRI25-168 (from 0 to 2 pmol left to right) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut (rAtMC9C147AC29A), and cleavage products were analyzed by Western blot with anti‐MBP and anti‐AtMC9 antibodies. Arrowheads in the anti‐MBP blot (from top to bottom) indicate MBP‐GRI25-168 (63.5 kDa), MBPMCS (53.5 kDa), rAtMC9‐cleaved MBP‐GRI25-168 (46 kDa). Arrowheads in the anti‐AtMC9 blot indicate: 1: N‐terminal domain + p20 + p10 subunits of AtMC9, 2: p20 + p10 subunits of AtMC9, 3: N‐terminal domain + p20 subunit of AtMC9, 4: p20 subunit of AtMC9.B Infiltration of wild‐type, prk5‐1 and atmc9‐1 leaves with 37 nM GRIp31-96, GRIp65-84, GRIp68-78 or GST. GRIp65-84 and GRIp68-78 but not GRIp31-96 were able to induce elevated ion leakage in the atmc9‐1 mutant.D Infiltration of Col‐0leaves with 37 nM of GRIp65-84, GRIp68-78, Y‐GRIp68-78, GRIp31-96 or GST. Y‐GRIp68-78 showed similar activity in cell death induction compared to the other GRI‐derived peptides.E 125I‐labeled Y‐GRIp68-78 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5‐1 and prk5‐2 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points).F Excess of GRIp68-78 and GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp68-78 to membrane extracts from Col‐0 (all bars show average of four samples, triangles show individual data points).G Saturation binding curve for 125I‐Y‐GRIp68-78 to Col‐0membrane extracts. Specific binding was calculated by subtracting non‐specific binding from the total binding. The affinity of 125I‐Y‐GRIp68-78 to the receptor (Kd = 1.9 nM) was calculated by non‐linear regression analysis. Scatchard plot is shown in Supplementary Fig SI7.


Figure 2D Immunofluorescence of DNA - damage - associated nuclear protein PARP . Images show adjacent normal and tumor crypts of tissue P009T , marked by N and T , respectively , and tumor tissue of patients P008 and P009 , as indicated . Scale bar 100µm . Significance was assessed by an unpaired t - test , after blinded analysis of 10 random images per tumor .

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Figure 2D Immunofluorescence of DNA-damage-associated nuclear protein PARP. Images show adjacent normal and tumor crypts of tissue P009T, marked by N and T, respectively, and tumor tissue of patients P008 and P009, as indicated. Scale bar 100µm. Significance was assessed by an unpaired t-test, after blinded analysis of 10 random images per tumor.


Figure 3E Activities of Wnt / β - catenin and MAPK target genes in organoid single cell transcriptomes , ordered along latent time . F Activities of TFF3 , FABP1 , MKI67 and MMP7 in organoid single cell transcriptomes , ordered along latent time .

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Figure 3E Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time.F Activities of TFF3, FABP1, MKI67 and MMP7 in organoid single cell transcriptomes, ordered along latent time.


Figure 4A Gene expression of LGR5 and EPHB2 , along activity gradients of LGR5 - ISC or MAPK target gene signatures .

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Figure 4A Gene expression of LGR5 and EPHB2, along activity gradients of LGR5-ISC or MAPK target gene signatures.


Figure 5C Heatmap of key CyTOF data . Average activities of selected analytes are given as log fold - change after normalization to DMSO control condition . Range of color scale was adjusted for each analyte . For relative changes between all analytes , see Appendix Fig . S7 ) .

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Figure 5C Heatmap of key CyTOF data. Average activities of selected analytes are given as log fold-change after normalization to DMSO control condition. Range of color scale was adjusted for each analyte. For relative changes between all analytes, see Appendix Fig. S7).


Figure 6F Scaled signature expression and single gene expression moments per cell , under control ( grey ) and preferred treatment ( color ) conditions , as indicated . 10 % of cells populating the end of latent time are displayed in bold . It is of note that latent time is linear and thus cannot capture multiple populations located at developmental end points . Densities at the side lines display expression in all cells .

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Figure 6F Scaled signature expression and single gene expression moments per cell, under control (grey) and preferred treatment (color) conditions, as indicated. 10% of cells populating the end of latent time are displayed in bold. It is of note that latent time is linear and thus cannot capture multiple populations located at developmental end points. Densities at the side lines display expression in all cells.


Figure 2C . Western blot analysis of CTRL # 1 or BRD9 - KO # 1 lysates from cells treated for the indicated times with 1000 IU / mL of IFN - α2 . The indicated proteins were detected with specific antibodies . Data are representative of at least two biological replicates . D . Immunofluorescence analysis of CTRL # 1 or BRD9 - KO # 1 cells treated for the indicated times with 1000 IU / mL of IFN - α2 . The indicated proteins were detected with specific antibodies . Data are representative of at least two biological replicates . Scale bars indicate 10μm . F . Western blot analysis of CTRL # 1 or BRD9 - KO # 1 lysates from cells treated or not for 16h with 1000 IU / mL of IFN - α2 . The indicated proteins were detected with specific antibodies . Data are representative of at least two biological replicates . I . CTRL # 1 or BRD9 - KO # 1 cells were stably - transduced with BRD9 - expressing , or control ( EV , empty vector ) , lentiviruses and treated with 1000 IU / mL of IFN - α2 for 16h prior to infection with IAV ( WSN / 33 ) at an MOI of 0 . 01 PFU / cell . Viral titers were determined after 24h by plaque assay . Data information : data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Statistical significance was determined by 1 - way ANOVA on log - transformed plaque counts ( * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ) . Dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN - α2 , respectively . Numbers above IFN - α2 - treated bars indicate their approximate difference to the respective untreated conditions .

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Figure 2C. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.D. Immunofluorescence analysis of CTRL#1 or BRD9-KO#1 cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates. Scale bars indicate 10μm.F. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated or not for 16h with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.I. CTRL#1 or BRD9-KO#1 cells were stably-transduced with BRD9-expressing, or control (EV, empty vector), lentiviruses and treated with 1000 IU/mL of IFN-α2 for 16h prior to infection with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay. Data information: data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed plaque counts (**p-value < 0.001; ****p-value < 0.0001). Dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.


Figure 3D . A549 cells were treated with 125nM dBRD9 - A ( + ) or DMSO ( - ) for 6h prior stimulation with 1000 IU / mL of IFN - α2 for 16h . Following lysis , western blot was used to detect levels of the indicated proteins . Data are representative of at least two biological replicates . I - L . Hep2 ( I ) , Huh - 7 ( J ) , MRC - 5 ( K ) , and undifferentiated HTBE ( L ) were treated and infected with VSV - GFP Data information : Total Integrated Green Fluorescent Intensities were determined using the Incucyte live - cell analysis system after 24h Data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Statistical significance was determined by 1 - way ANOVA on log - transformed intensity values ( * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ) . , dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN - α2 , respectively . Numbers above IFN - α2 - treated bars indicate their approximate difference to the respective untreated conditions .

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 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 O O O O O O O O O O O O O O O O O O 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 3D. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior stimulation with 1000 IU/mL of IFN-α2 for 16h. Following lysis, western blot was used to detect levels of the indicated proteins. Data are representative of at least two biological replicates.I-L. Hep2 (I), Huh-7 (J), MRC-5 (K), and undifferentiated HTBE (L) were treated and infected with VSV-GFP Data information: Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system after 24h Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001). , dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.


Figure 4C . Impact of dBRD9 - A pre - treatment on the induction of ISGs identified in ( B ) . Colors indicate IFN - induced transcripts significantly affected ( blue ) or unaffected ( gray ) by dBRD9 - A pretreatment ( FC > 2 , p - value ≤ 0 . 001 ) . F . RT - qPCR confirmation of transcriptomics results for selected transcripts ( MX1 , MX2 , IFITM1 , ISG15 and IFIT1 ) in A549 cells treated as outlined in ( A ) . GAPDH transcript levels were used for normalization . Data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Statistical significance was determined by unpaired 2 - tailed t - test on ΔCt values ( * p - value < 0 . 05 ; n . s . not significant ) .

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 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

Figure 4C. Impact of dBRD9-A pre-treatment on the induction of ISGs identified in (B). Colors indicate IFN-induced transcripts significantly affected (blue) or unaffected (gray) by dBRD9-A pretreatment (FC>2, p-value ≤ 0.001).F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).


Figure 5G . Western blot analysis of CTRL # 3 or BRD9 - KO # 3 cells stably transduced with lentiviruses expressing empty vector ( EV ) , or wt and chimeric constructsH . Western blot analysis of input lysates and anti - BRD9 immunoprecipitations ( IPs ) from cells described in ( G ) expressing wt BRD9 or the chimeric BRD7DUF . The indicated proteins were detected with specific antibodies . Data are representative of two biological replicates . I . The various CTRL # 3 or BRD9 - KO # 3 cells described in ( G ) were stimulated and infected as described in ( B ) . Data information : data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Numbers above IFN - α2 - treated bars indicate their approximate difference to the respective untreated conditions . Dotted lines are a visual guide for minimum virus replication in control cells in the presence of IFN - α2 . Statistical significance was determined by 1 - way ANOVA on log - transformed intensity values ( * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ; n . s . not significant ) .

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 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 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

Figure 5G. Western blot analysis of CTRL#3 or BRD9-KO#3 cells stably transduced with lentiviruses expressing empty vector (EV), or wt and chimeric constructsH. Western blot analysis of input lysates and anti-BRD9 immunoprecipitations (IPs) from cells described in (G) expressing wt BRD9 or the chimeric BRD7DUF. The indicated proteins were detected with specific antibodies. Data are representative of two biological replicates.I. The various CTRL#3 or BRD9-KO#3 cells described in (G) were stimulated and infected as described in (B). Data information: data represent means and standard deviations from n=3 biological experiments (individual data points shown). Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. Dotted lines are a visual guide for minimum virus replication in control cells in the presence of IFN-α2. Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (***p-value < 0.001; ****p-value < 0.0001; n.s. not significant).


Figure 6F . Western blot analysis of samples from independent small - scale streptavidin purifications ( termed ' Eluates ' ) following the proximity labeling approach outlined in ( B ) . The indicated proteins were detected with specific antibodies . M indicates the marker lane . Data are representative of at least two biological replicates .

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Figure 6F. Western blot analysis of samples from independent small-scale streptavidin purifications (termed 'Eluates') following the proximity labeling approach outlined in (B). The indicated proteins were detected with specific antibodies. M indicates the marker lane. Data are representative of at least two biological replicates.


Figure 1A Western blots of eEF2 expression in different brain regions , GAPDH was used as a loading control . B eEF2 expression levels were quantified and represented as fold changes of that in the hippocampus . n = 3 mice for each brain region . Data information : All data are shown as means ± s . e . m . All the t tests are two - tailed . C Immunofluorescent staining of eEF2 ( green ) and different nervous system cells ( red : NeuN / GFAP / IBA1 / OLIG2 / CaMKⅡα / GAD65 / 67 ) in somatosensory cortex of adult mice brains . Arrows indicate highly colocalized cells . Scale bar , 10 μm . D , E Colocalization of eEF2 and each cellular marker was quantified by calculating the Manders ' Colocalization Coefficients . 6 slices from 3 mice in each condition . ( D ) One - way ANOVA with Bonferroni ' s multiple comparisons post hoc , * * * * p < 0 . 0001 ( NeuN + / eEF2 + versus GFAP + / eEF2 + ) , * * * * p < 0 . 0001 ( NeuN + / eEF2 + versus IBA1 + / eEF2 + ) , * * * * p < 0 . 0001 ( NeuN + / eEF2 + versus OLIG2 + / eEF2 + ) . ( E ) Unpaired t test , * * p = 0 . 0065 ( CaMKⅡα + / eEF2 + versus GAD65 / 67 + / eEF2 + ) . Data information : All data are shown as means ± s . e . m . All the t tests are two - tailed .

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Figure 1A Western blots of eEF2 expression in different brain regions, GAPDH was used as a loading control. B eEF2 expression levels were quantified and represented as fold changes of that in the hippocampus. n=3 mice for each brain region. Data information: All data are shown as means ± s.e.m. All the t tests are two-tailed.C Immunofluorescent staining of eEF2 (green) and different nervous system cells (red: NeuN/GFAP/IBA1/OLIG2/CaMKⅡα/GAD65/67) in somatosensory cortex of adult mice brains. Arrows indicate highly colocalized cells. Scale bar, 10 μm.D, E Colocalization of eEF2 and each cellular marker was quantified by calculating the Manders' Colocalization Coefficients. 6 slices from 3 mice in each condition. (D) One-way ANOVA with Bonferroni's multiple comparisons post hoc, ****p<0.0001 (NeuN+/eEF2+ versus GFAP+/eEF2+), ****p<0.0001 (NeuN+/eEF2+ versus IBA1+/eEF2+), ****p<0.0001 (NeuN+/eEF2+ versus OLIG2+/eEF2+). (E) Unpaired t test, **p=0.0065 (CaMKⅡα+/eEF2+ versus GAD65/67+/eEF2+). Data information: All data are shown as means ± s.e.m. All the t tests are two-tailed.


Figure 2B Western blots of eEF2 in the PFC , the rest part of cortex , the hippocampus , and the striatum of Eef2 WT and HET mice . C Quantification analysis of ( B ) . n = 5 mice for cortex ( without PFC ) , n = 8 mice for PFC , n = 9 mice for HP , n = 3 mice for striatum . Unpaired t tests . Cortex ( without PFC ) , p = 0 . 9757 ; PFC , * * p = 0 . 0049 ; HP , p = 0 . 1338 ; Striatum , p = 0 . 7301 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed . D Representative merged immunofluorescent staining images of eEF2 ( green ) and CaMKⅡα ( red ) in the mPFC of Eef2 WT and HET mice . Scale bar , 10 μm . E Quantification of mean fluorescent intensity of eEF2 in CaMKⅡα + cells in WT and HET mPFC . n = 918 cells from 3 WT mice , n = 965 cells from 3 HET mice . Unpaired t test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed . F Representative merged immunofluorescent staining images of eEF2 ( green ) and GAD65 / 67 ( red ) in the mPFC of Eef2 WT and HET mice . Scale bar , 10 μm . G Quantification of mean fluorescent intensity of eEF2 in GAD65 / 67 + cells in WT and HET mPFC . n = 690 cells from 3 WT mice , n = 686 cells from 3 HET mice . Mann - Whitney test . p = 0 . 6757 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed . H Western blots of de novo global protein synthesis in PFC slices of WT and HET mice , measured by SUnSET . α - tubulin was used as a loading control . I Quantification analysis of puromycin - labeled protein levels . WT , n = 5 mice , HET , n = 4 mice . Unpaired t tests . * * p = 0 . 0011 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed . J Representative images of AHA incorporation ( green ) with CaMKⅡα + staining ( red ) in mPFC of WT and HET mice . Arrows indicate CaMKⅡα + cells . Scale bar , 20 μm . K Quantification of total AHA incorporation . 12 slices from 3 mice for each genotype . Unpaired t test . * p = 0 . 0268 . L Quantification of AHA incorporation in CaMKⅡα + cells . WT , n = 276 cells from 3 WT mice , n = 293 cells from 3 HET mice . Mann - Whitney test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

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 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 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 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 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 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

Figure 2B Western blots of eEF2 in the PFC, the rest part of cortex, the hippocampus, and the striatum of Eef2 WT and HET mice.C Quantification analysis of (B). n=5 mice for cortex (without PFC), n=8 mice for PFC, n=9 mice for HP, n=3 mice for striatum. Unpaired t tests. Cortex (without PFC), p=0.9757; PFC, **p=0.0049; HP, p=0.1338; Striatum, p=0.7301. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.D Representative merged immunofluorescent staining images of eEF2 (green) and CaMKⅡα (red) in the mPFC of Eef2 WT and HET mice. Scale bar, 10 μm. E Quantification of mean fluorescent intensity of eEF2 in CaMKⅡα+ cells in WT and HET mPFC. n=918 cells from 3 WT mice, n=965 cells from 3 HET mice. Unpaired t test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.F Representative merged immunofluorescent staining images of eEF2 (green) and GAD65/67 (red) in the mPFC of Eef2 WT and HET mice. Scale bar, 10 μm. G Quantification of mean fluorescent intensity of eEF2 in GAD65/67+ cells in WT and HET mPFC. n=690 cells from 3 WT mice, n=686 cells from 3 HET mice. Mann-Whitney test. p=0.6757. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.H Western blots of de novo global protein synthesis in PFC slices of WT and HET mice, measured by SUnSET. α-tubulin was used as a loading control. I Quantification analysis of puromycin-labeled protein levels. WT, n=5 mice, HET, n=4 mice. Unpaired t tests. **p=0.0011. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.J Representative images of AHA incorporation (green) with CaMKⅡα+ staining (red) in mPFC of WT and HET mice. Arrows indicate CaMKⅡα+ cells. Scale bar, 20 μm. K Quantification of total AHA incorporation. 12 slices from 3 mice for each genotype. Unpaired t test. *p=0.0268. L Quantification of AHA incorporation in CaMKⅡα+ cells. WT, n=276 cells from 3 WT mice, n=293 cells from 3 HET mice. Mann-Whitney test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


Figure 3G Representative images of Golgi ‐ stained dendrite spines in the mPFC from WT and HET mice . Scale bar , 5 μm . H Quantification of spine density per 10 μm of dendrites . n = 18 dendrites from 3 WT mice ; n = 23 dendrites from 3 HET mice . Unpaired t test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed . I Representative Western blots of expression levels of AMPAR and NMDAR subunits in the synaptosomal fraction of WT and HET PFC . α - tubulin was served as a loading control . J Quantification analysis of synaptosomal expression of different receptor subunits . Unpaired t test . GluA1 , p = 0 . 9346 ; GluA2 , * * p = 0 . 0075 ; GluN1 , p = 0 . 6946 ; GluN2A , p = 0 . 4091 ; GluN2B , p = 0 . 0796 . n . s . , not significant . n = 5 mice for each genotype . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed . L Representative immunofluorescent images of eEF2 ( magenta ) and PLA signals ( white ) in lentivirus ( LV ) - scramble - EGFP or LV - Eef2 shRNA - EGFP infected primary cortical neurons ( green , DIV 14 ) . Scale bar , 10 μm . M Quantification of mean fluorescent intensity of eEF2 in EGFP + cells in scramble and Eef2 shRNA groups . 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments . Mann - Whitney test . * * * * p < 0 . 0001 . N Quantification of mean fluorescent intensity of PLA signals in EGFP + cells in the two groups . The PLA signals represent newly synthesized GluA2 . 64 cells for scramble group , 51 cells for Eef2 shRNA group from 3 independent experiments . Mann - Whitney test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

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 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 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 O O O O O O O O O O O O O O O O O O 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 3G Representative images of Golgi‐stained dendrite spines in the mPFC from WT and HET mice. Scale bar, 5 μm. H Quantification of spine density per 10 μm of dendrites. n=18 dendrites from 3 WT mice; n=23 dendrites from 3 HET mice. Unpaired t test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.I Representative Western blots of expression levels of AMPAR and NMDAR subunits in the synaptosomal fraction of WT and HET PFC. α-tubulin was served as a loading control. J Quantification analysis of synaptosomal expression of different receptor subunits. Unpaired t test. GluA1, p=0.9346; GluA2, **p=0.0075; GluN1, p=0.6946; GluN2A, p=0.4091; GluN2B, p=0.0796. n.s., not significant. n=5 mice for each genotype. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.L Representative immunofluorescent images of eEF2 (magenta) and PLA signals (white) in lentivirus (LV)-scramble-EGFP or LV-Eef2 shRNA-EGFP infected primary cortical neurons (green, DIV 14). Scale bar, 10 μm. M Quantification of mean fluorescent intensity of eEF2 in EGFP+ cells in scramble and Eef2 shRNA groups. 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. N Quantification of mean fluorescent intensity of PLA signals in EGFP+ cells in the two groups. The PLA signals represent newly synthesized GluA2. 64 cells for scramble group, 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


Figure 7A PF - 4778574 ( PF ) directly potentiates EPSC amplitude , dashed line represents baseline value of normalized EPSC amplitude . Bottom : representative EPSC traces from baseline ( 1 ) , maximal potentiation ( 2 ) , and recovery ( 3 ) . B , C Quantifications of EPSC peak amplitude ( B ) and decay time ( C ) after PF ( 6 cells from 3 HET mice ) . ( B ) Wilcoxon signed - rank test , * p = 0 . 0313 ( PF v . s . baseline ) and p = 0 . 1600 ( Wash out v . s . baseline ) ; ( C ) Paired t test , * * * * p < 0 . 0001 ( PF v . s . baseline ) and p = 0 . 4423 ( Wash out v . s . baseline ) . Data information : Data are shown as means ± s . e . m . , dataD PF rescued AMPAR - EPSC in L5 neurons from HET mice . 25 - 30 % increase at strongest stimulation ( 7 cells from 3 HET mice ) , paired t test , * * p = 0 . 0014 . Bottom : representative traces of the effects of PF on AMPAR - EPSC evoked by different stimulus intensities . Data information : Data are shown as means ± s . e . m . E Representative heatmaps of the two social tasks ( left , social approach ; right , social novelty ) in the Three - chamber test performed by Eef2 HET mice intraperitoneal administered with PF - 4778574 or DMSO ( vehicle control ) . E : empty cup ; S1 : stranger # 1 contained cup ; S2 : stranger # 2 contained cup . F Discrimination score of social approach performed by two groups of mice , calculated by the difference of time spent in sniffing S1 and E . Unpaired t test . p = 0 . 7335 . WT , n = 9 mice ; HET , n = 8 mice . G Discrimination score of social novelty performed by two groups of mice , calculated by the difference of time spent in sniffing S2 and S1 . Unpaired t test . * p = 0 . 0492 . WT , n = 9 mice ; HET , n = 8 mice . Data information : Data F , G , are shown as interquartile range , with line across the box indicating median , whiskers show the highest and lowest values . n . s . , not significant . All the t tests are two - tailed . I Representative heatmaps of the two social tasks ( left , social approach ; right , social novelty ) performed by Eef2 HET mice with mPFC infusion of PF - 4778574 or DMSO ( vehicle control ) in the Three - chamber test . E : empty cup ; S1 : stranger # 1 contained cup ; S2 : stranger # 2 contained cup . J Discrimination score of social approach performed by two groups of mice , calculated by the difference of time spent in sniffing S1 and E . Unpaired t test . p = 0 . 3984 . n = 11 mice for each group . K Discrimination score of social novelty performed by two groups of mice , calculated by the difference of time spent in sniffing S2 and S1 . Unpaired t test . * p = 0 . 0489 . n = 11 mice for each group . Data information : Data of J and K are shown as interquartile range , with line across the box indicating median , whiskers show the highest and lowest values . n . s . , not significant . All the t tests are two - tailed .

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 O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7A PF-4778574 (PF) directly potentiates EPSC amplitude, dashed line represents baseline value of normalized EPSC amplitude. Bottom: representative EPSC traces from baseline (1), maximal potentiation (2), and recovery (3). B, C Quantifications of EPSC peak amplitude (B) and decay time (C) after PF (6 cells from 3 HET mice). (B) Wilcoxon signed-rank test, *p=0.0313 (PF v.s. baseline) and p=0.1600 (Wash out v.s. baseline); (C) Paired t test, ****p<0.0001 (PF v.s. baseline) and p=0.4423 (Wash out v.s. baseline). Data information: Data are shown as means ± s.e.m., dataD PF rescued AMPAR-EPSC in L5 neurons from HET mice. 25-30% increase at strongest stimulation (7 cells from 3 HET mice), paired t test, **p=0.0014. Bottom: representative traces of the effects of PF on AMPAR-EPSC evoked by different stimulus intensities. Data information: Data are shown as means ± s.e.m.E Representative heatmaps of the two social tasks (left, social approach; right, social novelty) in the Three-chamber test performed by Eef2 HET mice intraperitoneal administered with PF-4778574 or DMSO (vehicle control). E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. F Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.7335. WT, n=9 mice; HET, n=8 mice. G Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0492. WT, n=9 mice; HET, n=8 mice. Data information: Data F, G, are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.I Representative heatmaps of the two social tasks (left, social approach; right, social novelty) performed by Eef2 HET mice with mPFC infusion of PF-4778574 or DMSO (vehicle control) in the Three-chamber test. E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. J Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.3984. n=11 mice for each group. K Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0489. n=11 mice for each group. Data information: Data of J and K are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.


Figure 1 ( A ) Abnormal distribution of Cux1 - positive neurons in CAMDI - KO mice . Expression of Cux1 in the somatosensory cortex was compared between P2 wild - type ( WT ) and CAMDI - KO ( KO ) mice . Scale bar , 100 μm . ( B ) Quantification of the number of Cux1 - positive neurons . Note the abnormal distribution of neurons in deep cortical layers of CAMDI - KO mice . n = 3 mice / genotype ( WT = 788 cells , KO = 2 , 139 cells ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM . ( C ) Delay in neuronal migration by CAMDI deletion . Coronal sections through the somatosensory cortex of P21 WT and CAMDI - KO mice were analyzed following in utero electroporation of EGFP plasmid at E14 . 5 . Scale bar , 100 μm . ( D ) Quantification of the number of EGFP - positive neurons . Note the abnormal distribution of neurons in lower cortical layers of CAMDI - KO mice . n = 3 for WT mice ( 546 cells ) , n = 4 for KO mice ( 680 cells ) . * , p < 0 . 05 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM . ( E ) Aberrant axonal projection and terminal arbors by CAMDI deletion . Coronal sections through the somatosensory cortex of P21 WT and CAMDI - KO mice were analyzed following in utero electroporation of EGFP plasmid at E14 . 5 . Representative pictures show an aberrant axonal projection of corticostriatal neurons in CAMDI - KO mice . Sections were stained with Hoechst 33258 and anti - EGFP antibody . Scale bar , 1 mm . ( F ) Quantification of the striatal intensity . n = 3 mice / genotype . * * * , p < 0 . 001 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM .

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 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 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

Figure 1(A) Abnormal distribution of Cux1-positive neurons in CAMDI-KO mice. Expression of Cux1 in the somatosensory cortex was compared between P2 wild-type (WT) and CAMDI-KO (KO) mice. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT = 788 cells, KO = 2,139 cells). *, p<0.05, **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.(C) Delay in neuronal migration by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Scale bar, 100 μm.(D) Quantification of the number of EGFP-positive neurons. Note the abnormal distribution of neurons in lower cortical layers of CAMDI-KO mice. n = 3 for WT mice (546 cells), n = 4 for KO mice (680 cells). *, p<0.05, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.(E) Aberrant axonal projection and terminal arbors by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Representative pictures show an aberrant axonal projection of corticostriatal neurons in CAMDI-KO mice. Sections were stained with Hoechst 33258 and anti-EGFP antibody. Scale bar, 1 mm.(F) Quantification of the striatal intensity. n = 3 mice/genotype. ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


Figure 2 ( A , B ) Representative traces from adult control ( WT ) and CAMDI - KO ( KO ) in open field test . Locomotor activity ( A ) and distance traveled ( B ) for 15 min . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc test ( F ( 1 , 13 ) = 5 . 53 ) . Data are presented as mean ± SEM . ( C ) Repetitive jumping behavior in open field test . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc test ( F ( 1 , 13 ) = 5 . 09 ) . Data are presented as mean ± SEM . ( D ) Three - chamber social interaction test . The time spent sniffing with stranger mouse . n = 19 for WT mice , n = 15 for KO mice . * , p < 0 . 05 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 63 ) = 5 . 79 , main effect of chamber F ( 1 , 63 ) = 90 . 47 , interaction F ( 1 , 63 ) = 5 . 29 ) . Data are presented as mean ± SEM . ( E ) Resident - intruder test ( social sniffing ) . n = 7 for WT mice , n = 8 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc test ( F ( 1 , 13 ) = 5 . 15 ) . Data are presented as mean ± SEM . ( F ) Resident - intruder test ( digging ) . n = 7 for WT mice , n = 8 for KO mice . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc test ( F ( 1 , 13 ) = 10 . 97 ) . Data are presented as mean ± SEM . ( G ) Marble burying test . n = 9 for WT mice , n = 25 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc test ( F ( 1 , 32 ) = 5 . 39 ) . Data are presented as mean ± SEM . ( H ) Forced swimming test . n = 10 for WT mice , n = 13 for KO mice . * * , p < 0 . 01 , * * * , p < 0 . 001 Two - way ANOVA followed by Scheffe ' s post - hoc test ( main effect of genotype F ( 1 , 42 ) = 84 . 30 , main effect of session F ( 1 , 42 ) = 33 . 88 , interaction F ( 1 , 42 ) = 4 . 92 ) . Data are presented as mean ± SEM . ( I , J ) Light - dark test . Time in light chamber ( I ) and Latency to light chamber ( J ) . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc test , ( ( I ) F ( 1 , 13 ) = 8 . 75 , ( J ) F ( 1 , 13 ) = 9 . 73 ) . Data are presented as mean ± SEM . ( K ) Hot plate test . . n = 8 for WT mice , n = 12 for KO mice . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc test ( F ( 1 , 18 ) = 17 . 51 ) . Data are presented as mean ± SEM .

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 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 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 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 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 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 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 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 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 2(A, B) Representative traces from adult control (WT) and CAMDI-KO (KO) in open field test. Locomotor activity (A) and distance traveled (B) for 15 min. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.53). Data are presented as mean ± SEM.(C) Repetitive jumping behavior in open field test. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.09). Data are presented as mean ± SEM.(D) Three-chamber social interaction test. The time spent sniffing with stranger mouse. n = 19 for WT mice, n = 15 for KO mice. *, p<0.05, ***, p<0.001,Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 63)=5.79, main effect of chamber F(1, 63)=90.47, interaction F(1, 63)=5.29). Data are presented as mean ± SEM.(E) Resident-intruder test (social sniffing). n = 7 for WT mice, n = 8 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.15). Data are presented as mean ± SEM.(F) Resident-intruder test (digging). n = 7 for WT mice, n = 8 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 10.97). Data are presented as mean ± SEM.(G) Marble burying test. n = 9 for WT mice, n = 25 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 32) = 5.39). Data are presented as mean ± SEM.(H) Forced swimming test. n = 10 for WT mice, n = 13 for KO mice. **, p<0.01, ***, p<0.001 Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 42)=84.30, main effect of session F(1, 42)=33.88, interaction F(1, 42)=4.92). Data are presented as mean ± SEM.(I, J) Light-dark test. Time in light chamber (I) and Latency to light chamber (J). n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test, ((I) F(1, 13) = 8.75, (J) F(1, 13)=9.73). Data are presented as mean ± SEM.(K) Hot plate test.. n = 8 for WT mice, n = 12 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 18) = 17.51). Data are presented as mean ± SEM.


Figure 3 ( A ) Co - expressed FLAG - CAMDI and HDAC6 - HA interacted with each other in SH - SY5Y cells . n = 3 independent experiments . ( B ) Endogenous interaction of CAMDI with HDAC6 in the mousebrain . Co - IP assay was performed with the indicated antibodies using E16 mousebrain . n = 3 independent experiments . ( C , D ) N - terminal region of CAMDI recognizes the second HDAC domain of HDAC6 . SH - SY5Y cells were co - transfected with HDAC6 - HA and each FLAG - CAMDI fragment ( C ) ( fragment 1 , 1 - 250 ; 2 , 251 - 500 ; 3 , 501 - 750 ; 4 , 751 - 1000 ; 5 , 1001 - 1451 amino acids ) or co - transfected with FLAG - CAMDI and each HDAC6 - HA fragment ( D ) ( fragment 1 ( DD1 ) , 1 - 440 ; 2 ( DD2 ) , 441 - 840 ; 3 , 841 - 1215 amino acids ) . Co - IP assay was performed with the indicated antibodies . n = 3 independent experiments . * * , p < 0 . 01 , * * * , p < 0 . 001 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM .

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 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 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 O O O O O O O O O O O O O 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 3(A) Co-expressed FLAG-CAMDI and HDAC6-HA interacted with each other in SH-SY5Y cells. n=3 independent experiments.(B) Endogenous interaction of CAMDI with HDAC6 in the mousebrain. Co-IP assay was performed with the indicated antibodies using E16 mousebrain. n=3 independent experiments.(C, D) N-terminal region of CAMDI recognizes the second HDAC domain of HDAC6. SH-SY5Y cells were co-transfected with HDAC6-HA and each FLAG-CAMDI fragment (C) (fragment 1, 1-250; 2, 251-500; 3, 501-750; 4, 751-1000; 5, 1001-1451 amino acids) or co-transfected with FLAG-CAMDI and each HDAC6-HA fragment (D) (fragment 1 (DD1), 1-440; 2 (DD2), 441-840; 3, 841-1215 amino acids). Co-IP assay was performed with the indicated antibodies. n=3 independent experiments. **, p<0.01, ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


Figure 4 ( A ) CAMDI and CAMDI Fragment 1 overexpression enhanced α - tubulin acetylation . HeLa cells overexpressing FLAG - CAMDI and FLAG - CAMDI frag . 1 were immunostained with anti - FLAG or anti - Ac - tubulin antibodies . Ratio line - scans along the line ( from x - to y - axis ) from images . Scale bar , 10 μm . ( B - C ) CAMDI blocked HDAC6 - mediated α - tubulin deacetylation . ( B ) Centrosomal fraction was isolated from cell lysates of SH - SY5Y cells expressing each construct , and Ac - tubulin level was assessed by immunoblot analysis . n = 3 independent experiments . * * , p < 0 . 01 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM . ( B - C ) CAMDI blocked HDAC6 - mediated α - tubulin deacetylation . ( C ) CAMDI N - terminal domain alone increased Ac - tubulin level . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM . ( D ) Deletion of CAMDI N - terminal domain ( 1 - 250 amino acids ) failed to inhibit HDAC6 . n = 3 independent experiments . N . S . , not significant . Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM .

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 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 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 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

Figure 4(A) CAMDI and CAMDI Fragment 1 overexpression enhanced α-tubulin acetylation. HeLa cells overexpressing FLAG-CAMDI and FLAG-CAMDI frag.1 were immunostained with anti-FLAG or anti-Ac-tubulin antibodies. Ratio line-scans along the line (from x- to y-axis) from images. Scale bar, 10 μm.(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (B) Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and Ac-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (C) CAMDI N-terminal domain alone increased Ac-tubulin level. n=3 independent experiments. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.(D) Deletion of CAMDI N-terminal domain (1-250 amino acids) failed to inhibit HDAC6. n = 3 independent experiments. N.S., not significant. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.