Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . A mRNA expression of CD4 + cytokines Il2 , IL4 , IL8 and IL22 relative to endogenous controls , n = 13 / 11 / 10 / 8 biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. A mRNA expression of CD4+ cytokines Il2, IL4, IL8 and IL22 relative to endogenous controls, n=13/11/10/8 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . D mRNA expression of CD8 + cytokines IFNγ , PRF1 , TNFα , GZMB and CTLA4 in stimulated human T cells relative to internal controls , n = 15 / 14 / 10 / 14 / 9 biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. D mRNA expression of CD8+ cytokines IFNγ, PRF1, TNFα, GZMB and CTLA4 in stimulated human T cells relative to internal controls, n=15/14/10/14/9 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4 + CD25 + Foxp3 + regulatory T cells ( Treg ) following 5 days of Treg differentiation with a representative Foxp3 histogram plot ( right side ) , n = 7 / 11 biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n=7/11 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Human pan T cell RNA was isolated and cell culture supernatant was sampled . G IL10 and TGFβ1 mRNA and protein expression of human Treg cells , n = 8 / 9 ( IL10 ) , 5 / 9 ( TGFβ1 ) biological replicates . Data information : Data depicted as mean ± SEM ( protein data ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. G IL10 and TGFβ1 mRNA and protein expression of human Treg cells, n=8/9 (IL10), 5/9 (TGFβ1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human PBMCs were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan T cells , CD4 + and CD8 + T cells were isolated through magnetic cell separation . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . ( A - C ) OCR , maximum respiration , spare respiratory capacity and basal respiration were measured in A pan T cells , B CD4 + T cells and C CD8 + T cells , n = 9 ( pan T ) , n = 3 / 4 ( CD4 unstimulated ) , n = 7 / 8 / 9 ( CD4 stimulated ) , n = 5 ( CD8 unstimulated ) and n = 8 / 7 / 7 ( CD8 stimulated ) individual experiments . Data depicted as mean ± SEM ( OCR ) and box plots with median , twenty - fifth and seventy - fifth percentiles and range ( all other ) . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Human PBMCs were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan T cells, CD4+ and CD8+ T cells were isolated through magnetic cell separation. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. (A-C) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A pan T cells, B CD4+ T cells and C CD8+ T cells, n=9 (pan T), n=3/4 (CD4 unstimulated), n=7/8/9 (CD4 stimulated), n=5 (CD8 unstimulated) and n=8/7/7 (CD8 stimulated) individual experiments. Data depicted as mean ± SEM (OCR) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . A Quantification of cellular reactive oxygen species ( ROS ) using CellROX dye , indicated by mean fluorescence intensity ( MFI ) FITC in human pan / CD4 + / CD8 + T cells , n = 8 / 6 / 6 biological replicates . B Mitochondrial superoxide production quantified through MitoSOX staining , depicted as MFI Phycoerythrin ( PE ) in human pan / CD4 + / CD8 + T cells , n = 8 / 14 / 14 biological replicates . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. A Quantification of cellular reactive oxygen species (ROS) using CellROX dye, indicated by mean fluorescence intensity (MFI) FITC in human pan/CD4+/CD8+ T cells, n=8/6/6 biological replicates. B Mitochondrial superoxide production quantified through MitoSOX staining, depicted as MFI Phycoerythrin (PE) in human pan/CD4+/CD8+ T cells, n=8/14/14 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . C Confocal microscopy images of human CD4 + / CD8 + T cells , stained with MitoTracker green , representative of two individual experiments .

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

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. C Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.


Human peripheral blood mononuclear cells ( PBMCs ) were cultivated for 48h in RPMI containing 80mg / dl glucose ( NC ) and supplemented with 10mM beta - hydroxybutyrate ( BHB ) . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Flow cytometry analyses were performed using the indicated dyes and antibodies identifying T cells and CD4 + / CD8 + T cell subsets . K Flow cytometric quantification of memory T cells : CD4 + / CD8 + T cells were stained for CCR7 ( PE + / - ) and subsequently defined memory phenotype for CD45RO + ( Pacific Blue + ) and CD45RA - ( PerCP - ) staining . Histogram plots depicting the exemplary change of CD45RO and CD45RA distribution for CD8 + T cells following memory cell differentiation ( left side ; NC = black , BHB = red ) . Fractions of CCR7 + CD45RA - CD45RO + central memory ( CM ) and CCR7 - CD45RA - CD45RO + effector memory ( EM ) CD4 + / 8 + T cells ( right side ) , n = 12 / 11 / 8 / 7 biological replicates . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Flow cytometry analyses were performed using the indicated dyes and antibodies identifying T cells and CD4+/CD8+ T cell subsets. K Flow cytometric quantification of memory T cells: CD4+/CD8+ T cells were stained for CCR7 (PE+/-) and subsequently defined memory phenotype for CD45RO+ (Pacific Blue+) and CD45RA- (PerCP-) staining. Histogram plots depicting the exemplary change of CD45RO and CD45RA distribution for CD8+ T cells following memory cell differentiation (left side; NC=black, BHB=red). Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (right side), n=12/11/8/7 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . A IL2 , IL4 , and CTLA4 mRNA expression in stimulated CD4 + T cells , n = 17 / 18 / 17 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. A IL2, IL4, and CTLA4 mRNA expression in stimulated CD4+ T cells, n=17/18/17 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . B Tbet and GATA3 mRNA expression in CD4 + T cells , n = 7 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. B Tbet and GATA3 mRNA expression in CD4+ T cells, n=7 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . C Flow cytometric quantification of Th1 / Th2 cells and respective ratio of Th1 / Th2 cells , n = 7 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. C Flow cytometric quantification of Th1/Th2 cells and respective ratio of Th1/Th2 cells, n=7 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . D IL10 mRNA expression and flow cytometric quantification of CD4 + CD25 + Foxp3 + regulatory T cells ( Treg ) , n = 19 / 9individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. D IL10 mRNA expression and flow cytometric quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg), n=19/9individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . E IFNγ GZMB , PRF1 and CTLA4 mRNA expression in CD8 + T cells , n = 16 / 18 / 17 / 17 individual human subjects . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. E IFNγ GZMB, PRF1 and CTLA4 mRNA expression in CD8+ T cells, n=16/18/17/17 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to ( T0 ) and post KD ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . CD4 + / CD8 + T cells were separated via magnetic cell labeling . F relative CD8 + cell lysis activity as measured by calcein - fluorescence of isolated T cells , n = 4 individual experiments . Data information : Data depicted as box plots with median , twenty - fifth and seventy - fifth percentiles and range . Dots indicating individual values . * p < 0 . 05 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. F relative CD8+ cell lysis activity as measured by calcein-fluorescence of isolated T cells, n=4 individual experiments. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . ( A , B ) OCR , maximum respiration , spare respiratory capacity and basal respiration were measured in A CD4 + and B CD8 + T cells , n = 14 individual experiments , each performed in three technical replicates . Data information : Data depicted as mean ± SEM ( OCR ) or as box plots with median , twenty - fifth and seventy - fifth percentiles and range , crosses indicating mean Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

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

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. (A, B) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A CD4+ and B CD8+ T cells, n=14 individual experiments, each performed in three technical replicates. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range , crosses indicating mean Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . E Western Blot of mitochondrial oxidative phosphorylation proteins in CD4 + / CD8 + T cells as indicated , representative of five individual experiments .

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

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. E Western Blot of mitochondrial oxidative phosphorylation proteins in CD4+/CD8+ T cells as indicated, representative of five individual experiments.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . F Confocal microscopy images of human CD4 + / CD8 + T cells , stained with MitoTracker green , representative of two individual experiments .

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. F Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.


44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of < 30g per day . Blood was taken and analyzed prior to starting the diet ( T0 ) and again after three weeks of strict adherence to the diet ( T1 ) . PBMCs were isolated . T cell stimulation was performed through CD3 / CD28 Dynabeads at a bead : cell ratio of 1 : 8 . Pan / CD4 + / CD8 + T cells were separated via magnetic cell labeling . Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer . H TruCulture IFNγ , IL4 , IL6 , IL8 , IL12 subunit p40 , IL23 and TNFα protein quantification of LPS - stimulated whole blood samples , n = 11 / 12 / 11 / 10 / 11 / 10 / 11 individual human subjects . Data information : Data depicted as mean ± SEM ( OCR ) or as box plots with median , twenty - fifth and seventy - fifth percentiles and range Dots indicating individual values . * p < 0 . 05 , * * p < 0 . 01 , paired t - test / Wilcoxon matched - pairs signed rank test , as appropriate .

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. H TruCulture IFNγ, IL4, IL6, IL8, IL12 subunit p40, IL23 and TNFα protein quantification of LPS-stimulated whole blood samples, n=11/12/11/10/11/10/11 individual human subjects. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.


Tau phospho - variants generated by in vitro phosphorylation of non - phosphorylated Tau from E . coli with different kinases ( PKA , GSK3β , or Cdk5 ) show differences in phosphorylation at specific phospho - site ( pT205 , pS262 / pS356 , pS324 and pS404 ) tested by Western blot . Non - phosphorylated Tau and p - Tau from insect cells are loaded for comparison . Position of tested p - sites is indicated in the Tau sequence cartoon . Western blot for total Tau ( Tau13 antibody ) confirms equal loading ( 0 . 2 μg Tau per lane ) of the Tau preparations .

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

Tau phospho-variants generated by in vitro phosphorylation of non-phosphorylated Tau from E. coli with different kinases (PKA, GSK3β, or Cdk5) show differences in phosphorylation at specific phospho-site (pT205, pS262/pS356, pS324 and pS404) tested by Western blot. Non-phosphorylated Tau and p-Tau from insect cells are loaded for comparison. Position of tested p-sites is indicated in the Tau sequence cartoon. Western blot for total Tau (Tau13 antibody) confirms equal loading (0.2 μg Tau per lane) of the Tau preparations.


Phos - tag gel shows degree and heterogeneity of phosphorylation in the different Tau phospho - variants . Intensity along the lanes are plotted as colored lines next to gel image .

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Phos-tag gel shows degree and heterogeneity of phosphorylation in the different Tau phospho-variants. Intensity along the lanes are plotted as colored lines next to gel image.


Tubulin co - condensation with Tau . Addition of tubulin - Alexa647 ( 5 μM , 10 : 1 unlabeled : labeled tubulin ) to a Tau - Dy488 solution ( 25 μM ) induces Tau condensation . Addition of tubulin to Tau : PEG , Tau : RNA ( polyA - Cy3 ) , and Tau : PEG : RNA condensates leads to partitioning of tubulin into existing condensates and the occurrence of additional small Tau : tubulin condensates . Condensates with high tubulin - HiLyte647 intensity have low polyA - Cy3 intensity , and vice versa . In Tau : PEG : polyA condensates , individual condensates host RNA - and tubulin - rich domains ( arrow heads ) at homogeneous Tau - Dy488 intensity . Scale bars = 20 μm . Average intensity of RNA ( top graph ) normalized to Tau intensity in individual condensates . Addition of tubulin reduces the average amount of RNA in condensates . Intensity of tubulin ( middle graph ) normalized to Tau intensity in individual condensates . The presence of RNA increases the amount of tubulin in the condensates . Data shown as mean ± SD . Data + / - tubulin and + / - polyA were compared by two - tailed Student ' s t - tests . Tubulin and RNA intensity correlate negatively on an individual condensate level ( bottom graph ) . Line represent linear regression to all data . N = 3 experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Condensate size ( are on surface ) relative to tubulin ( yellow data points ) and RNA ( pink data points ) intensity . Smaller ( new ) condensates are high in tubulin , larger ( old ) condensates have more RNA . Lines represent linear regressions to tubulin ( yellow ) and RNA ( pink ) data .

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

Tubulin co-condensation with Tau. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) to a Tau-Dy488 solution (25 μM) induces Tau condensation. Addition of tubulin to Tau:PEG, Tau:RNA (polyA-Cy3), and Tau:PEG:RNA condensates leads to partitioning of tubulin into existing condensates and the occurrence of additional small Tau:tubulin condensates. Condensates with high tubulin-HiLyte647 intensity have low polyA-Cy3 intensity, and vice versa. In Tau:PEG:polyA condensates, individual condensates host RNA- and tubulin-rich domains (arrow heads) at homogeneous Tau-Dy488 intensity. Scale bars=20 μm. Average intensity of RNA (top graph) normalized to Tau intensity in individual condensates. Addition of tubulin reduces the average amount of RNA in condensates. Intensity of tubulin (middle graph) normalized to Tau intensity in individual condensates. The presence of RNA increases the amount of tubulin in the condensates. Data shown as mean±SD. Data +/- tubulin and +/- polyA were compared by two-tailed Student's t-tests. Tubulin and RNA intensity correlate negatively on an individual condensate level (bottom graph). Line represent linear regression to all data. N=3 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Condensate size (are on surface) relative to tubulin (yellow data points) and RNA (pink data points) intensity. Smaller (new) condensates are high in tubulin, larger (old) condensates have more RNA. Lines represent linear regressions to tubulin (yellow) and RNA (pink) data.


MT polymerization out of Tau : PEG and Tau : PEG : RNA condensates . Addition of tubulin - Alexa647 ( 5 μM , 10 : 1 unlabeled : labeled tubulin ) and GTP to Tau - Dy488 ( 25 μM ) diluted in BRB80 polymerization buffer induces MT polymerization . Images taken 30 min after addition of tubulin show thin partially curved MT bundles in Tau : PEG system , and thick bundles attached to persisting Tau : RNA condensates in the Tau : PEG : RNA system . Scale bar = 5 μm . Cross - sectional intensity plots ( line plots ) of MT bundles differ between Tau : PEG ( yellow / red line plots ) and Tau : PEG : RNA ( blue / green line plots ) systems . Four line plots at random positions are shown per condition .

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MT polymerization out of Tau:PEG and Tau:PEG:RNA condensates. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) and GTP to Tau-Dy488 (25 μM) diluted in BRB80 polymerization buffer induces MT polymerization. Images taken 30 min after addition of tubulin show thin partially curved MT bundles in Tau:PEG system, and thick bundles attached to persisting Tau:RNA condensates in the Tau:PEG:RNA system. Scale bar=5 μm. Cross-sectional intensity plots (line plots) of MT bundles differ between Tau:PEG (yellow/red line plots) and Tau:PEG:RNA (blue/green line plots) systems. Four line plots at random positions are shown per condition.


Nocodazole treatment ( 120 μM for 30 min ) of MTs formed from Tau : PEG : RNA and Tau : PEG condensates . MT formed in absence of Tau shown as control . Graphs show surface coverage ( % ) of fibrillar MT structures before and after nocodazole treatment . N = 3 experiments , 3 images analyzed per experiment and condition . Data shown as mean ± SD . Two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Scale bar = 20 μm .

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

Nocodazole treatment (120 μM for 30 min) of MTs formed from Tau:PEG:RNA and Tau:PEG condensates. MT formed in absence of Tau shown as control. Graphs show surface coverage (%) of fibrillar MT structures before and after nocodazole treatment. N=3 experiments, 3 images analyzed per experiment and condition. Data shown as mean±SD. Two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar=20 μm.


FRAP experiments of Tau : PEG , Tau : polyA , and Tau : PEG : polyA condensates at 1h , 4h , and 24h after LLPS induction . Tau : PEG and Tau : PEG : polyA condensates show a decrease in recovery rate and mobile fraction over time . Tau : polyA coacervates maintain fast recovery and a large mobile fraction even after 24h . Time until half maximum recovery : t ( y1 / 2 ) polyA : 1h ≈ 2s , 4h ≈ 3 . 5s , 24h ≈ 4 . 5s ; t ( y1 / 2 ) PEG : 1h ≈ 6s , 4h ≈ 8s , 24h ≈ 11s ; t ( y1 / 2 ) PEG + polyA : 1h ≈ 4 . 5s , 4h ≈ 9 . 5s , 24h ≈ 9s ) . Scale bars = 5 μm for Tau : polyA and Tau : PEG , and 10 μm for Tau : PEG : polyA . Data presented as error bars ( SEM ) , N = 15 to 20 condensates per condition and time point .

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FRAP experiments of Tau:PEG, Tau:polyA, and Tau:PEG:polyA condensates at 1h, 4h, and 24h after LLPS induction. Tau:PEG and Tau:PEG:polyA condensates show a decrease in recovery rate and mobile fraction over time. Tau:polyA coacervates maintain fast recovery and a large mobile fraction even after 24h. Time until half maximum recovery: t(y1/2)polyA: 1h≈2s, 4h≈3.5s, 24h≈4.5s; t(y1/2)PEG: 1h≈6s, 4h≈8s, 24h≈11s; t(y1/2)PEG+polyA: 1h≈4.5s, 4h≈9.5s, 24h≈9s). Scale bars=5 μm for Tau:polyA and Tau:PEG, and 10 μm for Tau:PEG:polyA. Data presented as error bars (SEM), N=15 to 20 condensates per condition and time point.


Microscopy of Tau : PEG : polyA condensates ( Tau - Dylight488 and polyA - Cy3 ) shows condensates with different Cy3 intensities . The bimodal fluorescence intensity distribution suggests two groups of condensates with high or low Cy3 fluorescence . Cy3 - intensities , normalized to Tau - DyLight488 intensities in the same condensates , were independent of condensate size . Scale bar = 10 μm .

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

Microscopy of Tau:PEG:polyA condensates (Tau-Dylight488 and polyA-Cy3) shows condensates with different Cy3 intensities. The bimodal fluorescence intensity distribution suggests two groups of condensates with high or low Cy3 fluorescence. Cy3-intensities, normalized to Tau-DyLight488 intensities in the same condensates, were independent of condensate size. Scale bar=10 μm.


FRAP of Tau - Dylight488 and polyA - Cy3 of 4h - old Tau : PEG : polyA condensates . The mean FRAP of polyA - Cy3 is lower than of Tau - Dy488 ( left ) . Data shown as mean ± SEM , n = 24 condensates . FRAP data of individual condensates ( bottom ; # 1 to # 3 ) shows a decrease of Tau = Dy488 ( middle ) and polyA - Cy3 ( right ) FRAP with an increase of polyA - Cy3 ( relative to Tau - Dy488 ) in the condensates . Scale bar = 5μm .

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

FRAP of Tau-Dylight488 and polyA-Cy3 of 4h-old Tau:PEG:polyA condensates. The mean FRAP of polyA-Cy3 is lower than of Tau-Dy488 (left). Data shown as mean±SEM, n=24 condensates. FRAP data of individual condensates (bottom; #1 to #3) shows a decrease of Tau=Dy488 (middle) and polyA-Cy3 (right) FRAP with an increase of polyA-Cy3 (relative to Tau-Dy488) in the condensates. Scale bar=5μm.


HEK sensor cell Tau aggregation assay . Cells were treated with 24 h - old condensates or aggregates ( 1 . 2 µM Tau in the presence of 0 . 8 % lipofectamine ) . The number of Tau aggregates was determined from low magnification ( 20x ) images taken in the green channel and normalized to the number of cells ( DAPI ) . Scale bar = 100 μm .

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HEK sensor cell Tau aggregation assay. Cells were treated with 24 h-old condensates or aggregates (1.2 µM Tau in the presence of 0.8% lipofectamine). The number of Tau aggregates was determined from low magnification (20x) images taken in the green channel and normalized to the number of cells (DAPI). Scale bar=100 μm.


Quantification of seeding assay in ( C ) . Data were collected from three images of 3 wells for each condition . Data shown as mean ± SD , N = 3 experiments , 1 - 3 images per experiment and condition . One - way ANOVA with Tukey test for multiple comparison . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Quantification of seeding assay in (C). Data were collected from three images of 3 wells for each condition. Data shown as mean±SD, N=3 experiments, 1-3 images per experiment and condition. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


Confocal microscopy of HEK sensor cells treated with 24h - old Tau condensates ( Tau : tRNA ) shows different types of intracellular CFP / YFP - Tau accumulations : spherical intranuclear Tau ( NUC ) , bright cytoplasmic accumulations ( CYT ) adjacent to the nucleus , and small , dimmer accumulations right at and along the nuclear envelope ( NE ) . Cells treated with Tau aggregates ( hepAGG ) had mainly large cytoplasmic ( CYT ) Tau inclusions . The different Tau accumulations coexisted in most cells , and also more than one accumulation of the same type could occur . Nuclei were stained with propidium iodide ( PI , pink ) . Scale bars = 10 μm .

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Confocal microscopy of HEK sensor cells treated with 24h-old Tau condensates (Tau:tRNA) shows different types of intracellular CFP/YFP-Tau accumulations: spherical intranuclear Tau (NUC), bright cytoplasmic accumulations (CYT) adjacent to the nucleus, and small, dimmer accumulations right at and along the nuclear envelope (NE). Cells treated with Tau aggregates (hepAGG) had mainly large cytoplasmic (CYT) Tau inclusions. The different Tau accumulations coexisted in most cells, and also more than one accumulation of the same type could occur. Nuclei were stained with propidium iodide (PI, pink). Scale bars=10 μm.


Fluorescence intensity of NE and CYT Tau compared to soluble Tau in the cytosol of cells without accumulations . N = 3 experiments , and 5 - 6 images per experiment . Data shown as mean ± SD . One - way ANOVA with Tukey test for multiple comparison . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Fluorescence intensity of NE and CYT Tau compared to soluble Tau in the cytosol of cells without accumulations. N=3 experiments, and 5-6 images per experiment. Data shown as mean±SD. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


Immunolabeling of lamin - B1 and nuclear pore complexes ( NPC ) shows the location of CYT and NE Tau in relation to the nuclear envelope in seeded HEK sensor cells . White arrows and boxes show position and width of cross - sectional profiles shown in ( D ) . Scale bar = 10 μm . Cross sectional profiles show the localization of Tau ( green ) in CYT accumulations adjacent to the nucleus in the cytosol , and the colocalization of NE Tau with lamin ( pink ) and NPCs ( blue ) . Notably , CYT - 1 also contains NPC signal . Dashed vertical lines indicate x - positions of highest intensity values for respective intensity profiles .

Immunolabeling of lamin-B1 and nuclear pore complexes (NPC) shows the location of CYT and NE Tau in relation to the nuclear envelope in seeded HEK sensor cells. White arrows and boxes show position and width of cross-sectional profiles shown in (D). Scale bar=10 μm. Cross sectional profiles show the localization of Tau (green) in CYT accumulations adjacent to the nucleus in the cytosol, and the colocalization of NE Tau with lamin (pink) and NPCs (blue). Notably, CYT-1 also contains NPC signal. Dashed vertical lines indicate x-positions of highest intensity values for respective intensity profiles.


Immunolabeling of phosphorylated Tau ( PHF1 = pS396 / 404 , AT8 = pS199 / pS202 / pT205 , AT180 = pT231 ) in AD brain section . Both 3 , 3 ′ - Diaminobenzidine ( DAB ) and fluorescence labeling show Tau accumulating in a layer at the nuclear envelope ( NE ) in non - tangle neurons with or without cytosolic ( CYT ) Tau accumulations . Control brain does not show Tau labeling in neuronal cell bodies . Scale bars = 20 and 10 μm , as indicated .

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

Immunolabeling of phosphorylated Tau (PHF1=pS396/404, AT8=pS199/pS202/pT205, AT180=pT231) in AD brain section. Both 3,3′-Diaminobenzidine (DAB) and fluorescence labeling show Tau accumulating in a layer at the nuclear envelope (NE) in non-tangle neurons with or without cytosolic (CYT) Tau accumulations. Control brain does not show Tau labeling in neuronal cell bodies. Scale bars= 20 and 10 μm, as indicated.


CFP - FLIM of seeded HEK sensor cells expressing only CFP - TauRDP301L . The dense packing of CFP - TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP - lifetime . Fit - free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters ( circles in phasor plot ) to different Tau accumulation types . LT - 2 = NE Tau , LT - 3 = CYT and NUC Tau , and LT - 1 = soluble CFP - TauRD in the cytosol of cells without accumulations . Graphs show lifetimes of CFP - lifetime components assigned to soluble , NE , and CYT + NUC Tau in 6 different images ( left graph , RM one - way ANOVA with Tukey post - test ) and lifetime reduction relative to LT - 1 in each respective image ( right graph , one - way ANOVA with Tukey post - test . ) . N = 2 experiments , 2 - 3 cells measured per experiments . Data shown as mean ± SD . Scale bars = 10 μm . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

CFP-FLIM of seeded HEK sensor cells expressing only CFP-TauRDP301L. The dense packing of CFP-TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP-lifetime. Fit-free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, and LT-1=soluble CFP-TauRD in the cytosol of cells without accumulations. Graphs show lifetimes of CFP-lifetime components assigned to soluble, NE, and CYT+NUC Tau in 6 different images (left graph, RM one-way ANOVA with Tukey post-test) and lifetime reduction relative to LT-1 in each respective image (right graph, one-way ANOVA with Tukey post-test.). N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. Scale bars=10 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


FLIM of 1h - and 24h - old CFP - Tau ( full - length ) condensates and heparin induced CFP - Tau aggregates . Lifetime components are indicated in phasor plots . Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP - Tau . N = 2 experiments , 2 - 3 cells measured per experiments . Data shown as mean ± SD . One - way ANOVA with Tukey post - test . Scale bar = 5 μm . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Bottom right : Schematic of lifetime quenching in condensates and aggregates .

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

FLIM of 1h- and 24h-old CFP-Tau (full-length) condensates and heparin induced CFP-Tau aggregates. Lifetime components are indicated in phasor plots. Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP-Tau. N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. One-way ANOVA with Tukey post-test. Scale bar=5 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Bottom right: Schematic of lifetime quenching in condensates and aggregates.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) or control followed by western blot analysis ( A ) The basal levels of p53 normalized to GAPDH were arbitrarily set at 1 . 0 and the fold change is shown in ( ii ) ( n = 4 ) .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I) or control followed by western blot analysis (A) The basal levels of p53 normalized to GAPDH were arbitrarily set at 1.0 and the fold change is shown in (ii) (n=4).


Treatment of HCT116 cells with siRNA targeting Ku70 or control ( A - I ) combined or not with a treatment with doxorubicin ( Doxo ) ( positive control for phosphorylation of ATM and p53 ) ( B ) followed by western blot analysis .

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

Treatment of HCT116 cells with siRNA targeting Ku70 or control (A-I) combined or not with a treatment with doxorubicin (Doxo) (positive control for phosphorylation of ATM and p53) (B) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , ASF ( C ) ( positive control for y - H2AX activation [ 29 ] ) or control ( A - I ) followed by western blot analysis .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), ASF (C) (positive control for y-H2AX activation [29])or control (A-I) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with wortmannin ( WTN ) ( D ) followed by western blot analysis .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with wortmannin (WTN) (D) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with NU - 7441 ( E , F ) followed by western blot analysis .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with NU-7441 (E,F) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , XRCC4 ( F ) or control ( A - I ) combined or not with a treatment with NU - 7441 ( E , F ) , followed by western blot analysis .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), XRCC4 (F) or control (A-I) combined or not with a treatment with NU-7441 (E,F), followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) followed by RT - qPCR analysis ( G ) . In RT - qPCR analysis ( G ) , mRNA levels were standardized against HPRT mRNA ( n = 3 ) . The two panels of Fig . 1C are partion of the same gel . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ) . All error bars reflect SEM .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) followed by RT-qPCR analysis (G). In RT-qPCR analysis (G), mRNA levels were standardized against HPRT mRNA (n=3). The two panels of Fig. 1C are partion of the same gel. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01). All error bars reflect SEM.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with MG132 ( H ) followed by western blot analysis ( A - F , H - I ) .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with MG132 (H) followed by western blot analysis (A-F, H-I).


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with cycloheximide ( CHX ) ( I ) followed by western blot analysis ( A - F , H - I ) .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with cycloheximide (CHX) (I) followed by western blot analysis (A-F, H-I).


( A ) RNA affinity chromatography using the p53 5 ' UTR ( 5 ' p53 ) or a portion of the p47 5 ' UTR ( 5 ' p47 ) ( depicted in ( i ) ) and HeLa cytoplasmic extracts ( CE ) , followed by western blot analysis ( ii ) .

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

(A) RNA affinity chromatography using the p53 5'UTR (5'p53) or a portion of the p47 5'UTR (5'p47) (depicted in (i)) and HeLa cytoplasmic extracts (CE), followed by western blot analysis (ii).


( B ) IP of in cellulo RNA - protein complexes ( RIP ) in HCT116 cells , followed either by RT - qPCR analysis . Bar plots represent the mean and the data of two independent experiments ( i ) or by western blot analysis ( ii ) . The relative p53 mRNA levels for each IP sample were normalized to the corresponding IP IgG and to the corresponding input sample and were plotted relatively to the HPRT mRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IP of in cellulo RNA-protein complexes (RIP) in HCT116 cells, followed either by RT-qPCR analysis. Bar plots represent the mean and the data of two independent experiments (i) or by western blot analysis (ii). The relative p53 mRNA levels for each IP sample were normalized to the corresponding IP IgG and to the corresponding input sample and were plotted relatively to the HPRT mRNA.


( C ) UV cross - linking of recombinant ( Ku70 / Ku80 ) with the in vitro transcribed 5 ' p53 with alpha 32P - GTP or alpha 32P - UTP ( i ) . UV cross - linking of recombinant Ku70 / Ku80 with the in vitro transcribed 5 ' p53 and the Ctrl ( 5 ' p53 antisense ) with alpha 32PGTP ( ii ) .

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(C) UV cross-linking of recombinant (Ku70/Ku80) with the in vitro transcribed 5'p53 with alpha 32P-GTP or alpha 32P-UTP (i). UV cross-linking of recombinant Ku70/Ku80 with the in vitro transcribed 5'p53 and the Ctrl (5'p53 antisense) with alpha 32PGTP (ii).


( D , E ) RNA affinity chromatography using the different RNA constructs depicted in ( i ) , as described in A .

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

(D,E) RNA affinity chromatography using the different RNA constructs depicted in (i), as described in A.


( A ) Non polysomal ( NP ) and polysomal ( P ) fractions were extracted from HCT116 cells transfected with the siRNA Ku70 and quantitative RT - qPCR was performed using specific primers for p53 and HPRT mRNAs . The p53 mRNA levels in P and NP fractions were normalized to the input ( n = 3 )

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

(A) Non polysomal (NP) and polysomal (P) fractions were extracted from HCT116 cells transfected with the siRNA Ku70 and quantitative RT-qPCR was performed using specific primers for p53 and HPRT mRNAs. The p53 mRNA levels in P and NP fractions were normalized to the input (n=3)


( B ) Western blot from H1299 ( p53 - null ) cells treated with siRNA Ku70 or control ( ii ) , followed by transfections of RNA constructs depicted in ( i ) . p53 protein levels from the WT and the ΔB2 reporters depicted in ( i ) normalized to firefly luciferase ( transfection control reporter ) ( iii , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot from H1299 (p53-null) cells treated with siRNA Ku70 or control (ii), followed by transfections of RNA constructs depicted in (i). p53 protein levels from the WT and the ΔB2 reporters depicted in (i) normalized to firefly luciferase (transfection control reporter) (iii, n=3).


( C ) AnnexinV / PI staining and flow cytometry experiments performed as in ( B ) to assess cell death ( n = 3 ) .

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

(C) AnnexinV/PI staining and flow cytometry experiments performed as in (B) to assess cell death (n=3).


( D ) RT - qPCR analysis of Bax , TP53I3 , BTG2 , GADD45α and p21 after siRNA - mediated Ku or control depletion in HCT116p53 + / + or HCT116p53 - / - ( n = 3 ) . Relative mRNA levels were standardized against GAPDH mRNA . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 NS , non significant ) . All error bars reflect SEM .

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

(D) RT-qPCR analysis of Bax, TP53I3, BTG2, GADD45α and p21 after siRNA-mediated Ku or control depletion in HCT116p53+/+ or HCT116p53-/- (n=3). Relative mRNA levels were standardized against GAPDH mRNA. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001 NS, non significant). All error bars reflect SEM.


( A ) Western blot analysis after treatment with Ku70 or control siRNA , followed by incubation with DMSO , etoposide ( ETO ) or bleomycin ( BLEO ) for 16 h ( i ) . p53 levels after Ku70 depletion normalized to siRNA control for each condition ( ii , n = 3 ) . All the experiments were performed with HCT116 cells . GAPDH : loading control in A , B , C . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

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

(A) Western blot analysis after treatment with Ku70 or control siRNA, followed by incubation with DMSO, etoposide (ETO) or bleomycin (BLEO) for 16 h (i). p53 levels after Ku70 depletion normalized to siRNA control for each condition (ii, n=3). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( B ) Western blot analysis as in A , except that tricostatin A ( TSA ) was used after Ku / control knockdown ( n = 4 ) . All the experiments were performed with HCT116 cells . GAPDH : loading control in A , B , C . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

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

(B) Western blot analysis as in A, except that tricostatin A (TSA) was used after Ku/control knockdown (n=4). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( C ) Western blot in cells treated either with DMSO , TSA , or ETO for 16 or 24 h . All the experiments were performed with HCT116 cells . GAPDH : loading control in A , B , C . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

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

(C) Western blot in cells treated either with DMSO, TSA, or ETO for 16 or 24 h. All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( D ) RNA affinity chromatography using the 5 ' p53RNA and total extracts from cells treated with TSA , ETO or BLEO for 24 h , followed by western blot analysis of Ku70 quantified and normalized to the input ( ii , n = 3 ) . All the experiments were performed with HCT116 cells . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

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

(D) RNA affinity chromatography using the 5'p53RNA and total extracts from cells treated with TSA, ETO or BLEO for 24 h, followed by western blot analysis of Ku70 quantified and normalized to the input (ii, n=3). All the experiments were performed with HCT116 cells. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( E ) RNA affinity chromatography using the 5 ' p53RNA and total extracts from cells transfected or not with PCAF encoding plasmid for 48 h , followed by western blot analysis . All the experiments were performed with HCT116 cells .

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

(E) RNA affinity chromatography using the 5'p53RNA and total extracts from cells transfected or not with PCAF encoding plasmid for 48 h, followed by western blot analysis. All the experiments were performed with HCT116 cells.


( F ) RNA affinity chromatography using the 5 ' p53RNA or a dsDNA and total extracts from cells transfected with HA - tagged Ku70 wild - type ( WT ) or mutated ( Mut6E ) Ku70 [ 25 ] for 48 h , followed by western blot analysis . All the experiments were performed with HCT116 cells .

(F) RNA affinity chromatography using the 5'p53RNA or a dsDNA and total extracts from cells transfected with HA-tagged Ku70 wild-type (WT) or mutated (Mut6E) Ku70 [25] for 48 h, followed by western blot analysis. All the experiments were performed with HCT116 cells.


( G ) RNA affinity chromatography as in F , but with two additional Ku mutants ( K282 / 338Q and K282 / 338R ) , followed by western blot of Ku70 quantified and normalized to the input and to the loading control , PTB ( ii , n = 3 ) All the experiments were performed with HCT116 cells .

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

(G) RNA affinity chromatography as in F, but with two additional Ku mutants (K282/338Q and K282/338R), followed by western blot of Ku70 quantified and normalized to the input and to the loading control, PTB (ii, n=3) All the experiments were performed with HCT116 cells.


C . ) Live epifluorescence microscopy of PfMev parasites that are expressing api - SFG ( green ) and the P . falciparum DPCK - mCherry protein ( red ) with DAPI marking the nuclei ( blue ) . The borders of the red blood cell and parasite are marked with dotted blue and white lines , respectively . Microscopy images represent fields that are 10µm long by 10µm wide .

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

C.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and the P. falciparum DPCK-mCherry protein (red) with DAPI marking the nuclei (blue). The borders of the red blood cell and parasite are marked with dotted blue and white lines, respectively. Microscopy images represent fields that are 10µm long by 10µm wide.


B . ) Live epifluorescence microscopy of PfMev parasites that are expressing api - SFG ( green ) and api - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . Data information : All microscopy images represent fields that are 10µm long by 10µm wide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


D . ) Live epifluorescence microscopy of the PfMev api - EcDPCK - mCherry Δdpck parasite line that is expressing api - SFG ( green ) and api - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . E . ) Live epifluorescence microscopy of the PfMev api - EcDPCK - mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate . This parasite line expresses api - SFG ( green ) and api - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . Data information : All microscopy images represent fields that are 10µm long by 10µm wide .

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

D.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck parasite line that is expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). E.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate. This parasite line expresses api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


F . ) Attempted PCR detection of the ldh , sufB , and cox1 genes from the nuclear ( N ) , apicoplast ( A ) , and mitochondrial ( M ) genomes , respectively . We failed to amplify sufB from the azithromycin - treated ( + Azith ) PfMev api - EcDPCK - mCherry Δdpck parasites , indicating the loss of the apicoplast organellar genome .

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

F.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We failed to amplify sufB from the azithromycin-treated (+ Azith) PfMev api-EcDPCK-mCherry Δdpck parasites, indicating the loss of the apicoplast organellar genome.


B . ) Live epifluorescence microscopy of the PfMev EcDPCK - mCherry line . This parasite line expresses api - SFG ( green ) and EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . D . ) Live epifluorescence microscopy of the PfMev EcDPCK - mCherry Δdpck line . This parasite line expresses api - SFG ( green ) and EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . Data information : All microscopy images represent fields that are 10µm long by 10µm wide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). D.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry Δdpck line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


E . ) Attempted PCR detection of the ldh , sufB , and cox1 genes from the nuclear ( N ) , apicoplast ( A ) , and mitochondrial ( M ) genomes , respectively . We were successful in amplifying sufB from the PfMev EcDPCK - mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate , indicating retention of the apicoplast organellar genome .

E.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were successful in amplifying sufB from the PfMev EcDPCK-mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate, indicating retention of the apicoplast organellar genome.


C . ) Live epifluorescence microscopy of the PfMev CLD - EcDPCK - mCherry - apt Δdpck line . This parasite line expresses api - SFG ( green ) and CLD - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . This parasite line was grown in the presence of 0 . 5μM aTc . D . ) The PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown in the absence of aTc for 48 hours . The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite . E . ) The PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown in the presence of 0 . 5μM aTc , and in the presence of 0 . 5μM Shield1 ( Shld ) for 48 hours . F . ) The PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown in the absence of aTc , and in the presence of 0 . 5μM Shield1 ( Shld ) for 48 hours . The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite . Data information : All microscopy images represent fields that are 10µm long by 10µm wide .

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

C.) Live epifluorescence microscopy of the PfMev CLD-EcDPCK-mCherry-apt Δdpck line. This parasite line expresses api-SFG (green) and CLD-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). This parasite line was grown in the presence of 0.5μM aTc. D.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. E.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the presence of 0.5μM aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. F.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


G . ) Growth curve of the PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown either in the presence of 0 . 5μM aTc and the absence 0 . 5μM Shield1 ( permissive condition , blue ) , or the absence of 0 . 5μM aTc and the presence of 0 . 5μM Shield1 ( non - permissive condition , red ) . Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1 : 5 on day 4 . Treatment under non - permissive conditions resulted in reduced parasite growth beginning at day 1 ( two - way ANOVA , followed by Bonferroni ' s correction ; * , P < 0 . 05 ) . Error bars represent the standard error of the mean from two independent experiments , each conducted in quadruplicate .

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

G.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 1 (two-way ANOVA, followed by Bonferroni's correction; *, P < 0.05). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.


A . ) Attempted PCR detection of the ldh , sufB , and cox1 genes from the nuclear ( N ) , apicoplast ( A ) , and mitochondrial ( M ) genomes , respectively . We were unsuccessful in amplifying sufB from the PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line after treatment with 100nM azithromycin ( Azith ) in the presence of 50μM mevalonate , indicating the disruption of the apicoplast organelle .

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

A.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were unsuccessful in amplifying sufB from the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line after treatment with 100nM azithromycin (Azith) in the presence of 50μM mevalonate, indicating the disruption of the apicoplast organelle.


B . ) Live epifluorescence microscopy of the apicoplast - negative PfMev CLD - EcDPCK - mCherry - apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate . This parasite line expresses api - SFG ( green ) and the CLD - EcDPCK - mCherry construct ( red ) with DAPI marking the nuclei ( blue ) . Images represent fields that are 10µm long by 10µm wide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.) Live epifluorescence microscopy of the apicoplast-negative PfMev CLD-EcDPCK-mCherry-apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate. This parasite line expresses api-SFG (green) and the CLD-EcDPCK-mCherry construct (red) with DAPI marking the nuclei (blue). Images represent fields that are 10µm long by 10µm wide.


C . ) Growth curve of the PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0 . 5μM aTc and the absence 0 . 5μM Shield1 ( permissive condition , blue ) , or the absence of 0 . 5μM aTc and the presence of 0 . 5μM Shield1 ( non - permissive condition , red ) . Pantothenate ( 50nM ) and mevalonate ( 50μM ) were supplied in both conditions and the cultures were diluted 1 : 5 on day 4 . Treatment under non - permissive conditions resulted in reduced parasite growth beginning at day 2 ( two - way ANOVA , followed by Bonferroni ' s correction ; * * , P < 0 . 01 ) . Error bars represent the standard error of the mean from two independent experiments , each conducted in quadruplicate .

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

C.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate (50nM) and mevalonate (50μM) were supplied in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 2 (two-way ANOVA, followed by Bonferroni's correction; **, P < 0.01). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.


a . Fold change in mRNA expression of members of the PI3K / AKT / Foxo pathway in IFN + as compared to IFN - Tregs in the microarray analysis .

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

a. Fold change in mRNA expression of members of the PI3K/AKT/Foxo pathway in IFN+ as compared to IFN- Tregs in the microarray analysis.


b . mRNA expression of members of the PI3K / AKT / Foxo signaling pathway in sorted IFN + and IFN - Tregs .

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b. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway in sorted IFN+ and IFN- Tregs.


c . Sorted CD4 + CD25highCD127 - Tregs were stimulated in the presence of IL - 2 ( white ) or IL - 2 + IL - 12 ( black dots ) and RNA was isolated every 24 hours for 4 days . mRNA expression of members of the PI3K / AKT / Foxo signaling pathway .

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c. Sorted CD4+CD25highCD127- Tregs were stimulated in the presence of IL-2 (white) or IL-2+IL-12 (black dots) and RNA was isolated every 24 hours for 4 days. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway.


d . Representative example of the protein expression of AKT1 , AKT2 , AKT3 , PTEN and FOXO3 on sorted CD4 + CD25highCD127 - Tregs stimulated in the presence of IL - 2 ( grey line ) or IL - 2 + IL - 12 ( black line ) and day 2 for AKT1 , AKT2 , AKT3 and PTEN and day 3 for FOXO3 . Grey histogram represents the isotype . e . Statistical analysis of AKT1 , AKT2 , AKT3 , FOXO3 and PTEN gMFI of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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d. Representative example of the protein expression of AKT1, AKT2, AKT3, PTEN and FOXO3 on sorted CD4+CD25highCD127- Tregs stimulated in the presence of IL-2 (grey line) or IL-2+IL-12 (black line) and day 2 for AKT1, AKT2, AKT3 and PTEN and day 3 for FOXO3. Grey histogram represents the isotype. e. Statistical analysis of AKT1, AKT2, AKT3, FOXO3 and PTEN gMFI of n=6 experiments performed. * p<0.05, ** p<0.005, *** p<0.0005.


Sorted CD4 + CD25hiCD127 - Tregs were stimulated with anti - CD3 , anti - CD28 and IL - 2 in the presence or absence of IL - 12 . a . Tregs were stimulated for 50 minutes with IL - 12 or vehicle . Histograms show a representative example of n = 8 independent experiments performed of the expression of phosphorylated molecules at 30 minutes after stimulation for pAKT and pFoxo1 , and at 40 minutes for pFoxo3 in vehicle - ( grey open histogram ) or IL - 12 - treated ( dark open histogram ) Tregs . Grey histograms represent the isotype . The time points selected were based on kinetic experiments performed for a total of 50

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

Sorted CD4+CD25hiCD127- Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. a. Tregs were stimulated for 50 minutes with IL-12 or vehicle. Histograms show a representative example of n=8 independent experiments performed of the expression of phosphorylated molecules at 30 minutes after stimulation for pAKT and pFoxo1, and at 40 minutes for pFoxo3 in vehicle- (grey open histogram) or IL-12-treated (dark open histogram) Tregs. Grey histograms represent the isotype. The time points selected were based on kinetic experiments performed for a total of 50 minutes


Sorted CD4 + CD25hiCD127 - Tregs were stimulated with anti - CD3 , anti - CD28 and IL - 2 in the presence or absence of IL - 12 . b . Tregs were stimulated in the presence ( black ) or absence ( grey ) of IL - 12 and the expression of phosphorylated molecules was examined 16 hours later . c . gMFI of pAKT , pFoxo1 and pFoxo3 of n = 6 independent experiments performed .

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

Sorted CD4+CD25hiCD127- Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. b. Tregs were stimulated in the presence (black) or absence (grey) of IL-12 and the expression of phosphorylated molecules was examined 16 hours later. c. gMFI of pAKT, pFoxo1 and pFoxo3 of n=6 independent experiments performed.


d . Foxo1 and Foxo3 transcriptional targets expression on sorted CD4 + CD25hiCD127 - Tregs stimulated in the presence of vehicle or IL - 12 at day 2 after stimulation ( n = 8 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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d. Foxo1 and Foxo3 transcriptional targets expression on sorted CD4+CD25hiCD127-Tregs stimulated in the presence of vehicle or IL-12 at day 2 after stimulation (n=8). *p < 0.05, **p < 0.005, ***p < 0.0005.


a . Sorted CD4 + CD25hiCD127 - Tregs were stimulated in the presence of vehicle ( white dots ) or either SF1670 ( first row ) , SC - 79 ( second row ) , 740 Y - P ( third row ) or AS1842856 ( fourth row , black dots ) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours . Statistical analysis of n = 4 independent experiments performed .

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

a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white dots) or either SF1670 (first row), SC-79 (second row), 740 Y-P (third row) or AS1842856 (fourth row, black dots) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=4 independent experiments performed.


b . Representative example of the frequency of IFN + Foxp3 + cells in Tregs stimulated in the presence of vehicle , SF1670 , SC - 79 , 740 Y - P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin . Numbers in brackets represent total cell numbers c . Statistical analysis of the frequency of IFN + Foxp3 + cells in Tregs stimulated in the presence of vehicle , SF1670 , SC - 79 , 740 Y - P or AS1842856 of n = 5 experiments performed .

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

b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin. Numbers in brackets represent total cell numbers c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 of n=5 experiments performed.


d . Proliferation of CFSE - labeled responder T cells alone ( left histogram ) or co - cultured with Tregs that have been pre - incubated with either vehicle , SF1670 , SC - 79 or 740 Y - P at day 4 after co - culture at a 1 : 2 Treg : Tresp ratio . e . Statistical analysis of the percentage of suppression at different Treg : Tresp ratios of n = 6 experiments performed .

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

d. Proliferation of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that have been pre-incubated with either vehicle, SF1670, SC-79 or 740 Y-P at day 4 after co-culture at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression at different Treg:Tresp ratios of n=6 experiments performed.


a . Sorted CD4 + CD25hiCD127 - were pre - incubated with increasing concentrations of AS1842856 , washed and co - cultured with CFSE - labeled responder T cells . Representative histograms of proliferation of Tresp alone ( left ) or in co - culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1 : 2 Treg : Tresp ratio . b . Statistical analysis of the suppression of proliferation at different Treg : Tresp ratios ( n = 6 ) on Tregs pretreated with vehicle ( white dots ) or 10 nM ( black dots , left diagram ) or 50 nM AS1842856 ( black dots , right diagram ) .

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

a. Sorted CD4+CD25hiCD127- were pre-incubated with increasing concentrations of AS1842856, washed and co-cultured with CFSE-labeled responder T cells. Representative histograms of proliferation of Tresp alone (left) or in co-culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1:2 Treg:Tresp ratio. b. Statistical analysis of the suppression of proliferation at different Treg:Tresp ratios (n=6) on Tregs pretreated with vehicle (white dots) or 10 nM (black dots, left diagram) or 50 nM AS1842856 (black dots, right diagram).


c . Representative dot plots of the frequency of IFN + Foxp3 + Tregs in the co - cultures in a at a 1 : 2 Treg : Tresp ratio after adding GolgiStop for the last 6 hours .

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c. Representative dot plots of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a at a 1:2 Treg:Tresp ratio after adding GolgiStop for the last 6 hours.


d . Statistical analysis of the frequency of IFN + Foxp3 + Tregs in the co - cultures in a of n = 6 experiments performed .

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d. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a of n=6 experiments performed.


e . Correlation between frequency of IFN + Foxp3 + Tregs in co - cultures and percentage of suppression in Tregs pre - incubated with different doses of AS1842856 at a 1 : 4 Treg : Tresp ratio .

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

e. Correlation between frequency of IFN+Foxp3+ Tregs in co-cultures and percentage of suppression in Tregs pre-incubated with different doses of AS1842856 at a 1:4 Treg:Tresp ratio.


f . IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT - shRNA control , measured at day 6 after 4 hours of stimulation with PMA and ionomycin .

f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin.


g . Percentage of suppression of CFSE - labeled Tresp proliferation co - cultured with Tregs transduced with a non - target ( white dots ) , Foxo1 ( left diagram , black dots ) or Foxo3 shRNA ( right diagram , black dots ) at different Treg : Tresp ratios ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p < 0.05, **p < 0.005, ***p < 0.0005.


Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 and shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control and resorted based on GFP expression at day 5 . a . IFNG , TBX21 , IL10 , FOXP3 gene expression after sort at day 5 ( n = 5 ) .

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Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. a. IFNG, TBX21, IL10, FOXP3 gene expression after sort at day 5 (n=5).


Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 and shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control and resorted based on GFP expression at day 5 . b . Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 ( n = 4 ) .

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Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4).