Figure 3Human 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Human 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.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.


Figure 5Healthy 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Healthy 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.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.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.


Figure 644 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 . 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 .

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

Figure 644 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.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.


Figure 1 ( D ) HaloTag pulse - chase analysis in WT MEF monitoring fluorescent Halo - AATNNN during up to 6 h of chase in the presence of BafA1 . The fluorescent JF646 - Halo ligand and the polymer - specific 2C1 antibody stains are shown . ( E ) Same as ( D ) for fluorescent Halo - ATZNNN . ( F ) Quantification of accumulation of fluorescent Halo - AATNNN and Halo - ATZNNN within LAMP1 - positive endolysosomes . Mean ± SEM , n = 20 , 11 , 10 , 13 for Halo - AATNNN , n = 18 , 12 , 12 , 13 for Halo - ATZNNN . Two - way ANOVA and Sidak ' s multiple comparison test , ns P > 0 . 05 , * * * P < 0 . 001 .

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

Figure 1(D) HaloTag pulse-chase analysis in WT MEF monitoring fluorescent Halo-AATNNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646-Halo ligand and the polymer-specific 2C1 antibody stains are shown. (E) Same as (D) for fluorescent Halo-ATZNNN. (F) Quantification of accumulation of fluorescent Halo-AATNNN and Halo-ATZNNN within LAMP1-positive endolysosomes. Mean ± SEM, n=20, 11, 10, 13 for Halo-AATNNN, n= 18, 12, 12, 13 for Halo-ATZNNN. Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, *** P<0.001.


Figure 2A Confocal laser scanning microscopy analyses in cells treated with BafA1 . Total HA - ATZNNN ( red ) or HA - ATZNNN polymers ( magenta ) in LAMP1 - positive endolysosome ( green ) in WT MEF . ( C ) Same as ( A ) in Cnx - KO MEF . ( D ) Same as ( A ) in WT MEF exposed to kifunensine ( KIF ) . ( E ) Same as ( A ) in WT MEF exposed to Castanospermine ( CST ) . ( F ) Same as ( A ) in sCNX MEF . ( G ) Same as ( A ) in Uggt1 - KO MEF . ( J ) Halo - ATZNNN in Uggt1 - KO MEF mock - transfected ( empty pcDNA3 plasmid ) . ( K , L ) ( K ) Same as ( J ) , with a plasmid for expression of active or ( L ) inactive UGGT1 . ( M ) Quantification of Halo - ATZNNN - positive endolysosomes ( mean , n = 22 , 10 , 15 cells for panels J - L , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 ) .

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

Figure 2A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF. (C) Same as (A) in Cnx-KO MEF. (D) Same as (A) in WT MEF exposed to kifunensine (KIF). (E) Same as (A) in WT MEF exposed to Castanospermine (CST). (F) Same as (A) in sCNX MEF. (G) Same as (A) in Uggt1-KO MEF. (J) Halo-ATZNNN in Uggt1-KO MEF mock-transfected (empty pcDNA3 plasmid). (K, L) (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1. (M) Quantification of Halo-ATZNNN-positive endolysosomes (mean, n=22, 10, 15 cells for panels J-L, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001).


Figure 3 ( B - I ) Confocal laser scanning microscopy analyses ( as in Fig . 2 ) in WT MEF treated with BafA1 . ( J , K ) ( J ) Quantification of ATZxxx - positive endolysosomes and ( K ) of ATZxxx polymers - positive endolysosomes ( mean , n = 18 , 30 , 24 , 14 , 27 , 22 , 27 , 18 cells for HA stain and n = 12 , 14 , 14 , 11 , 14 , 14 , 17 , 11 cells for 2C1 stain of panels B - I , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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Figure 3(B-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1. (J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.


Figure 4 ( C ) Co - immunoprecipitation of ATZXXX ( Bait , lower panel ) with CNX ( upper panel ) and BiP ( middle panel ) . Protein content in the total cell extracts ( TCE ) is shown . ( D ) Quantification of ( C ) for CNX association ( n = 3 ) . Unpaired two - tailed t - test , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( E ) Quantification of ( C ) for BiP association ( n = 3 for ATZNNN and ATZQQN ; n = 4 for ATZQNQ and ATZQQQ ) . Unpaired two - tailed t - test , * P < 0 . 005 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( G ) Confocal laser scanning microscopy in Flp - In 3T3 cells stably expressing ATZNNN in the absence of BafA1 ( to allow clearance of misfolded model proteins delivered within endolysosomes ) . Co - localization with Calnexin ( CNX ) . ( H ) Same as ( G ) for BiP co - localization . ( I ) Same as ( G ) for ATZQQQ . ( J ) Same as ( H ) for ATZQQQ . ( K ) HaloTag pulse - chase analysis in MEF monitoring fluorescent Halo - ATZNNN during up to 24 h . ( L ) Same as ( K ) for fluorescent Halo - ATZQQQ . Arrowheads show select clusters of JF646 - labeled Halo - ATZQQQ . Arrowheads show co - localization Halo - ATZQQQ : BiP .

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Figure 4(C) Co-immunoprecipitation of ATZXXX (Bait, lower panel) with CNX (upper panel) and BiP (middle panel). Protein content in the total cell extracts (TCE) is shown. (D) Quantification of (C) for CNX association (n=3). Unpaired two-tailed t-test, ** P<0.01*** P<0.001, **** P<0.0001. (E) Quantification of (C) for BiP association (n=3 for ATZNNN and ATZQQN; n=4 for ATZQNQ and ATZQQQ). Unpaired two-tailed t-test, * P<0.005, ** P<0.01, *** P<0.001, **** P<0.0001. (G) Confocal laser scanning microscopy in Flp-In 3T3 cells stably expressing ATZNNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co-localization with Calnexin (CNX). (H) Same as (G) for BiP co-localization. (I) Same as (G) for ATZQQQ. (J) Same as (H) for ATZQQQ. (K) HaloTag pulse-chase analysis in MEF monitoring fluorescent Halo-ATZNNN during up to 24 h. (L) Same as (K) for fluorescent Halo-ATZQQQ. Arrowheads show select clusters of JF646-labeled Halo-ATZQQQ. Arrowheads show co-localization Halo-ATZQQQ:BiP.


Figure 1 ( A ) Quantification of AURKB transcripts in non - IPF lung derived resident - fibroblasts treated with indicated mitogens for 16hrs . * P < 0 . 05 , * * * P < 0 . 0005 , and * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 4 ) . ( B ) Quantification of AURKB gene transcripts in isolated fibroblasts from non - IPF and IPF lung stromal cell cultures . * * P < 0 . 005 , unpaired t - test , ( n = 6 ) . ( C ) IPF and non - IPF lung sections were immunostained using AURKB antibody ( n = 4 ) . Representative images were obtained at 40X magnification . Scale bar : 40µm . Dotted area : black ; myofibroblastic core , blue ; active fibrotic front . Arrows indicate AURKB positive cells . ( D ) Immunoblots of Aurkb and Aurka in total lung lysates of normal ( CCSP / - ) and fibrotic ( CCSP / TGFα ) mice fed with Dox for 6wks . Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH . * P < 0 . 05 , unpaired t - test , ( n = 4 ) . ( E ) Immunostaining was performed using AURKB antibody in lung sections of control ( CCSP / - ) and TGFα ( CCSP / TGFα ) mice on Dox for 6wks ( n = 6 ) . Representative images were obtained at 20X ( low ; Scale bar : 100µm ) and 63X ( high ; Scale bar : 30µm ) magnification . Dashed box indicates area of the section showing in high magnification . ( F ) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice . Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh . * * P < 0 . 005 , unpaired t - test , ( n = 4 - 5 ) .

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

Figure 1(A) Quantification of AURKB transcripts in non-IPF lung derived resident-fibroblasts treated with indicated mitogens for 16hrs. *P < 0.05, ***P < 0.0005, and ****P < 0.00005, 1-way ANOVA, (n=4).(B) Quantification of AURKB gene transcripts in isolated fibroblasts from non-IPF and IPF lung stromal cell cultures. **P < 0.005, unpaired t-test, (n=6).(C) IPF and non-IPF lung sections were immunostained using AURKB antibody (n=4). Representative images were obtained at 40X magnification. Scale bar: 40µm. Dotted area: black; myofibroblastic core, blue; active fibrotic front. Arrows indicate AURKB positive cells.(D) Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/-) and fibrotic (CCSP/TGFα) mice fed with Dox for 6wks. Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH. *P < 0.05, unpaired t-test, (n=4).(E) Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/-) and TGFα (CCSP/TGFα) mice on Dox for 6wks (n=6). Representative images were obtained at 20X (low; Scale bar: 100µm) and 63X (high; Scale bar: 30µm) magnification. Dashed box indicates area of the section showing in high magnification.(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).


Figure 2 ( A ) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) . ( B ) Lung - resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) . ( C ) Immunoblot analysis of AURKB and WT1 in the lysates of non - IPF fibroblasts transduced with control or WT1 - adenoviral particles for 72hrs . * * P < 0 . 005 , unpaired t - test , ( n = 3 ) . ( D ) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα ( 100ng / mL ) for 16hrs . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) . ( E ) Primary lung - resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks . Cell lysates were prepared , and the ChIP assay was performed with anti - WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter - specific PCR primers . Non - immunoprecipitated DNA is represented as input DNA ( product size , 104 bp ) . * * * P < 0 . 0005 , unpaired t - test , ( n = 2 ) . ( F ) AURKB - promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing ( OE ) vector . * * * * P < 0 . 00005 , one - way - ANOVA , ( n = 6 ) .

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

Figure 2(A) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).(B) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).(C) Immunoblot analysis of AURKB and WT1 in the lysates of non-IPF fibroblasts transduced with control or WT1-adenoviral particles for 72hrs. **P < 0.005, unpaired t-test, (n=3).(D) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100ng/mL) for 16hrs. ****P < 0.00005, unpaired t-test, (n=4).(E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2).(F) AURKB-promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one-way-ANOVA, (n=6).


Figure 3 ( C ) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB ( green ) and Ki - 67 ( red ) . Merged image shows cells that co - express AURKB and Ki - 67 ( yellow ) . ( D ) Quantification of Ki - 67 + and AURKB + double positive cells was performed using five confocal images per mice in control and TGFα mice . Images were obtained at 40X magnification . Scale bar : 50µm . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) . ( E ) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA . * * * * P < 0 . 00005 , unpaired t - test , ( n = 9 - 11 ) . ( F ) Proliferation was measured in primary lung - resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα ( 20ng / mL ) for 24hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) . ( G ) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) . ( H ) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB ( green ) and αSMA ( red ) . Images were obtained at 40X magnification . Scale bar : 50µm . ( n = 4 ) . ( I ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in lung - resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h . * * P < 0 . 005 , * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) . ( J ) Quantification of BAK , BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) . ( K ) Quantification of Bak , Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs . * P < 0 . 005 , * * P < 0 . 005 , unpaired t - test , ( n = 3 ) .

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

Figure 3(C) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB (green) and Ki-67(red). Merged image shows cells that co-express AURKB and Ki-67 (yellow).(D) Quantification of Ki-67+ and AURKB+ double positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40X magnification. Scale bar: 50µm. ****P < 0.00005, unpaired t-test, (n=4).(E) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t-test, (n=9-11).(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).(G) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).(J) Quantification of BAK, BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).(K) Quantification of Bak, Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs. *P < 0.005, **P < 0.005, unpaired t-test, (n=3).


Figure 4 ( A ) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) . ( B ) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs . * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 9 - 11 ) . ( C ) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody . Images were obtained at 40X magnification . Scale bar : 50µm . The number of PCNA - positive cells and total DAPI - positive cells were quantified using MetaMorph image analysis software . Proliferation is indicated as the percentage of proliferating cells in total DAPI - positive cells . * * * * P < 0 . 00005 , unpaired t - test , ( n = 3 - 4 ) . ( D ) Proliferation was measured in primary fibroblasts treated with TGFα ( 20ng / mL ) and indicated doses of barasertib for 48hrs . . * * P < 0 . 005 , 1 - way ANOVA , ( n = 9 - 11 ) . ( E ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib . * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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

Figure 4(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).


Figure 5 ( B ) Representative images of Masson ' s trichrome - stained lung sections from the vehicle and barasertib treated mice . Images were obtained at 10X magnification . Scale bar : 200μm . ( C ) Quantification of right lung weight of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) . ( D ) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) . ( E ) Quantification of Col1α , Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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Figure 5(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


Figure 6 ( A ) Lung sections from vehicle and barasertib treated mice were immunostained using Ki - 67 antibody . Representative images were obtained at 20X magnification . Scale bar : 50μm . ( B ) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody . Gapdh is used as loading control . * * P < 0 . 005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) . ( C ) Quantification of Aurkb , Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib . * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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

Figure 6(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


Figure 7 ( B ) Quantification of right lung weight of mice treated with vehicle and barasertib . * * P < 0 . 005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) . ( C ) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies . Gapdh is used as loading control . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) . ( D ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm . ( E ) Quantification of lung mechanics in mice treated with vehicle and barasertib . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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

Figure 7(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


Figure 8 ( B ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm . ( C ) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 7 - 8 mice / group ) . ( D ) Quantification of Col1α1 , Col3α , Col5α , Col14 , Col15 , αSma , Ccna2 , and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib . * P < 0 . 05 , * * P < 0 . 005 * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 6 mice / group ) . ( E ) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice . Gapdh is used as loading control . Data are presented as mean ± SEM . * * P < 0 . 005 , unpaired t - test ( n = 5mice / group ) .

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

Figure 8(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).(D) Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P< 0.05, **P<0.005 ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=6 mice/group).(E) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P<0.005, unpaired t-test (n=5mice /group).


Figure 1 ( D ) Western blot of whole zebrafish larvae , Smyhc1 stained with the F59 antibody . smyhc1 + / + larvae express Smyhc1 at 24hpf , but not at 48hpf . smyhc1 - / - larvae do not express Smyhc1 . ( E ) Smyhc1 immunohistochemistry stain of 24 hpf ( hour post fertilization ) zebrafish larvae . Filamentous actin is stained with phalloidin ( red ) . Nuclei are stained with DAPI ( blue ) . Smyhc1 is stained with the F59 antibody ( green ) ( Elworthy et al , 2008 ) . smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae display Smyhc1 in the muscle fibers at 24hpf . smyhc1 - / - larvae at 24 do not stain positively for Smyhc1 . Scale bar represents 50 µm .

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Figure 1(D) Western blot of whole zebrafish larvae, Smyhc1 stained with the F59 antibody. smyhc1+/+ larvae express Smyhc1 at 24hpf, but not at 48hpf. smyhc1-/- larvae do not express Smyhc1.(E) Smyhc1 immunohistochemistry stain of 24 hpf (hour post fertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae display Smyhc1 in the muscle fibers at 24hpf. smyhc1-/- larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 µm.


Figure 2 ( A ) Morphologies of smyhc1 + / + , smyhc1 - / + and smyhc1 - / - embryos are grossly normal at 2 days post fertilization ( dpf ) , despite complete paralysis of smyhc1 - / - embryos . In contrast , the smyhc1R673H / + embryos have notochord kinks in larval stages ( arrow head ) , while smyhc1R673H / R673H embryos have much more severe notochord kinks that compress and distort the body axis ( arrow heads ) . The smyhc1R673H / - embryos completely phenocopy smyhc1R673H / R673H embryos . ( B ) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf .

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

Figure 2(A) Morphologies of smyhc1+/+, smyhc1-/+ and smyhc1-/- embryos are grossly normal at 2 days post fertilization (dpf), despite complete paralysis of smyhc1-/- embryos. In contrast, the smyhc1R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/- embryos completely phenocopy smyhc1R673H/R673H embryos. (B) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf.


Figure 3 ( B ) The predicted mendelian ( 1 : 2 : 1 ) and experimental ratio of genotypes of progeny from smyhc1R673H / + in - crosses demonstrates survival at the indicated ages . This ratio quickly skewed toward an overrepresentation of smyhc1 + / + fish with no representation of smyhc1R673H / R673H after 5 dpf . There is also dropout of smyhc1 + / R673H fish into adulthood . Number of fish counted per group is indicated .

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Figure 3(B) The predicted mendelian (1:2:1) and experimental ratio of genotypes of progeny from smyhc1R673H/+ in-crosses demonstrates survival at the indicated ages. This ratio quickly skewed toward an overrepresentation of smyhc1+/+ fish with no representation of smyhc1R673H/R673H after 5 dpf. There is also dropout of smyhc1+/R673H fish into adulthood. Number of fish counted per group is indicated.


Figure 4 ( A ) Gross morphology of smyhc1 mutant adults . Most smyhc1 - / - adult fish display dorsal tail curvature , while smyhc1R673H / + adults have a shortened body axis and variable spinal curves . ( B ) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1 - / - adults , in contrast to the compression and fusion of vertebrae seen in smyhc1R673H / + adults . Distal tail regions are highlighted and enlarged . ( C ) Quantification of skeletal phenotype severity in adults . Scale bars represent a length of 5 mm .

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Figure 4(A) Gross morphology of smyhc1 mutant adults. Most smyhc1-/- adult fish display dorsal tail curvature, while smyhc1R673H/+ adults have a shortened body axis and variable spinal curves.(B) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1-/- adults, in contrast to the compression and fusion of vertebrae seen in smyhc1R673H/+ adults. Distal tail regions are highlighted and enlarged.(C) Quantification of skeletal phenotype severity in adults. Scale bars represent a length of 5 mm.


Figure 5 ( A ) Time course of light - triggered larval movements were manually counted at indicated times between 24 and 48 hpf . The smyhc1 - / - larvae ( n = 36 ) were paralyzed up to 48 hpf . smyhc1R673H / + mutants ( n = 15 ) twitched slightly less than their smyhc1 + / + siblings ( n = 21 ) at 34hpf . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( B ) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software ( Noldus Ethovision ) . At 6 dpf , both smyhc1 - / - ( n = 24 ) and smyhc1R673H / + ( n = 11 ) larvae travelled significantly less than smyhc1 + / + larvae ( n = 13 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( C ) Diagram of swim tunnel used to quantify adult swimming behavior . Water flow was gradually increased until the fish fatigued and were collected at the grate . ( D ) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization ( mpf ) . smyhc1 + / + ( n = 6 ) , smyhc1 - / - ( n = 5 ) , smyhc1R673H / + ( n = 5 ) .

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Figure 5(A) Time course of light-triggered larval movements were manually counted at indicated times between 24 and 48 hpf. The smyhc1-/- larvae (n = 36) were paralyzed up to 48 hpf. smyhc1R673H/+ mutants (n = 15) twitched slightly less than their smyhc1+/+ siblings (n = 21) at 34hpf. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.(B) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software (Noldus Ethovision). At 6 dpf, both smyhc1-/- (n = 24) and smyhc1R673H/+ (n = 11) larvae travelled significantly less than smyhc1+/+ larvae (n = 13). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.(C) Diagram of swim tunnel used to quantify adult swimming behavior. Water flow was gradually increased until the fish fatigued and were collected at the grate.(D) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization (mpf). smyhc1+/+ (n = 6), smyhc1-/- (n = 5), smyhc1R673H/+ (n = 5).


Figure 6 ( A ) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae , stained with phalloidin - rhodamine to detect actin . Puncta of filamentous actin are indicated with white arrowheads , misshapen myofibers are indicated with white arrows and outlined . ( B ) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae , stained with phalloidin - rhodamine . Puncta of filamentous actin are indicated with white arrowheads , distorted myofibers are indicated with arrows and outlined , and frayed myofibers are indicated with yellow arrowhead . ( C ) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , and DAPI ( blue ) . ( D ) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , and DAPI ( blue ) . Bundle of actin ringed with α - actinin indicated with white - bordered black arrowhead . ( E ) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 54 ) , smyhc1 - / - ( n = 31 ) , smyhc1R673H / + ( n = 45 ) , and smyhc1R673H / R673H ( n = 49 ) . Distance between myosepta was measured perpendicular to rostral - caudal body axis at defined mid - body regions in phalloidin stained slow skeletal muscle fluorescence images . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( F ) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 51 ) , smyhc1 - / - ( n = 32 ) , smyhc1R673H / + ( n = 64 ) , and smyhc1R673H / R673H ( n = 38 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( G ) Z - disc intervals ( sarcomere length ) of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 380 ) , smyhc1R673H / + ( n = 293 ) , and smyhc1R673H / R673H ( n = 268 ) . Distance between z - discs was measured in anti - α - actinin stained slow skeletal muscle fluorescence images . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( H ) Z - disc intervals ( sarcomere length ) of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 249 ) , smyhc1 - / - ( n = 278 ) , smyhc1R673H / + ( n = 246 ) , and smyhc1R673H / R673H ( n = 252 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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

Figure 6(A) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin-rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, misshapen myofibers are indicated with white arrows and outlined. (B) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin-rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead. (C) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). (D) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α-actinin indicated with white-bordered black arrowhead. (E) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 54), smyhc1-/- (n = 31), smyhc1R673H/+ (n = 45), and smyhc1R673H/R673H (n = 49). Distance between myosepta was measured perpendicular to rostral-caudal body axis at defined mid-body regions in phalloidin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (F) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 51), smyhc1-/- (n = 32), smyhc1R673H/+ (n = 64), and smyhc1R673H/R673H (n = 38). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (G) Z-disc intervals (sarcomere length) of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 380), smyhc1R673H/+ (n = 293), and smyhc1R673H/R673H (n = 268). Distance between z-discs was measured in anti-α-actinin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (H) Z-disc intervals (sarcomere length) of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 249), smyhc1-/- (n = 278), smyhc1R673H/+ (n = 246), and smyhc1R673H/R673H (n = 252). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


Figure 7 ( A ) Representative morphologies of smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae at 2 dpf after 24 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . All larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype . All para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) and smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike the untreated larvae which have markedly curved or distorted body axis . ( B ) Representative images of smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae at 3dpf after 48 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . All larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype . Para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) and smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike the untreated larvae which have markedly curved or distorted body axis .

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

Figure 7(A) Representative morphologies of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 2 dpf after 24 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. All para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis. (B) Representative images of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 3dpf after 48 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. Para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis.


Figure 1ATPase staining of GAS and SOL muscles of WT and DKO mice ( left ) . Corresponding percentage of type I fibers were determined ( right ) ( n = 5 ) . Scale bars : 100 μm . Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice ( n = 3 for PGC1α analysis ; n = 5 for PLIN5 analysis ) . Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR - 183 and miR - 96 agomirs ( Ago - 183 / 96 ) or antagomirs ( Ant - 183 / 96 ) ( n = 3 ) . Mitochondrial respiration profile of C2C12 cells transfected with Ago - 183 / 96 ( J ) or Ant - 183 / 96 ( K ) . Cellular oxygen consumption rate ( OCR ) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR ( n = 3 or 4 per group ) .

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

Figure 1ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm.Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice (n=3 for PGC1α analysis; n=5 for PLIN5 analysis).Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3).Mitochondrial respiration profile of C2C12 cells transfected with Ago-183/96 (J) or Ant-183/96 (K). Cellular oxygen consumption rate (OCR) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR (n=3 or 4 per group).


Figure 2Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in GAS muscles of WT and DKO mice . Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) . Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice . Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) . LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR - 183 / 96 ( N ) or antagomir - 183 / 96 ( P ) stained with Nile red ( n = 5 ) . Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR - 183 / 96 ( O ) or antagomir - 183 / 96 ( Q ) . Scale bars : 5 μm .

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

Figure 2Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in GAS muscles of WT and DKO mice. Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice. Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR-183/96 (N) or antagomir-183/96 (P) stained with Nile red (n=5). Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR-183/96 (O) or antagomir-183/96 (Q). Scale bars: 5 μm.


Figure 3Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 agomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 . Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 antagomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 . Relative mRNA expression ( I , left ) and western blot analysis ( J , top ) of ATGL in C2C12 cells transfected with agomir for miR - 183 ( Ago - 183 ) or miR - 96 ( Ago - 96 ) . Relative mRNA expression ( I , right ) and western blot analysis ( J , bottom ) of ATGL in C2C12 cells transfected with antagomir for miR - 183 ( Ant - 183 ) or miR - 96 ( Ant - 96 ) . ( n = 5 per group for RT - PCR analysis )

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

Figure 3Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96.Relative mRNA expression (I, left) and western blot analysis (J, top) of ATGL in C2C12 cells transfected with agomir for miR-183 (Ago-183) or miR-96 (Ago-96). Relative mRNA expression (I, right) and western blot analysis (J, bottom) of ATGL in C2C12 cells transfected with antagomir for miR-183 (Ant-183) or miR-96 (Ant-96). (n=5 per group for RT-PCR analysis)


Figure 4Relative mRNA expression of ATGL , PGC1α , PLIN5 , and HSL in C2C12 cells transfected with ATGL ( n = 5 ) . Relative mRNA expression of PGC1α , PLIN5 , and HSL in C2C12 cells transfected with Ant - 183 / 96 and siRNA of PPARδ ( siPPARδ ) .

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Figure 4Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5).Relative mRNA expression of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siRNA of PPARδ (siPPARδ).


Figure 5Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice ( n = 3 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice . Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months ( n = 5 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months .

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Figure 5Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice (n=3). Western blot analysis of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice.Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months (n=5). Western blot analysis of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months.


Figure 6Concentration of serum triglyceride ( TAG ) ( left ) and non - esterified fatty acid ( NEFA ) ( right ) in WT and DKO mice after HFD feeding for 2 months ( n = 5 ) . Western blot analysis of FoxO1 , PDK4 , p - PDHA1 , PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months . Glucose tolerance test ( GTT ) and area under the curve ( AUC ) data for GTT in WT and DKO mice after HFD feeding ( n = 10 - 11 ) .

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

Figure 6Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5).Western blot analysis of FoxO1, PDK4, p-PDHA1, PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months.Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).


Figure 1B Growth curves of the indicated strains shown as population doublings ( pds ) on the y axis and hours in liquid culture on the x axis . Before inoculation in liquid YES medium , single cells underwent approximately 25 pds to produce visible colonies on solid medium . For each strain , averages and SDs ( error bars ) of three biological replicates are shown . Arrows indicate times of harvesting and corresponding pds for experiments shown in ( C ) and ( D ) . C Telo - PCR analysis of telomere length in wt ( ter1 + and est1 + ) and two independent clones ( a and b ) deleted for ter1 + or est1 + grown for ~ 25 + 10 ( ~ 35 ) , ~ 25 + 14 ( ~ 39 ) and ~ 25 + 18 ( ~ 43 ) pds in YES medium . PCRs were performed with the subtelomeric oligonucleotide oF5 , located 620 bp upstream of the first telomeric repeat , and the oligo ( dG ) - containing oligonucleotide odG18 . Marker ( m ) molecular weights are on the left in kilobases . tels : telomere - containing Telo - PCR products . D qRT - PCR analysis of G - rich TERRA , polyA + TERRA and ARRET / αARRET levels in cells as in ( B ) . Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains ( ter1 + and est1 + ) grown in parallel . Bars and error bars are averages and SD from 4 ( ter1Δ clones ) or 3 ( est1Δ clones ) independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to wt ; two - tailed Student ' s t test ) . E ChIP experiments with anti - RNA polymerase II ( RPII ) antibodies and extracts from ter1 + and ter1Δ cells grown for ~ 43 pds . Quantitative PCRs were performed using oligonucleotides oF1 + oR1 ( subtel ) or oligonucleotides amplifying a region from the actively transcribed act1 + locus . Values are expressed as fold increase over ter1 + . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 ( relative to ter1 + ; two - tailed Student ' s t test ) . The western blot to the right shows no difference in RNPII levels between ter1 + and ter1Δ cells used in ChIP experiments . Ponceau Sstaining serves as loading control . G Western blot analysis of cellular fractions from ter1 + and ter1Δ cells grown for ~ 43 pds . Total proteins ( tot ) and equivalents of soluble ( sol ) and insoluble ( ins ) proteins were loaded . Actin serves as a control for soluble proteins , H3K9ac for chromatin - bound proteins . H qRT - PCR analysis of total TERRA and polyA + TERRA in cellular fractions as in ( G ) . Values are expressed after normalization to total cellular extracts . Bars and error bars are averages and SD from 3 independent experiments .

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

Figure 1B Growth curves of the indicated strains shown as population doublings (pds) on the y axis and hours in liquid culture on the x axis. Before inoculation in liquid YES medium, single cells underwent approximately 25 pds to produce visible colonies on solid medium. For each strain, averages and SDs (error bars) of three biological replicates are shown. Arrows indicate times of harvesting and corresponding pds for experiments shown in (C) and (D).C Telo-PCR analysis of telomere length in wt (ter1+ and est1+) and two independent clones (a and b) deleted for ter1+ or est1+ grown for ~ 25+10 (~ 35), ~ 25+14 (~ 39) and ~ 25+18 (~ 43) pds in YES medium. PCRs were performed with the subtelomeric oligonucleotide oF5, located 620 bp upstream of the first telomeric repeat, and the oligo(dG)-containing oligonucleotide odG18. Marker (m) molecular weights are on the left in kilobases. tels: telomere-containing Telo-PCR products.D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student's t test).E ChIP experiments with anti-RNA polymerase II (RPII) antibodies and extracts from ter1+ and ter1Δ cells grown for ~ 43 pds. Quantitative PCRs were performed using oligonucleotides oF1 + oR1 (subtel) or oligonucleotides amplifying a region from the actively transcribed act1+ locus. Values are expressed as fold increase over ter1+. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to ter1+; two-tailed Student's t test). The western blot to the right shows no difference in RNPII levels between ter1+ and ter1Δ cells used in ChIP experiments. Ponceau Sstaining serves as loading control.G Western blot analysis of cellular fractions from ter1+ and ter1Δ cells grown for ~ 43 pds. Total proteins (tot) and equivalents of soluble (sol) and insoluble (ins) proteins were loaded. Actin serves as a control for soluble proteins, H3K9ac for chromatin-bound proteins.H qRT-PCR analysis of total TERRA and polyA+ TERRA in cellular fractions as in (G). Values are expressed after normalization to total cellular extracts. Bars and error bars are averages and SD from 3 independent experiments.


Figure 2A RIP experiments were performed using anti - myc antibodies and extracts from Trt1 - myc expressing strains followed by qRT - PCR to detect G - rich TERRA , polyA + TERRA , ARRET , TER1 and ACT1 . Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged ( unt ) wt strain . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - tailed Student ' s t test ) . B RIP experiments as in ( A ) where , prior to RNA extraction , washed beads were resuspended in DNaseI buffer ( DNbuff ) and incubated for 1 hour at 30 ° C in presence or absence of DNaseI . Values represent fractions of input polyA + TERRA detected in immunoprecipitated material expressed as fold increase over an untagged ( unt ) wt strain . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - tailed Student ' s t test ) . C RIP experiments were performed using anti - myc antibodies and extracts from Trt1 - myc expressing strains ( either ter1 + or ter1Δ ) cultured in YES medium for ~ 43 pds . Values represent polyA + TERRA detected in immunoprecipitated material expressed as a fraction of the input ( graph on the left ) or after normalization to ACT1 RNA in the corresponding input ( on the right ) , and expressed as fold increase over an untagged wt strain ( unt ter1 + ) . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to unt ter1 + ; two - tailed Student ' s t test ) . D Western blot analysis of Trt1 - myc in cells used in ( C ) . Ponceau S staining serves as a loading control .

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

Figure 2A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test).D Western blot analysis of Trt1-myc in cells used in (C). Ponceau S staining serves as a loading control.


Figure 3B Genomic DNA from wt cells ( CAF13 ) and cells carrying two tiTELs ( CAF110 ) maintained in YES medium was digested with HindIII and Southern blot - hybridized consecutively with the indicated probes . RET refers to telomeric sequences ( tiTELs or natural telomeres , nTELs ) retained in the upper part of the gel ; REL refers to telomeric sequences released in the lower part of the gel . The black arrow points to the natural nmt1 + sequence on chromosome III . Marker molecular weights are on the left in kilobases . D qRT - PCR analysis of tiTERRA in cells carrying no tiTEL ( CAF13 ) , one tiTEL ( CAF545 ) or two tiTELs ( CAF110 ) cultured in EMM medium in presence or absence of Thiamine ( THI ; repressed and induced condition , respectively ) for 24 h . RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA ( graph on the left ) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA ( on the right ) . Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions ( THI + ) . Bars and error bars are averages and SD from 3 independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to CAF545 THI + ; two - tailed Student ' s t test ) . E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs ( CAF110 ) treated with combinations of THI and Trichostatin A ( TSA ) for 24 h . Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C - rich ( oC ) or G - rich ( oG ) telomeric repeats . To confirm similar hybridization efficiencies of the two oligonucleotides , northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA ( upper right insets ) . After signal detection , RNAmembranes were stripped and re - hybridized with U6 probes to assure equal loading . Marker molecular weights are on the left in kilobases .

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

Figure 3B Genomic DNA from wt cells (CAF13) and cells carrying two tiTELs (CAF110) maintained in YES medium was digested with HindIII and Southern blot-hybridized consecutively with the indicated probes. RET refers to telomeric sequences (tiTELs or natural telomeres, nTELs) retained in the upper part of the gel; REL refers to telomeric sequences released in the lower part of the gel. The black arrow points to the natural nmt1+ sequence on chromosome III. Marker molecular weights are on the left in kilobases.D qRT-PCR analysis of tiTERRA in cells carrying no tiTEL (CAF13), one tiTEL (CAF545) or two tiTELs (CAF110) cultured in EMM medium in presence or absence of Thiamine (THI; repressed and induced condition, respectively) for 24 h. RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA (graph on the left) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA (on the right). Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions (THI+). Bars and error bars are averages and SD from 3 independent experiments. * P < 0.05, ** P < 0.01 (relative to CAF545 THI+; two-tailed Student's t test).E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs (CAF110) treated with combinations of THI and Trichostatin A (TSA) for 24 h. Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C-rich (oC) or G-rich (oG) telomeric repeats. To confirm similar hybridization efficiencies of the two oligonucleotides, northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA (upper right insets). After signal detection, RNAmembranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.