Figure 4D ) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in ( C ) WT : wild - type , MT : mutant .

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Figure 4D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant.


Figure 5C ) Representative genotyping outcomes of hCD34 + cells nucleofected sgRNA - 1 , xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS . Data information : The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion . A dash inside a gray box indicates a deleted base pair .

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Figure 5C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS. Data information: The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.


Figure 6B ) Telomere restriction fragment assay for the five subjects shown in ( A ) . Mean telomere length is indicated .

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Figure 6B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.


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 .

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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 ) .

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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 1 ( C - E ) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate ( TCL ) and further increases by IFN - γ activation ( C , E ) . Equal protein amounts were loaded for phagosomal and TCL samples . Vimentin serves as loading control ; EEA1 as purity marker . ( D ) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome ( 30 min pulse , 0 , 30 , and 180 min chase , respectively ) . LAMP1 serves as purity marker . Representative images of three replicates . ( H ) Ubiquitin Chain Restriction ( UbiCRest ) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs . UT = untreated .

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Figure 1(C-E) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate (TCL) and further increases by IFN-γ activation (C, E). Equal protein amounts were loaded for phagosomal and TCL samples. Vimentin serves as loading control; EEA1 as purity marker. (D) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome (30 min pulse, 0, 30, and 180 min chase, respectively). LAMP1 serves as purity marker. Representative images of three replicates.(H) Ubiquitin Chain Restriction (UbiCRest) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs. UT = untreated.


Figure 3 ( B ) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264 . 7 cells and its increased abundance in response to IFN - γ activation . Rab7 serves as purity control , iNOS as activation control and vimentin as loading control . Representative image of three replicates . Relative mobilities of reference proteins ( masses in kDa ) are shown on the right of each blot .

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Figure 3(B) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264.7 cells and its increased abundance in response to IFN-γ activation. Rab7 serves as purity control, iNOS as activation control and vimentin as loading control. Representative image of three replicates. Relative mobilities of reference proteins (masses in kDa) are shown on the right of each blot.


Figure 4 ( A - B ) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells . ( C ) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes , shows that RNF115 ligase activity is required for the increase of phagosome maturation .

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Figure 4(A-B) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells. (C) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes, shows that RNF115 ligase activity is required for the increase of phagosome maturation.


Figure 5 ( C ) Immunofluorescence micrographs of VAMP8 ( green ) and Syntaxin - 7 ( STX7 ; red ) show strong colocation around 3 µm silica bead phagosomes ( white ) in BMA macrophages . Nuclei are stained with Dapi . Scale bar is 5 µm . To account for people with red - green colour - blindness , ( D ) Colocation of VAMP8 ( green ) and STX7 ( red ) on individual phagosomes between WT and RNF115 KO cells represented by intersection , Manders ' M1 and overlap coefficients . Data is represented as a box and whisker plot of total values across biological duplicate experiments , where the whiskers represent the minimum to maximum values and the central band indicates the median .

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Figure 5(C) Immunofluorescence micrographs of VAMP8 (green) and Syntaxin-7 (STX7; red) show strong colocation around 3 µm silica bead phagosomes (white) in BMA macrophages. Nuclei are stained with Dapi. Scale bar is 5 µm. To account for people with red-green colour-blindness, (D) Colocation of VAMP8 (green) and STX7 (red) on individual phagosomes between WT and RNF115 KO cells represented by intersection, Manders' M1 and overlap coefficients. Data is represented as a box and whisker plot of total values across biological duplicate experiments, where the whiskers represent the minimum to maximum values and the central band indicates the median.


Figure 7 ( D ) Box and whisker plot represents the quantification of the percentage ( % ) of the hepatic lesion area in the livers from six wild - type ( WT ) and six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Haematoxylin and eosin ( H & E ) - stained liver sections of one representative animal per genotype are shown on the right , dotted line depicts the damaged area . ( E - F ) Box and whisker plots represent the average cell counts per high power field ( HPF ) of CD3 positive T - cells ( E ) , and CD68 positive macrophages ( F ) in the livers from six wild - type ( WT ) and six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Representative immunohistochemistry sections are shown on the right .

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Figure 7(D) Box and whisker plot represents the quantification of the percentage (%) of the hepatic lesion area in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Haematoxylin and eosin (H&E)-stained liver sections of one representative animal per genotype are shown on the right, dotted line depicts the damaged area.(E-F) Box and whisker plots represent the average cell counts per high power field (HPF) of CD3 positive T-cells (E), and CD68 positive macrophages (F) in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Representative immunohistochemistry sections are shown on the right.


Figure 2C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains . Data represent means ± SE . M : Marker ; arrowhead : FIX protein ; Note : the bands of FIX in F9383STOP lanes are faint with small molecular weight smears . D Measurement of coagulation activity by aPTT in mice at 8 weeks of age . n = 5 for each group , Data represent means ± SE . E Measurement of coagulation activity by PT in mice at 8 weeks of age . n = 5 for each group , Data represent means ± SE . F Measurement of blood loss over a 5 - minute period after tail transection in mice at 8 weeks of age . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 . G Survival rate of mice after the tail - clip challenge . The mice were monitored for two days after tail clipping . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

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Figure 2C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears.D Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.E Measurement of coagulation activity by PT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.G Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


Figure 3B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids ( or 120 µg ssODN ) per mouse . C Survival rate of mice after the tail - clip challenge . F9Y381D mice were treated with or without Cas9 / sgRNA / donor DNA for 3 months . After tail - clip challenge , the survival rate of each group over 2 days was determined . Wild - type , n = 9 ; untreated F9Y381D , n = 13 ; DNA treated F9Y381D , n = 14 . D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing ( Cas9 / donor vector treated group ) or by TA - clone sequencing ( Cas9 / ssODN group ) . F The genome editing efficiency of individual mice is presented . G Plasma aspartate aminotransferase ( AST ) and alanine aminotransferase ( ALT ) level was determined in F9Y381D mice 8 weeks after HTV injection ( n = 5 ) . H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real - time PCR . Data represent means ± SE .

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Figure 3B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids (or 120 µg ssODN) per mouse.C Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n=9; untreated F9Y381D, n=13; DNA treated F9Y381D, n=14.D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group).F The genome editing efficiency of individual mice is presented.G Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n=5).H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real-time PCR. Data represent means ± SE.


Figure 4B In vivo infection efficiency 4 days after Adv injection . Inserts : negative control from untreated mouse liver tissue . Scale bar : 100 μm . C T7E1 assay was performed on liver DNA obtained from samples in ( B ) , and the frequency of Cas9 - induced indels is indicated as ' % ' below the lane . D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors . Data represent means ± SE . E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv ( Cas9 + G / T ) . ( Left ) HDR donor used for correction of Y381D mutation , and Illumina sequencing reads ( rn ) are presented . Red text indicates the correction of the F9 mutation , whereas blue text indicates the intended synonymous mutations . ( Right ) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA - clones ( low dose ) or Illumina sequencing ( high dose ) of treated mouseliver tissues .

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Figure 4B In vivo infection efficiency 4 days after Adv injection. Inserts: negative control from untreated mouse liver tissue. Scale bar: 100 μm.C T7E1 assay was performed on liver DNA obtained from samples in (B), and the frequency of Cas9-induced indels is indicated as '%' below the lane.D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors. Data represent means ± SE.E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouseliver tissues.


Figure 1Immunofluorescence of early pluripotent marker Sall4 in DMSO - and R406 - treated cells on d12 . n = 5 . Scale bar , 100 μm . RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in MEFs , intermediate cells on d12 treated with DMSO and R406 , and R1 ( mESCs ) . n = 3 . Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk . n = 3 . Scale bar , 100 μm . ciPSCs express key pluripotent markers Oct4 , Sox2 and Nanog . Scale bar , 100 μm .

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Figure 1Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm.ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm.


Figure 2Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12 . n = 6 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca . n = 3 . Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12 . n = 3 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1 . n = 3 .

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Figure 2Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3.Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.


Figure 3RT - qPCR analysis of Gldc , Cbs and Tdh expression in R406 - and shSyk - treated cells ( E ) and FK506 - , shPpp3ca - and shNfatc1 - treated cells ( F ) on d8 . n = 3 .

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Figure 3RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.


Figure 4Metabolite abundance of SAM , SAH in samples treated with DMSO and R406 . n = 6 .

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Figure 4Metabolite abundance of SAM, SAH in samples treated with DMSO and R406. n = 6.


Figure 5Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12 . n = 3 . Scale bar , 100 μm . Immunofluorescence of Sall4 in NAC - and DPBS - treated cells on d12 . n = 5 . Scale bar , 100 μm . RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and HA - treated cells on d12 . n = 3 . RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and NAC - treated cells on d12 . n = 3 . Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12 . n = 4 . Scale bar , 100 μm .

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Figure 5Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.


Figure 6Quantification of oxygen consumption rate ( OCR ) of reprogramming intermediates treated with small molecules ( A , C ) and the indicated OCR parameters ( B , D ) . n = 9 . Fluorescence images of DCFH - DA staining for ROS levels in cells treated with DMSO , HA , NAC , and R406 on d12 ( E ) and the quantification of the ROS levels ( F ) . n = 4 . Scale bar , 100 μm . MDA quantification in cells treated with DMSO , HA , NAC , and R406 on d12 . n = 9 .

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Figure 6Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9.Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9.


figf1 ( a , b ) Immunoblot analysis of LAMP - 2A in total lysates ( a ) and quantitative PCR analysis of Lamp2a mRNA ( b ) of naive CD4 + T cells and TH1 and TH2 cells following activation with anti - CD3 and anti - CD28 . β - actin ( a ) serves as a loading control throughout ; mRNA results ( b ) are presented relative to those of resting cells . ( c ) Immunofluorescence microscopy of LAMP - 2A ( green ) and nuclei ( stained with the DNA - binding dye DAPI ; blue ) in resting TH1 cells ( Rest ) and TH1 cells activated for 24 h as in a , b ( Act ) ( left ) , and quantification of lysosomes in cells as at left ( right ) . Original magnification , × 630 ; scale bar , 10 μm . * P = 0 . 013 ( t - test ) . ( d ) Electron microscopy ( left ) of resting TH1 cells and TH1 cells activated for 24 h as in a , b ( top row ) ; below , enlargement of areas outlined above , showing individual lysosomes ( arrows ) . Original magnification ( top row ) , × 12 , 000 ; scale bars ( bottom row ) , 1 μm . Right , morphometric analysis of cells as at left . * P = 0 . 037 and * * P = 0 . 034 ( t - test ) . ( e ) Distribution of KFERQ - PA - mCherry - 1 in TH1 cells transiently transfected with a CMA reporter vector , photoactivated , and left unstimulated ( Rest ) or stimulated with anti - CD3 and anti - CD28 in the presence ( 3 - MA ) or absence ( vehicle ( veh ) ) of 3 - methyladenine ( left ) ; arrows indicate mCherry + puncta . Original magnification , × 630 . Right , quantification of the results for the cells at left . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) . ( f ) Immunoblot analysis of hsc70 in lysosomal fractions of resting ( Lys - R ) and stimulated ( Lys - A ) TH1 cells and in cytosolic fractions of TH1 cells activated as in a , b ( Cyto ) ; LAMP - 2A and LAMP - 1 serve as controls . ( g ) Immunoblot analysis of LAMP - 2A in resting TH1 cells and TH1 cells activated in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( h ) Quantitative PCR analysis of Lamp2a mRNA in cells as in g ( results presented as in b ) . * P = 0 . 007 ( t test ) . ( i ) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0 , 12 or 24 h with plate - bound anti - CD3 and anti - CD28 ; results are presented relative to those of resting cells ( time 0 ) . ( j ) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated ( control ( Ctrl ) ) or stimulated for 16 h with 50 μM paraquat ( Pq ) or 1 mM H2O2 ; results are presented in relative light units ( RLU ) . ( k ) Immunoblot analysis of LAMP - 2A in total lysates of resting TH1 cells ( − ) and TH1 cells activated for 24 h as in a , b ( + ) , transfected with a control nontargeting siRNA ( with a scrambled sequence ( Scr ) ) or siRNA specific for mRNA encoding Duox1 ( either of two siRNAs : Dx ( 1 ) and Dx ( 2 ) ) or RISP ( Uq ( 1 ) ) . ( l ) Immunoblot analysis of LAMP - 2A in lysates of resting or activated TH1 cells in the presence ( right half ) or absence ( left half ) of cyclosporine A ( CsA ) . ( m ) Chromatin - immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 ( negative control ) ( left ) or to Il2 ( positive control ) ( right ) in resting and activated TH1 cells ; results are presented relative to input chromatin precipitated with anti - NFAT1 .

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 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 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 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 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 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 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

figf1(a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28. β-actin (a) serves as a loading control throughout; mRNA results (b) are presented relative to those of resting cells.(c) Immunofluorescence microscopy of LAMP-2A (green) and nuclei (stained with the DNA-binding dye DAPI; blue) in resting TH1 cells (Rest) and TH1 cells activated for 24 h as in a,b (Act) (left), and quantification of lysosomes in cells as at left (right). Original magnification, ×630; scale bar, 10 μm. *P = 0.013 (t-test).(d) Electron microscopy (left) of resting TH1 cells and TH1 cells activated for 24 h as in a,b (top row); below, enlargement of areas outlined above, showing individual lysosomes (arrows). Original magnification (top row), ×12,000; scale bars (bottom row), 1 μm. Right, morphometric analysis of cells as at left. *P = 0.037 and **P = 0.034 (t-test).(e) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test).(f) Immunoblot analysis of hsc70 in lysosomal fractions of resting (Lys-R) and stimulated (Lys-A) TH1 cells and in cytosolic fractions of TH1 cells activated as in a,b (Cyto); LAMP-2A and LAMP-1 serve as controls.(g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test).(i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0).(j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU).(k) Immunoblot analysis of LAMP-2A in total lysates of resting TH1 cells (−) and TH1 cells activated for 24 h as in a,b (+), transfected with a control nontargeting siRNA (with a scrambled sequence (Scr)) or siRNA specific for mRNA encoding Duox1 (either of two siRNAs: Dx(1) and Dx(2)) or RISP (Uq(1)).(l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA).(m) Chromatin-immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 (negative control) (left) or to Il2 (positive control) (right) in resting and activated TH1 cells; results are presented relative to input chromatin precipitated with anti-NFAT1.


figf2 ( a ) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA ( Scr ) or Lamp2a mRNA - specific shRNA ( shL2A ) ( left ) , and immunoblot analysis of LAMP - 2A in sorted GFP + T cells ( right ) . Numbers above bracketed lines ( left ) indicate percent GFP + cells . ( b ) Enzyme - linked immunosorbent assay of IL - 2 and interferon - γ ( IFN - γ ) in TH1 cells transduced as in a and then left unstimulated ( Rest ) or stimulated for 24 h with anti - CD3 and anti - CD28 ( Act ) . * P = 0 . 038 and * * P = 0 . 031 ( t - test ) . ( c ) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b , assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm ( A495 ) . * P = 0 . 013 ( t - test ) . ( d ) Quantification of annexin V - positive ( AnnV + ) cells among resting or stimulated TH1 cells transduced as in a .

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 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 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

figf2(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells.(b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test).(c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test).(d) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.


figf3 ( a ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti - CD3 and anti - CD28 ; LAMP - 1 serves as a control . ( b ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with ( + ) or without ( − ) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin ( Lys inh ) ; LAMP - 1 serves as a control . ( c ) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( d ) Immunoblot analysis of lysates of TH1 cells transfected with GFP - Itch or GFP - mu - Itch ( top ) or with Myc - RCAN1 or Myc - mu - RCAN1 ( bottom ) and cotransfected with a GFP expression plasmid ( to assess transfection efficiency ) , then activated for 24 h as in a . ( e , f ) Proliferation ( e ) and IL - 2 production ( f ) of TH1 cells transfected with plasmid expressing GFP ( Ctrl ) or plasmids as in c and left unstimulated ( Rest ) or activated for 48 h ( e ) or 24 h ( f ) as in a ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 019 and * * P = 0 . 0034 ( e ) or * P = 0 . 046 and * * P = 0 . 044 ( f ) ( t test ) . Data are representative of four ( a , b ) , two ( c ) or three ( d ) independent experiments or are from three independent experiments ( e , f ; mean and s . e . m . ) .

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

figf3(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.(d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a.(e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).


figf4 ( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells ( left ) and liver , kidneys , thymus and lungs ( right ) of L2A - cKO mice ( KO ) and their control littermates ( Ctrl ) . ( b ) Quantitative PCR analysis of Lamp2a , Lamp2b and Lamp2c mRNA in T cells from L2A - cKO mice ( right ) ; results are presented relative to those of their control littermates ( left ) , set as 1 . ( c ) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form ( LC3 - I ) and its autophagosome membrane - associated form ( LC3 - II ) in total lysates of CD4 + T cells obtained from L2A - cKO mice or their control littermates and then left unstimulated ( Rest ) or activated for 24 h with anti - CD3 and anti - CD218 ( Act ) , cultured for the final 4 h in the presence ( + ) or absence ( − ) of NH4Cl and leupeptin ( NH4Cl + Leup ) . ( d ) Spleens from an L2A - cKO mouse and its control littermate ( left ) , and flow cytometry of peripheral T cells from such mice ( right ) . Numbers in quadrants ( right ) indicate percent CD4 − CD8 + T cells ( top left ) or CD4 + CD8 − T cells ( bottom right ) . ( e , f ) Proliferation ( e ) and IL - 2 production ( f ) by CD4 + T cells isolated from the spleen of L2A - cKO mice and their control littermates and stimulated ex vivo for 48 h with anti - CD3 and anti - CD28 ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 0062 ( e ) or 0 . 006 ( f ) ( t - test ) . ( g ) Immunoblot analysis of LAMP - 2A , Itch and RCAN1 in total lysates of CD4 + T cells obtained from L2A - cKO mice ( n = 3 ( KO1 , KO2 and KO3 ) ) and their control littermates ( n = 3 ) and activated for 24 h as in e , f . * , nonspecific band . ( h ) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4 + T cells isolated from L2A - cKO mice and their control littermates and activated for 24 h as in e , f ; results are presented relative to those of resting cells . ( i , j ) Recall responses to OVA ( 323 - 339 ) in draining lymph nodes isolated from L2A - cKO mice and their control littermates after immunization with OVA ( 323 - 339 ) , assessed as proliferation ( i ) and IL - 2 production ( j ) ( as in e , f ) . * P = 0 . 0054 ( i ) or 0 . 0052 ( j ) ( t - test ) . ( k , l ) Recall responses to L . monocytogenes in CD4 + T cells isolated from L2A - cKO mice and their control littermates ( n = 6 per group ) previously uninfected ( UI ) or previously infected with L . monocytogenes ( Inf ) , assessed as IFN - γ production ( k ) and proliferation ( l ) ( as in e , f ) . * P = 0 . 032 ( k ) or 0 . 02 ( l ) ( t - test ) . ( m ) L . monocytogenes in spleen and liver of L2A - cKO mice and their control littermates ( n = 3 per genotype ) immunized with L . monocytogenes and then reinfected with a high dose of L . monocytogenes . CFU , colony - forming units . * P = 0 . 008 and * * P = 0 . 001 ( t - 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 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 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 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 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 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 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 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

figf4(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl).(b) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1.(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).(d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).(e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).(g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.(h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).


figf5 ( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells obtained from 4 - month - old or 22 - month old mice , then left resting or stimulated for 24 h with anti - CD3 plus anti - CD28 . ( b ) Immunoblot analysis of RCAN1 and Itch in CD4 + T cells obtained from 4 - month - old or 22 - month old mice ( n = 3 per group ) , then stimulated for 24 h as in a ( left ) , and quantification of protein in 22 - month old mice , presented relative to that of 4 - month - old mice , set as 100 % . * P = 0 . 044 ( t - test ) . ( c ) Immunoblot analysis of LAMP - 2A in total lysates of naive and memory CD4 + T cells left resting or activated for 24 h as in a . ( d ) Immunoblot analysis ( bottom ) of LAMP - 2A in total lysates of naive and memory CD4 + T cells obtained from 4 - or 22 - month - old mice and left resting ( R ) or activated for 24 h as in a ( A ) ; above , quantification from results below ( n = 3 mice per group ) . * P = 0 . 009 ( t - test ) . ( e ) Electrophoretic mobility - shift assay of the binding of NFAT ( N ) and free probe ( FP ) in nuclear extracts of CD4 + T cells isolated from 3 - , 12 - or 22 - month - old mice and activated for 4 h as in a . ( f ) ROS production in CD4 + T cells isolated from 4 - or 22 - month - old mice ( n = 3 per group ) and left resting or activated for 12 h as in a , assessed by flow cytometry and presented as the mean fluorescence intensity ( MFI ) of a fluorogenic probe for oxidative stress ( CellROX Green ) . ( g - i ) Immunoblot analysis of LAMP - 2A ( g ) and analysis of the proliferation ( h ) and IL - 2 production ( i ) of TH1 cells obtained from 4 - or 22 - month - old mice and transduced with retrovirus expressing GFP alone ( RV - GFP ) or human LAMP - 2A and GFP ( RV - LAMP2A ) , sorted by GFP expression , cultured for 2 weeks and then stimulated for 24 - 48 h as in a ( assessed as in Fig . 2c ( h ) or Fig . 2b ( i ) ) . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) . ( j ) Immunoblot analysis of LAMP - 2A in total lysates of CD4 + T cells obtained from two human donors ( A and B ) and left resting or activated for 24 or 48 h as in a ( above lanes ) . ( k ) Real - time PCR analysis of LAMP2A mRNA in naive and memory CD4 + T cells obtained from young human donors ( 26 ± 2 years of age ; n = 3 ) or old human donors ( 72 ± 6 years of age ; n = 6 ) and activated for 24 h as in a ; results were calculated by the change - in - cycle - threshold method ( 2 − ΔCt ) relative to those of the gene encoding β - actin . * P = 0 . 047 and * * P = 0 . 0033 ( Mann - Whitney ) .

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

figf5(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.(b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).(c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.(d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).(g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).


Figure 1 ( B ) The degree of polymerisation of Z α1 - antitrypsin after 72 hours at 37 ° C , as determined by an end - point immunoassay using the 2C1 monoclonal antibody , in varying concentrations of compound ( shown in panel A ) . Modification of the phenyl and indole heterocycle of GSK425 ( pIC50 6 . 5 ) resulted in an ~ 100 fold increase in potency and the discovery of the 2 - oxindole GSK716 ( pIC50 8 . 3 ) . Data presented as mean ± SD , n = 2 ( GSK425 ) and n = 25 ( GSK716 ) . ( C ) GSK716 binds to Z α1 - antitrypsin with a high affinity mean pKD of 8 . 5 ± 0 . 12 ( n = 18 ) as determined by a competition binding assay with a fluorescently labelled derivative . There was a 50 - fold lower affinity for plasma - purified wild - type M α1 - antitrypsin , with a mean pKD of 6 . 8 ± 0 . 18 ( n = 10 ) . Data presented as mean ± SD . ( D ) The compound bound to monomeric but not polymeric Z α1 - antitrypsin ( Z α1 - AT ) as reported by fluorescence polarisation of an Alexa - 488 - labelled variant of GSK716 . ( E ) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1 - antitrypsin based on changes in intrinsic tryptophan fluorescence . Based on the concentration dependence ( inset ) , the second - order rate constant of association was found to be 4 . 1 x 104 M - 1 s - 1 . ( F ) The association of GSK716 with M α1 - antitrypsin , giving a second - order rate constant of 2 . 1 x 102 M - 1 s - 1 .

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 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 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

Figure 1(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).(C) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.


Figure 2 ( G ) CHO - K1 Tet - On cells expressing Z α1 - antitrypsin were induced with doxycycline ( 0 . 5μg / ml ) for 48 h . Cells were incubated with 10μM GSK716 ( or 0 . 1 % v / v DMSO for the control ) during the induction . Culture media containing either the experimental compound or DMSO were changed every 24 h . After the induction , cells were labelled for 10 minutes with 35S Met / Cys and chased at the indicated times . Culture media were collected and cells lysed in 1 % v / v NP - 40 buffer . Intracellular fractions and culture media from cells expressing Z α1 - antitrypsin were immunoprecipitated either with a mAb against total α1 - antitrypsin ( 3C11 ) or with a polymer - specific mAb ( 2C1 ) . Samples were resolved by 4 - 12 % w / v acrylamide SDS - PAGE and detected by autoradiography . ( H ) The graphs show the effect of GSK716 on intracellular and extracellular Z α1 - antitrypsin ( mean ± SEM , n = 2 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2)


Figure 4 ( B ) M ( above ) and Z ( below ) α1 - antitrypsin , at a concentration of 0 . 2mg / ml in PBS + 5 % v / v glycerol , were heated at a range of temperatures between 48 ° C - 65 ° C for 4 hours in the presence and absence of GSK716 , as indicated . The oligomerisation state was determined by non - denaturing PAGE electrophoresis ; the lane denoted ' M ' contains the unheated monomeric control . ( C ) M and Z α1 - antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride ( gdnhcl ) in the presence and absence of GSK716 , with bis - ANS dye added to report the presence of the unfolded intermediate . The normalised fluorescence intensity data were fitted with an equation describing a three - state unfolding curve . Values shown are the mean of three independent experiments and the error bars represent ± SEM . ( D ) Top panel , the association of GSK716 induced a marked quenching and blue - shift of the α1 - antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein ( inset graph ) . The association of 10µM GSK716 with four α1 - antitrypsin variants that vary in their propensity to polymerise , ranging from inert ( M ) , to mild ( S ) , moderate ( Baghdad denoted ' B ' ) and severe ( Z ) . Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0 . 2mg / ml protein are shown . Bottom panel , the half - time of association calculated from three such independent experiments ( error bars are ± SEM ) show a correspondence with the polymerisation propensity of the four variants . The change in Z α1 - antitrypsin fluorescence was faster than the dead - time of the apparatus ( ~ 10s ) and scaling was with reference to unbound intensity .

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 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 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

Figure 4(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.


Figure 5 ( B ) M α1 - antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS - PAGE . The position of covalent α1 - antitrypsin - chymotrypsin complex ( cpx ) , native ( nat ) and cleaved ( clv ) is shown . ( C ) The inhibitory activity of α1 - antichymotrypsin against chymotrypsin ( ach + chym ) , neuroserpin against trypsin ( neus + tryp ) , and antithrombin against thrombin ( atiii + thrombin ) was determined in the presence and absence of GSK716 . Error bars reflect ± SD of two independent experiments .

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

Figure 5(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.


Figure 6Z α1 - antitrypsin transgenic animals were dosed with 10 , 30 or 100 mg / kg GSK716 three times a day . Total concentrations of GSK716 were determined by LC - MS / MS ( Table 2 ) . ( A ) 100mg / kg GSK716 resulted in a mean 7 - fold increase in circulating monomeric Z α1 - antitrypsin levels demonstrating robust target engagement in the liver . The observed steady state total and free compound levels of GSK716 in the transgenic Z α1 - antitrypsin mouse were well - predicted by the in silico PK model built on : ( 1 ) in vitro metabolic clearance data , ( 2 ) plasma protein binding data , ( 3 ) in vivo PK data from wild type mice and ( 4 ) a term comprising a 5μM circulating sink for drug with an affinity of 1 . 5 nM , representing the Z α1 - antitrypsin within blood . The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay . Interestingly , 30mg / kg and 10mg / kg groups also gave significant , dose - dependent increases in circulating Z α1 - antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period . Significance at p < 0 . 05 by a student ' s t test is denoted by * , shown as pairwise comparisons ( each animal is compared with itself , treated vs pre - treatment ) and between groups ( compound treated vs vehicle ) , with n = 7 , 8 and 8 per group for day 6 dose level 10 , 30 and 100mg / Kg respectively ; day 15 , n = 10 ; day 21 , n = 5 . Data presented as mean ± SD .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.


Figure 1A . Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry . B . Cardiac troponin I ( cTnI ) and α - Sarcomeric actinin ( α - SA ) staining of the TMRM + + population ( CMs ) and Vimentin staining of the TMRM + population ( NCs ) . The scale bar represents 100 µm . D . EGFP expression in iPSC - like colonies derived from CMs or NCs isolated from triple transgenic mice . E . EGFP expression combined with immunofluorescence staining of SSEA - 1 and DAPI of iPSC - like colonies derived from CMs isolated from triple transgenic mice . F . Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6 . The scale bar represents 100 µm .

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 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 B-PANEL_START 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 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

Figure 1A. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.


Figure 2A . Gene profiling of CM and NC at D0 - D6 as shown clustered into 8 categories . Red indicates up - regulated genes while green indicates down - regulated genes ( n = 3 , n = 2 for NC - D6 ) . B . Expression patterns of all genes per cluster by grey lines . Black line shows the mean value . D . Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2 , 4 and 6 . Red indicates closely related groups , while green indicates significantly different groups .

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Figure 2A. Gene profiling of CM and NC at D0-D6 as shown clustered into 8 categories. Red indicates up-regulated genes while green indicates down-regulated genes (n=3, n=2 for NC-D6).B. Expression patterns of all genes per cluster by grey lines. Black line shows the mean value.D. Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2, 4 and 6. Red indicates closely related groups, while green indicates significantly different groups.


Figure 3A . RNA expression ratio ( D2 - to - D0 ) of candidates selected from day 2 microarray data . C . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with control ( Ctrl ) , or combinations of a FoxM1 up - regulation adenovirus ( F ) , Id1 up - regulation adenovirus ( I ) , Hmgb2 up - regulation adenovirus ( H ) or a small molecule Jnk3 inhibitor ( Ji ) . D . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3 - shRNAs in place of the small molecule ( Ji ) . The arrow head indicates Ki67 + / α - MHC + proliferated CMs . E . Morphology and quantification of FIJs - treated CMs undergoing cytokinesis by H3P and Aurora kinase B ( AURKB ) staining . G . RNA expression ratio of Oct4 , FoxM1 , Id1 , or Jnk3 in doxycycline - injected versus control adult CMs isolated from reprogrammable mice . H . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice .

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

Figure 3A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.


Figure 4B . The size of hearts isolated from Ctrl or FIJs - treated mice ; heart / body weight ratio of Ctrl or FIJs - treated mice . C . H3P , cTnI , and DAPI staining and quantification of heart tissues in Ctrl or FIJs - treated mice . The arrowhead indicates H3P + / cTnI + proliferated CMs and the scale bar represents 50 µm . E . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno - Ctrl or adeno - FIJs injected mice . F . Quantification of BrdU + / EGFP + population % in isolated CMs from adeno - Ctrl or adeno - FIJs injected M / Z mice by flow cytometry .

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Figure 4B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.F. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.