( B - C ) Represented of interaction between TLR2 TLR6 ( A ) between F4 / 80 TLR2 ( B ) in BM - derived macrophages with indicated treatments were observed by Resonance Energy Transfer ( FRET ) using ImageStreamX . Scale bars 10μm . were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed incubated with 500ng / ml LTA for another 0 . 5h at 370C . were probed with anti TLR6 - PE or anti F4 / 80 - PE conjugated antibody ( donor ) anti TLR2 antibody following by with APC - labeled secondary antibody ( acceptor ) . PE ( middle panel ) FRET ( right panel ) were measured via ImageStreanX imaging flow cytometer . FRET shown are mean ± S . E . M of two independent experiments ( n = 2579 - 15567 ) .

O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567).


( D ) Represented image of ERK1 , 2 phosphorylation in CD115 + BM monocytes upon LTA exposure . were pre - treated for 0 . 5h with 20µM of TLR2 - p peptide , scrTLR2 - p peptide or untreated , then washed incubated with 500ng / ml LTA for indicated times . ERK1 , 2 phosphorylation total ERK1 , 2 were detected by western blotting . Equal loading was detected by measuring anti tubulin . Results are representative data of 2 independent experiments . bands was quantified for p - ERK after 15min of LTA exposure . Results are normalized to tubulin are mean of 2 experiments ± S . D . M .

O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O

(D) Represented image of ERK1,2 phosphorylation levels in CD115+BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.


( B ) Representative colonoscopy of indicated treatments ( day 7 ) . C57BL mice were treated with 2 % of dextran sulfate sodium ( DSS ) in drinking water for 7 days . An amount of 5mg / Kg of TLR2 - p scrTLR2 - p peptides were injected IP every following day from day one . ( C ) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only , DSS with TLR2 - p peptide , DSS with scrTLR2 - p peptide untreated mice ( n = 19 , 21 , 22 19 respectively ) . Results represent 3 independent experiments , scale bars 100µm 1000μm .

O O O O B-EXP_ASSAY O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5mg/Kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one.(C) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide and untreated mice (n= 19, 21, 22 and 19 respectively). Results represent 3 independent experiments, scale bars 100µm and 1000μm.


( D ) A graphical summary of changes in ( day 6 ) of indicated groups .

(D) A graphical summary of changes in body weight (day 6) of the indicated groups.


( E ) Representative H & amp ; amp ; E histological for distal colon section ( day 7 ) of 9 - 10 sections from 2 independent experiments . Black asterisk indicates on large collection of blood in lumen , arrowheads indicate on widespread mucosal collapse ulceration arrows indicates on edema in sub mucosa . ( F ) A graphical summary of histological severity score of indicated treatments .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

(E) Representative H&amp;E histological images for distal colon section (day 7) of 9-10 sections from 2 independent experiments. Black asterisk indicates on large collection of blood in the lumen, arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa.(F) A graphical summary of histological severity score of the indicated treatments.


( A - B ) samples of colon tissue were extracted from mice with indicated treatments . ( A ) Cytokine of IL - 6 , IL - 1β INF - λ were determined by ELISA ( n = 4 - 7 ) . Results represent two or more independent experiments .

O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β and INF-λ were determined by ELISA (n=4-7). Results represent two or more independent experiments.


( A - B ) samples of colon tissue were extracted from mice with indicated treatments . ( B ) mRNA expression of IL - 6 , IL - 12p35 IL - 23p19 were assessed by quantitative RT - PCR normalized to TBP ( n = 4 - 6 ) . Results represent two or more independent experiments .

O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O

(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (B) mRNA expression levels of IL-6, IL-12p35 and IL-23p19 were assessed by quantitative RT-PCR and normalized to TBP (n=4-6). Results represent two or more independent experiments.


( A ) Flow cytometry of colonic lamina propria CD11c - CD11b + LY6Chi MHCII - monocytes from steady - state DSS day 7 treated with TLR2 peptide or scrTLR2 peptide , showing comparable monocytes infiltrates in to colitic colon . Plots were pre gated on live CD45 + . Results are representative of three independent experiments ( n = 3 per group ) . ( B ) Graphical summary of monocytes presented as % out of CD11b + .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O

(A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.


( B ) loss of DSS challenged mice on day 6 .

O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O

(B) Weight loss of DSS challenged mice on day 6.


( C ) Real - time PCR showing mRNA expression of IL - 6 , IL - 12p35 IL - 23p19 of FACS isolated monocytes collected from mice with indicated treatments . Data are obtained from two to three independent experiments ( n = 6 - 7 mice pooled per group ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(C) Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35 and IL-23p19 of FACS isolated monocytes collected from mice with the indicated treatments. Data are obtained from two to three independent experiments (n=6-7 mice pooled per group).


E , F : EM of negatively stained Dam1c alone ( E ) in presence of Bim1 ( F ) , at low salt conditions . final concentrations of Dam1c Bim1 were 0 . 3 µM 0 . 1 µM , respectively . scale bars are 100 nm , boxes mark representative complexes shown as magnified inserts .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.


B : Chromatogram SDS - PAGE of elution fractions from gel filtration . Binding of Bim1 to Dam1 ∆ SxIPc lacking SxIP motif in C - terminus of Duo1 was examined by analytical exclusion chromatography . Elution fractions were analyzed by SDS - PAGE .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

B: Chromatogram and SDS-PAGE of elution fractions from gel filtration. Binding of Bim1 to Dam1∆SxIPc lacking the SxIP motif in the C-terminus of Duo1 was examined by analytical size exclusion chromatography. Elution fractions were analyzed by SDS-PAGE.


C : Solid phase binding to for binding of different Dam1 complexes to Bim1 . Recombinant GST - Bim1185 - 344 was immobilized on glutathione sepharose beads incubated with either recombinant Dam1WTc or Dam1 ∆ SxIPc . Input pull down samples were analyzed by SDS - PAGE .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

C: Solid phase binding assay to test for binding of different Dam1 complexes to Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.


D : Pull down to compare binding of Dam1WTc Dam1 ∆ SxIPc to immobilized GST - Bim1185 - 344 . Soluble lysates from yeast strains expressing respective Duo1 - 6xFlag alleles were incubated with GST - Bim1185 - 344 immobilized on beads . Duo1 - 6xFlag bound to GST - Bim1185 - 344 was analyzed by western blot . An anti - Pgk1 antibody was used to confirm equal amounts in input samples .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O

D: Pull down assay to compare binding of Dam1WTc and Dam1∆SxIPc to immobilized GST-Bim1185-344. Soluble cell lysates from yeast strains expressing the respective Duo1-6xFlag alleles were incubated with GST-Bim1185-344 immobilized on beads. Duo1-6xFlag bound to GST-Bim1185-344 was analyzed by western blot. An anti-Pgk1 antibody was used to confirm equal protein amounts in input samples.


C : Representative from live microscopy of Dad1 - GFP strains with Duo1WT - 6xFlag or Duo1 ∆ SxIP - 6xFlag . Displayed are in either metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . D : of signal intensities of Dad1 - GFP at metaphase anaphase kinetochore clusters of Duo1WT - 6xFlag Duo1 ∆ SxIP - 6xFlag strains . Bars represent mean + / - standard deviation . Each spot represents value measured for a single kinetochore cluster . Values were normalized to mean value of metaphase clusters of Duo1WT - 6xFlag . A one - way ANOVA with Sidak ' s multiple comparisons was used to calculate p values . n ≥ 76 kinetochore clusters .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm. D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters.


F : of cycle progression of yeast strains with different Duo1 alleles . Accumulation degradation of Pds1 after release from α factor arrest was analyzed by western blot ( left ) . An antibody against Pgk1 was used to confirm equal amounts in samples . content of population at indicated time points after release from α factor arrest was analyzed by FACS ( right ) . in G1 phase mitosis are characterized by a 1C 2C content , respectively . An intermediate content is detected for in S phase .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F: Analysis of cell cycle progression of yeast strains with different Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content, respectively. An intermediate DNA content is detected for cells in S phase.


B : Representative from live microscopy of Dam1WT dam1 - 19 strains with Dad1 - GFP . Displayed are in metaphase ( left ) or anaphase ( right ) . Scale bar : 2 μm . C : of Dad1 - GFP signal intensities at metaphase anaphase kinetochore clusters of Dam1WT dam1 - 19 strains . Bars represent mean + / - standard deviation . Each spot represents value measured for a single kinetochore cluster . Values were normalized to mean value of metaphase clusters of Dam1WT . p - values were obtained by a one - way ANOVA with Sidak ' s multiple comparisons . n ≥ 86 kinetochore clusters .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O 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: Representative images from live cell microscopy of Dam1WT and dam1-19 strains with Dad1-GFP. Displayed cells are in metaphase (left) or anaphase (right). Scale bar: 2 μm. C: Quantification of Dad1-GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1-19 strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Dam1WT strain. p-values were obtained by a one-way ANOVA test with Sidak's multiple comparisons test. n ≥ 86 kinetochore clusters.


E , F : GST - Bim1185 - 344 pull down assays . Binding of immobilized GST - Bim1185 - 344 to Duo1 - 6xFlag was analyzed in context of ( E ) selective inhibition of Mps1 ( mps1 - 737 ) or overexpression of Mps1 from a galactose - inducible promotor ( F ) . For inhibition of Mps1 , were grown at restrictive of 37 ° C . Overexpression of Mps1 was achieved by growing in medium containing galactose . GST - Bim1185 - 344 immobilized on glutathione sepharose beads were incubated with soluble lysates of respective strains grown under mentioned conditions . Binding of Duo1 was analyzed by western blot . Pgk1 indicates equal amounts in samples .

O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O

E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1 (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing galactose. GST-Bim1185-344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by western blot. Pgk1 indicates equal protein amounts in all samples.


G : Live microscopy of a Dad1 - GFP under conditions of Mps1 overexpression . localization of Dam1c was visualized by Dad1 - GFP in either an Mps1WT or Mps1 overexpression setup ( pGal - Mps1 ) . Ndc80 - mRuby2 was used as reference point for localization of kinetochores . show representative metaphase from both strains . in dashed box is shown as magnification in lower left corner . arrowheads point at microtubules decorated by Dad1 - GFP . Please note that brightness contrast of Dad1 - GFP image in lower row were optimized to make microtubules clearly visible . Scale bar : 2 μm .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O

G: Live cell microscopy of a Dad1-GFP strain under conditions of Mps1 overexpression. The localization of Dam1c was visualized by Dad1-GFP in either an Mps1WT or Mps1 overexpression setup (pGal-Mps1). Ndc80-mRuby2 was used as reference point for the localization of kinetochores. Images show representative metaphase cells from both strains. The area in the white dashed box is shown as magnification in the lower left corner. White arrowheads point at nuclear microtubules decorated by Dad1-GFP. Please note that brightness and contrast of the Dad1-GFP image in the lower row were optimized to make the nuclear microtubules clearly visible. Scale bar: 2 μm.


A , B : 5 µM , 7 . 5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1 - Bik1 were analyzed by exclusion chromatography . SDS - PAGE of elution fractions is shown in B . High molecular species of Dam1WTc were eluted after 0 . 9 - 1 . 0 ml ( see chromatogram in A ) .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O

A, B: 5 µM, 7.5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1-Bik1 complex were analyzed by size exclusion chromatography. SDS-PAGE analysis of elution fractions is shown in B. High molecular weight species of Dam1WTc were eluted after 0.9 - 1.0 ml (see chromatogram in A).


B : Pull down for of Dam1c - Bim1 binding to Ndc80c . Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre - formed Dam1c - Bim1 with increasing amounts of Bim1 . Input elution samples were analyzed by SDS - PAGE . LC HC denote heavy chains of M2 anti - Flag antibody used for immobilization of Ndc80c on beads . names in blue , red green refer to Dam1c , Ndc80c Bim1 , respectively .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O

B: Pull down assay for analysis of Dam1c-Bim1 binding to Ndc80c. Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre-formed Dam1c-Bim1 complex with increasing amounts of Bim1. Input and elution samples were analyzed by SDS-PAGE. LC and HC denote the light and heavy chains of the M2 anti-Flag antibody used for immobilization of Ndc80c on beads. Protein names in blue, red and green refer to Dam1c, Ndc80c and Bim1, respectively.


D : Serial dilution of yeast strains with different Duo1 Ndc80 alleles in an Ndc80 - FRB - GFP background . Ndc80WT or Ndc80 ∆ 490 - 510 integration constructs were integrated at LEU2 locus combined with either Duo1WT or Duo1 ∆ SxIP allele ( at DUO1 locus ) . were spotted on YEPD medium without with 1 µg / ml Rapamycin incubated at indicated temperatures .

O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

D: Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures.


B : Serial dilution of yeast strains with 6xFlag - tagged Duo1WT or Duo1 ∆ SxIP Ask1WT , Ask12A or Ask12D . were grown at indicated temperatures were taken two days ( 25 ° C , 30 ° C 37 ° C ) or four days ( 16 ° C ) after spotting .

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B: Serial dilution assay of yeast strains with 6xFlag-tagged Duo1WT or Duo1∆SxIP and Ask1WT, Ask12A or Ask12D. Cells were grown at the indicated temperatures and images were taken two days (25 °C, 30 °C and 37 °C) or four days (16 °C) after spotting.


a , alpha - KG extends lifespan of adult worms in metabolite longevity screen . metabolites were given at a of 8 mM .

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a, alpha-KG extends the lifespan of adult worms in the metabolite longevity screen. All metabolites were given at a concentration of 8 mM.


c , Dose - response curve of α - KG effect on longevity .

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c, Dose-response curve of the α-KG effect on longevity.


d , e , α - KG extends lifespan of worms fed that have been ampicillin arrested , mean lifespan ( days of adulthood ) with vehicle treatment ( mveh ) = 19 . 4 ( n = 80 tested ) , mα - KG = 25 . 1 ( n = 91 ) , P 0 . 0001 ( log - rank ) ( d ) ; or γ - irradiation - killed , mveh = 19 . 0 ( n = 88 ) , mα - KG = 23 . 0 ( n = 46 ) , P 0 . 0001 ( log - rank ) ( e ) . OP50 , E . coli OP50 .

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d, e, α-KG extends the lifespan of worms fed bacteria that have been ampicillin arrested, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 19.4 (n = 80 animals tested), mα-KG = 25.1 (n = 91), P  0.0001 (log-rank test) (d); or γ-irradiation-killed, mveh = 19.0 (n = 88), mα-KG = 23.0 (n = 46), P  0.0001 (log-rank test) (e). OP50, E. coli OP50 strain.


f alpha - KG does not further extend lifespan of ogdh - 1 RNAi worms , mveh = 21 . 2 ( n = 98 ) , mα - KG = 21 . 1 ( n = 100 ) , P = 0 . 65 ( log - rank ) .

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f alpha-KG does not further extend the lifespan of ogdh-1 RNAi worms, mveh = 21.2 (n = 98), mα-KG = 21.1 (n = 100), P = 0.65 (log-rank test).


a , DARTS identifies ATP5B as an alpha - KG - binding . Red arrowhead , protected band .

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a, DARTS identifies ATP5B as an alpha-KG-binding protein. Red arrowhead, protected band.


b , DARTS confirms alpha - KG binding specifically to ATP5B . IB , immunoblot .

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b, DARTS confirms alpha-KG binding specifically to ATP5B. IB, immunoblot.


c , Inhibition of ATP synthase by alpha - KG ( released from octyl alpha - KG ; Supplementary Notes ) . This inhibition was reversible ( data not shown ) .

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c, Inhibition of ATP synthase by alpha-KG (released from octyl alpha-KG; Supplementary Notes). This inhibition was reversible (data not shown).


d , e , Reduced ATP in octyl alpha - KG - treated normal humanfibroblasts ( * * P = 0 . 0016 , * * * * P 0 . 0001 ; by t - , two - , two - sample unequal variance ) ( d )

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d, e, Reduced ATP levels in octyl alpha-KG-treated normal humanfibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d)


d , e , Reduced ATP in octyl alpha - KG - treated worms ( day 2 , P = 0 . 969 ; day 8 , * P = 0 . 012 ; by t - , two - , two - sample unequal variance ) ( e ) . RLU , relative luminescence units .

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d, e, Reduced ATP levels in octyl alpha-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units.


f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated ( * * * P = 0 . 0004 , * * * * P 0 . 0001 ; by t - , two - , two - sample unequal variance ) ( f )

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f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f)


f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated worms ( P 0 . 0001 ; by t - , two - , two - sample unequal variance ) ( g ) .

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f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g).


h , alpha - KG , released from octyl alpha - KG ( 800 µM ) , decreases state 3 , but not state 4o or 3u ( P = 0 . 997 ) , respiration in mitochondria isolated from mouse liver . respiratory control is decreased in octyl alpha - KG - ( 3 . 1 ± 0 . 6 ) versus vehicle - treated mitochondria ( 5 . 2 ± 1 . 0 ) ( * P = 0 . 015 ; by t - , two - , two - sample unequal variance ) . Oligo , oligomycin ; FCCP , carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ; AA , antimycin A .

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h, alpha-KG, released from octyl alpha-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl alpha-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A.


i , Eadie - Hofstee plot of steady - state inhibition kinetics of ATP synthase by alpha - KG ( produced by in situ hydrolysis of octyl alpha - KG ) . [ S ] is substrate ( ADP ) , V is initial velocity of ATP synthesis in presence of 200 µM octanol ( vehicle control ) or octyl alpha - KG . alpha - KG ( produced from octyl alpha - KG ) decreases apparent Vmax ( 53 . 9 to 26 . 7 ) Km ( 25 . 9 to 15 . 4 ) , by nonlinear regression least - squares fit . c - i , Results were replicated in two independent experiments . Mean ± standard deviation ( s . d . ) is plotted in cases .

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i, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by alpha-KG (produced by in situ hydrolysis of octyl alpha-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl alpha-KG. alpha-KG (produced from octyl alpha-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c-i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.


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a-g, Effect of alpha-KG on the lifespan of mutant or RNAi worms. a, atp-2 RNAi, mveh = 22.8 (n = 97), mα-KG = 22.5 (n = 94), P = 0.35; or RNAi control, mveh = 18.6 (n = 94), mα-KG = 23.4 (n = 91), P  0.0001. b, daf-2(e1370), mveh = 38.0 (n = 72), mα-KG = 47.6 (n = 69), P  0.0001. c, eat-2(ad1116), mveh = 22.8 (n = 59), mα-KG = 22.9 (n = 40), P = 0.79. d, let-363 RNAi, mveh = 25.1 (n = 96), mα-KG = 25.7 (n = 74), P = 0.95; or gfp RNAi control, mveh = 20.2 (n = 99), mα-KG = 27.7 (n = 81), P  0.0001. e, daf-16(mu86), mveh = 13.4 (n = 71), mα-KG = 17.4 (n = 72), P  0.0001; or N2, mveh = 13.2 (n = 100), mα-KG = 22.3 (n = 104), P  0.0001. f, pha-4(zu225), mveh = 14.2 (n = 94), mα-KG = 13.5 (n = 109), P = 0.55. g, hif-1(ia4), mveh = 20.5 (n = 85), mα-KG = 26.0 (n = 71), P  0.0001; or N2, mveh = 21.5 (n = 101), mα-KG = 24.6 (n = 102), P  0.0001. P values were determined by the log-rank test. Number of independent experiments: RNAi control (6), atp-2 (2), let-363 (3), N2 (5), daf-2 (2), eat-2 (2), pha-4 (2), daf-16 (2), hif-1 (5).


a , Decreased phosphorylation of mammalian TOR substrates in U87 treated with octyl alpha - KG or oligomycin . Similar results were obtained in HEK - 293 , normal human fibroblasts mouse embryonic fibroblasts ( data not shown ) . P , phospho .

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a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl alpha-KG or oligomycin. Similar results were obtained in HEK-293 cells, normal human fibroblasts and mouse embryonic fibroblasts (data not shown). P, phospho.


b , Increased autophagy in treated with alpha - KG or RNAi for atp - 2 or let - 363 . were taken at × 100 magnification . c , GFP : : LGG - 1 quantified using ImageJ ( Methods ) . Data show results of 2 - 3 independent experiments . Bars indicate mean . * * * * P 0 . 0001 ; NS , not significant ( t - , two - , two - sample unequal variance ) .

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b, Increased autophagy in animals treated with alpha-KG or RNAi for atp-2 or let-363. Photographs were taken at ×100 magnification. c, GFP::LGG-1 puncta quantified using ImageJ (Methods). Data show results of 2-3 independent experiments. Bars indicate the mean. ****P  0.0001; NS, not significant (t-test, two-tailed, two-sample unequal variance).


d , alpha - KG are increased in starved worms . * * P 0 . 01 ( t - , two - , two - sample unequal variance ) . Mean ± s . d . is plotted .

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d, alpha-KG levels are increased in starved worms. **P  0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted.


A SHP1immunoprecipitation ( IP ) from Jurkat 1G4 ‐ CD8 . Upper panel of immunoblots shows expression in input lysates , isolated complexes are shown at bottom . Rabbit IgGIP ( isotype ) is shown as control .

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A SHP1immunoprecipitation (IP) from Jurkat 1G4‐CD8 cells. Upper panel of immunoblots shows expression levels in the input lysates, and isolated protein complexes are shown at the bottom. Rabbit IgGIP (isotype) is shown as control.


B Lentiviral knock ‐ down / re ‐ expression constructs for THEMIS ‐ wt ‐ Strep THEMIS ‐ dPRR1 ‐ Strep in 1G4 ‐ CD8 . Knock ‐ down / re ‐ expression constructs are on shTHEMIS ‐ 128476 . Red arrowheads indicate specific signal for endogenous THEMIS re ‐ expressed THEMIS ‐ Strep . higher molecular band observed in lanes is a non ‐ specific signal .

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B Lentiviral knock‐down/re‐expression constructs for THEMIS‐wt‐Strep and THEMIS‐dPRR1‐Strep in 1G4‐CD8 cells. Knock‐down/re‐expression constructs are based on shTHEMIS‐128476. Red arrowheads indicate the specific signal for endogenous THEMIS and re‐expressed THEMIS‐Strep. The higher molecular weight band observed in all lanes is a non‐specific signal.


C Streptactinpull ‐ downs ( PDs ) of THEMIS ‐ Strep from 1G4 ‐ CD8 . as described in ( B ) were stimulated with 6V tetramers , followed by Streptactin PDs of THEMIS immunoblot of isolated complexes . Separate experiments for SHP1 SHP2 are shown .

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C Streptactinpull‐downs (PDs) of THEMIS‐Strep from 1G4‐CD8 cells. Cells as described in (B) were stimulated with 6V tetramers, followed by Streptactin PDs of THEMIS and immunoblot analysis of isolated protein complexes. Separate experiments for SHP1 and SHP2 are shown.


D SHP1IP in presence absence of SHP1 tail phosphorylation . SHP1 was immunoprecipitated from J . CaM1 . 6LCK ‐ Tet . Where indicated , LCK expression was induced with doxycycline prior to experiment or Src ‐ kinase inhibitor PP2 was used to abolish residual SHP1 ‐ pY564 . upper panel of immunoblots shows effect of LCK activity on SHP1 ‐ pY564 . red arrowhead indicates residual pY564 . Isolated complexes are shown in lower panels .

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D SHP1IP in the presence and absence of SHP1 tail phosphorylation. SHP1 was immunoprecipitated from J.CaM1.6LCK‐Tet cells. Where indicated, LCK expression was induced with doxycycline prior to the experiment or Src‐kinase inhibitor PP2 was used to abolish residual SHP1‐pY564. The upper panel of immunoblots shows the effect of LCK activity on SHP1‐pY564 levels. The red arrowhead indicates residual pY564. Isolated protein complexes are shown in the lower panels.


E In vitro phosphatase treatment of THEMIS : GRB2 : SHP1 . Bead ‐ bound THEMIS ‐ Strep complexes from 1G4 ‐ CD8 were incubated in alkaline phosphatase ( AP ) buffer in absence ( lane 1 ) or presence of AP ( lane 2 ) , or left completely untreated ( lane 3 ) , prior to washing elution .

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E In vitro phosphatase treatment of the THEMIS:GRB2:SHP1 complex. Bead‐bound THEMIS‐Strep complexes from 1G4‐CD8 cells were incubated in alkaline phosphatase (AP) buffer in the absence (lane 1) or presence of AP (lane 2), or left completely untreated (lane 3), prior to washing and elution.


F Anti ‐ HAIP from HEK293 transfected with HA ‐ SHP1 GRB2 ‐ Myc constructs . GRB2 mutants used : W36K , N ‐ SH3 mutant ; W193K , C ‐ SH3 mutant . Upper panels show expression in input ; isolated complexes are shown at bottom . Relative amounts of GRB2 ‐ Myc are normalized to bait HA ‐ SHP1 .

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F Anti‐HAIP from HEK293 cells transfected with HA‐SHP1 and GRB2‐Myc constructs. GRB2 mutants used: W36K, N‐SH3 mutant; W193K, C‐SH3 mutant. Upper panels show expression levels in the input; isolated proteins complexes are shown at the bottom. Relative amounts of GRB2‐Myc are normalized to the bait HA‐SHP1.


G Far ‐ Western blot of GRB2 ‐ SH3 domains binding to full ‐ SHP1 . HA IPs from empty vector or HA ‐ SHP1 ‐ transfected HEK293 were subjected to far ‐ Western blotting using recombinant GST ‐ tagged N ‐ or C ‐ SH3 domains of GRB2 . GST alone served as a background control . Blots were re ‐ probed for SHP1 loading for normalization of anti ‐ GST signals . Data shown are representative of three independent experiments .

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G Far‐Western blot of GRB2‐SH3 domains binding to full‐length SHP1. HA IPs from empty vector or HA‐SHP1‐transfected HEK293 cells were subjected to far‐Western blotting using recombinant GST‐tagged N ‐or C‐SH3 domains of GRB2. GST alone served as a background control. Blots were re‐probed for SHP1 loading for normalization of anti‐GST signals. Data shown are representative of three independent experiments.


A Lentiviral knock ‐ down ( KD ) of THEMIS expression in 1G4 ‐ CD8 . efficiency of THEMIS KD was assessed using immunoblotting .

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A Lentiviral knock‐down (KD) of THEMIS expression in 1G4‐CD8 cells. The efficiency of the THEMIS KD was assessed using immunoblotting.


B Phospho ‐ flow cytometry of pERK responses in THEMIS KD . 1G4 ‐ CD8 control THEMIS KD were stimulated with NY ‐ ESO ‐ 1 pMHC tetramers analysed for ERK phosphorylation using flow cytometry . Data shown are representative of three independent experiments . C of pERK ‐ positive from ( B ) . Data from three independent experiments were used in . n = 3 , means ± are shown ; two ‐ unpaired Student ' s t ‐ , * P 0 . 05 , * * P 0 . 01 .

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B Phospho‐flow cytometry analysis of pERK responses in THEMIS KD cells. 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for ERK phosphorylation using flow cytometry. Data shown are representative of three independent experiments.C Quantification of pERK‐positive cells from (B). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01.


D Phospho ‐ flow cytometry of pERK responses against titrated concentrations of 9V tetramer . Time point was fixed at 60 s . Data shown are representative of three independent experiments . E of pERK ‐ positive from ( D ) . Data from three independent experiments were used in . n = 3 , means ± are shown .

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D Phospho‐flow cytometry analysis of pERK responses against titrated concentrations of 9V tetramer. Time point was fixed at 60 s. Data shown are representative of three independent experiments.E Quantification of pERK‐positive cells from (D). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown.


A 1G4 ‐ CD8 control THEMIS KD were stimulated with NY ‐ ESO ‐ 1 pMHC tetramers analysed for pY142 ‐ CD3 ‐ ζ phosphorylation by flow cytometry . Data shown are representative of three independent experiments . B of pY142 ‐ CD3 ‐ ζ responses of from ( A ) . Data from three independent experiments were used in . Responses are expressed as of positive control sodium pervanadate . n = 3 , means ± are shown ; two ‐ unpaired Student ' s t ‐ , * * P 0 . 01 .

O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for pY142‐CD3‐ζ phosphorylation by flow cytometry. Data shown are representative of three independent experiments.B Quantification of pY142‐CD3‐ζ responses of cells from (A). Data from three independent experiments were used in the analysis. Responses are expressed as percentage of the positive control sodium pervanadate. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, **P 0.01.


A Lentiviral knock ‐ down of THEMIS expression in humanCD4 + T . Following transduction , were expanded on CD3 / CD28 beads in presence of exogenous IL ‐ 2 analysed for THEMIS KD by immunoblotting on day 4 post ‐ transduction . Data shown are representative of three independent experiments .

O B-ORGANISM O O O O B-GENEPROD O O B-ORGANISM O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

A Lentiviral knock‐down of THEMIS expression in primary humanCD4+T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2 and analysed for THEMIS KD by immunoblotting on day 4 post‐transduction. Data shown are representative of three independent experiments.


B pERK response in THEMIS KD humanCD4 + T . Transduced CD4 + were removed from beads , rested overnight stimulated with CD3 mAb for indicated time points . ERK phosphorylation was assessed by immunoblotting . Data shown are representative of three independent experiments .

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B pERK response in THEMIS KD humanCD4+T cells. Transduced CD4+ cells were removed from beads, rested overnight and stimulated with CD3 mAb for the indicated time points. ERK phosphorylation was assessed by immunoblotting. Data shown are representative of three independent experiments.


C Expression of CD25 CD69 surface markers of THEMIS KD humanCD4 + T was assessed on day 4 post ‐ transduction by flow cytometry . Data shown are representative of three independent experiments .

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C Expression of CD25 and CD69 surface markers of THEMIS KD humanCD4+T cells was assessed on day 4 post‐transduction by flow cytometry. Data shown are representative of three independent experiments.


D CD69 up ‐ regulation on re ‐ stimulated THEMIS KD CD4 + T . CD3 / CD28 beads were removed on day 1 post ‐ transduction , were cultured in IL ‐ 2 IL ‐ 7 . After removal of debris at day 4 , were restimulated with CD3 / CD28 beads at indicated bead to . CD69 surface expression was assessed by flow cytometry 24 h later . Data shown are representative of two independent experiments . n = 3 , means ± SD are shown ; two ‐ unpaired Student ' s t ‐ , * P 0 . 05 , * * * P 0 . 001 .

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D CD69 up‐regulation on re‐stimulated THEMIS KD CD4+T cells. CD3/CD28 beads were removed on day 1 post‐transduction, and cells were cultured in IL‐2 and IL‐7. After removal of cell debris at day 4, cells were restimulated with CD3/CD28 beads at the indicated bead to cell ratio. CD69 surface expression was assessed by flow cytometry 24 h later. Data shown are representative of two independent experiments. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, ***P 0.001.


A Donor ‐ derived humanCD4 + T transduced as in Fig A were analysed for apoptosis by Annexin ‐ V surface on day 4 post ‐ transduction . Data from three independent experiments were used in . n = 3 , means ± SD are shown ; two ‐ unpaired Student ' s t ‐ , * * P 0 . 01 .

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A Donor‐derived humanCD4+T cells transduced as in Fig A were analysed for apoptosis by Annexin‐V surface staining on day 4 post‐transduction. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, **P 0.01.


B Annexin ‐ V of THEMIS KD 1G4 ‐ CD8 . THEMIS KD control 1G4 ‐ CD8 were stimulated for 24 h with plate ‐ bound 6V tetramers . Annexin ‐ V surface expression was analysed by flow cytometry is shown as fold ‐ increase over non ‐ stimulated . Data from three independent experiments were used in . n = 3 , means ± SD are shown ; two ‐ unpaired Student ' s t ‐ , * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; ns , not significant .

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B Annexin‐V staining of THEMIS KD 1G4‐CD8 cells. THEMIS KD and control 1G4‐CD8 cells were stimulated for 24 h with plate‐bound 6V tetramers. Annexin‐V surface expression was analysed by flow cytometry and is shown as fold‐increase over non‐stimulated. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01, ***P 0.001; ns, not significant.


C Poly ‐ caspase activity in THEMIS KD 1G4 ‐ CD8 . were stimulated for 24 h with NY ‐ ESO ‐ 1 pMHC tetramers . Camptothecin was used as a positive control for caspase activation . A FAM ‐ FLICA detection probe was used to assess poly ‐ caspase activity by flow cytometry . Data shown are a representative example of three independent experiments . D of poly ‐ caspase activity of from ( C ) . Data from three independent experiments were used in . n = 3 , means ± are shown ; two ‐ unpaired Student ' s t ‐ , * P 0 . 05 , * * P 0 . 01 ; ns , not significant .

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C Poly‐caspase activity in THEMIS KD 1G4‐CD8 cells. Cells were stimulated for 24 h with NY‐ESO‐1 pMHC tetramers. Camptothecin was used as a positive control for caspase activation. A FAM‐FLICA detection probe was used to assess poly‐caspase activity by flow cytometry. Data shown are a representative example of three independent experiments.D Quantification of poly‐caspase activity of cells from (C). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01; ns, not significant.


A Lentiviral KD of SHP1 expression in 1G4 ‐ CD8 . KD efficiency was assessed using immunoblotting .

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A Lentiviral KD of SHP1 expression in 1G4‐CD8 cells. KD efficiency was assessed using immunoblotting.


B Annexin ‐ V of THEMIS SHP1 KD 1G4 ‐ CD8 . Control , THEMIS SHP1 knock ‐ down Jurkat 1G4 ‐ CD8 were stimulated for 24 h with plate ‐ bound 6V tetramers . Annexin ‐ V surface expression was analysed by flow cytometry is shown as fold ‐ increase over non ‐ stimulated . Data from three independent experiments were used in . n = 3 , means ± SD are shown ; two ‐ way ANOVA , Bonferroni post ‐ , * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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B Annexin‐V staining of THEMIS and SHP1 KD 1G4‐CD8 cells. Control, THEMIS and SHP1 knock‐down Jurkat 1G4‐CD8 cells were stimulated for 24 h with plate‐bound 6V tetramers. Annexin‐V surface expression was analysed by flow cytometry and is shown as fold‐increase over non‐stimulated. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐way ANOVA, Bonferroni post‐test, *P 0.05, **P 0.01, ***P 0.001.


C Lentiviral knock ‐ down of THEMIS SHP1 expression in humanCD4 + T . Following transduction , were expanded on CD3 / CD28 beads in presence of exogenous IL ‐ 2 . Knock ‐ down efficiency was analysed by immunoblotting on day 4 post ‐ transduction .

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C Lentiviral knock‐down of THEMIS and SHP1 expression in primary humanCD4+T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2. Knock‐down efficiency was analysed by immunoblotting on day 4 post‐transduction.


D Expression of CD25 , CD69 Annexin ‐ V surface markers of from ( C ) was assessed by flow cytometry . Data shown are representative of three independent experiments .

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D Expression of CD25, CD69 and Annexin‐V surface markers of cells from (C) was assessed by flow cytometry. Data shown are representative of three independent experiments.


E Annexin ‐ V of 1G4 ‐ CD8 re ‐ expressing a SHP1 tail tyrosine mutant . Jurkat 1G4 ‐ CD8 were transduced with Knock ‐ down / re ‐ expression constructs for HA ‐ SHP1 wild ‐ type ( wt ) or Tyr536 / 564 to Phe double mutant ( YYFF ) . were stimulated analysed by flow cytometry as in ( B ) . Data shown are representative of three independent experiments . n = 3 , means ± SD are shown .

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E Annexin‐V staining of 1G4‐CD8 cells re‐expressing a SHP1 tail tyrosine mutant. Jurkat 1G4‐CD8 cells were transduced with Knock‐down/re‐expression constructs for HA‐SHP1 wild‐type (wt) or Tyr536/564 to Phe double mutant (YYFF). Cells were stimulated and analysed by flow cytometry as in (B). Data shown are representative of three independent experiments. n = 3, means ± SD are shown.


A CD8 + T from OT ‐ I Rag2 − / − mice expressing wild ‐ type ( LCK ) or LCKS59A were stimulated with H ‐ 2Kb ‐ positive antigen ‐ presenting pulsed with serial dilutions of OVA ( 257 - 264 ) peptide , OVA peptide variants Q4 T4 , VSV , a peptide that is not recognized by OT ‐ I TCR . CD69 up ‐ regulation ( left ) TCR down ‐ regulation ( right ) were analysed . Data are representative of three independent experiments .

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A CD8+T cells from OT‐I Rag2−/−mice expressing wild‐type (LCK) or LCKS59A proteins were stimulated with H‐2Kb‐positive antigen‐presenting cells pulsed with serial dilutions of the OVA (257-264) peptide, the OVA peptide variants Q4 and T4, and VSV, a peptide that is not recognized by the OT‐I TCR. CD69 up‐regulation (left) and TCR down‐regulation (right) were analysed. Data are representative of three independent experiments.


B CD8 + T from OT ‐ I Rag2 − / − mice expressing wild ‐ type ( LCK ) or LCKS59A were labelled with CFSE stimulated with serial dilution of H ‐ 2Kb ‐ OVA or H ‐ 2Kb ‐ Q4R7 peptide ‐ MHC tetramers . After 3 days , absolute numbers of divided T were determined . Data are representative of three independent experiments .

O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

B CD8+T cells from OT‐I Rag2−/−mice expressing wild‐type (LCK) or LCKS59A proteins were labelled with CFSE and stimulated with serial dilution of H‐2Kb‐OVA or H‐2Kb‐Q4R7 peptide‐MHC tetramers. After 3 days, the absolute numbers of divided T cells were determined. Data are representative of three independent experiments.


Immunofluorescent detection of Nanog Esrrb in wild - type E14Tg2a ESCs cultured for three days in GMEMβ / FCS / LIF . ( A ) Widefield of a representative colony .

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Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. (A) Widefield images of a representative colony.


: Immunofluorescent detection of Nanog Esrrb in wild - type E14Tg2a ESCs cultured for three days in GMEMβ / FCS / LIF . of mean Nanog Esrrb measured in nuclei identified by automatic segmentation of single optical plane obtained by confocal microscopy . Values are expressed in Arbitrary Units ( AU ) . Oct4 negative , also identified by immunostaining , were excluded from . red represents moving average of data distribution . Representative of three independent experiments each including at least 1000 nuclei .

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: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Values are expressed in Arbitrary Units (AU). Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. The red line represents the moving average of the data distribution. Representative of three independent experiments each including at least 1000 nuclei.


C : Colonies of TNG E - tdT ESCs showing GFP expression from Nanog Esrrb - tdTomato fusion expression from Esrrb after three days in GMEMβ / FCS / LIF .

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C: Colonies of TNG E-tdT ESCs showing GFP expression from Nanog and Esrrb-tdTomato fusion protein expression from Esrrb after three days in GMEMβ/FCS/LIF.


D : Flow cytometric of Esrrb Nanog reporter expression in SSEA1 + TNG E - tdT ESCs cultured for three days in GMEMβ / FCS / LIF with or without puromycin .

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D: Flow cytometric analysis of Esrrb and Nanog fluorescent reporter expression in SSEA1+ TNG E-tdT ESCs cultured for three days in GMEMβ/FCS/LIF with or without puromycin.


E : Immunofluorescent detection of total Nanog Esrrb in E14Tg2a ESCs overexpressing Nanog ( left ) or Esrrb ( right ) cultured for three days in GMEMβ / FCS / LIF . Parental E14Tg2as are shown as a reference . red lines indicate negative thresholds defined by ESCs differentiatied for 3 days in absence of LIF presence of retinoic acid .

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E: Immunofluorescent detection and quantification of total Nanog and Esrrb protein in E14Tg2a ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental E14Tg2as are shown as a reference. The red lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid.


F : Flow cytometric of Esrrb - GFP Nanog - mCherry fusion expression in SSEA1 + NER ESCs cultured for three days in GMEMβ / FCS / LIF .

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F: Flow cytometric analysis of Esrrb-GFP and Nanog-mCherry fusion protein expression in SSEA1+ NER ESCs cultured for three days in GMEMβ/FCS/LIF.


G : Histograms showing expression of Esrrb - GFP Nanog - mCherry from respective targeted endogenous alleles in SSEA1 + NER ESCs overexpressing Nanog ( left ) or Esrrb ( right ) cultured for three days in GMEMβ / FCS / LIF . Parental NER ESCs are shown as a reference , wild - type E14Tg2a set negative thresholds .

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G: Histograms showing expression levels of Esrrb-GFP and Nanog-mCherry from the respective targeted endogenous alleles in SSEA1+ NER ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental NER ESCs are shown as a reference, and wild-type E14Tg2a set the negative thresholds.


H : Comparative flow cytometric of Esrrb - 2a - tdTomato Nanog : GFP expression in undifferentiated SSEA - 1 + TNG E - 2a - tdT or Nanog - null ES ∆ N E - 2a - tdT ESCs cultured for three days in GMEMβ / FCS / LIF . Data are shown as dot plots ( top ) histogram ( bottom ) .

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H: Comparative flow cytometric analysis of Esrrb-2a-tdTomato and Nanog:GFP expression in undifferentiated SSEA-1+ TNG E-2a-tdT or Nanog-null ES∆N E-2a-tdT ESCs cultured for three days in GMEMβ/FCS/LIF. Data are shown as dot plots (top) and histogram (bottom).


A : SSEA - 1 + TNG E - tdT ESCs were cultured for three days in GMEMβ / FCS / LIF sorted into GFP + / tdThigh ( Nanoghigh / Esrrbhigh ) or GFP - / tdTlow fractions ( Nanoglow / Esrrblow ) . purity of sorted populations ( > 99 . 5 % ) is shown ( day 0 ) . After replating in GMEMβ / FCS / LIF , SSEA - 1 + were analysed daily for Esrrb - tdTomato Nanog : GFP expression .

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A: SSEA-1+ TNG E-tdT ESCs were cultured for three days in GMEMβ/FCS/LIF and sorted into GFP+/tdThigh (Nanoghigh/Esrrbhigh) or GFP-/tdTlow fractions (Nanoglow/Esrrblow). The purity of the sorted populations (>99.5%) is shown (day 0). After replating in GMEMβ/FCS/LIF, SSEA-1+ cells were analysed daily for Esrrb-tdTomato and Nanog:GFP expression.


B , C : Immunofluorescent detection of Nanog Esrrb in wild - type E14Tg2a ESCs cultured in N2B27 / LIF / 2i released in GMEMβ / FCS / LIF for 1 , 2 or 3 days . of mean Nanog Esrrb measured in nuclei identified by automatic segmentation of single optical plane obtained by confocal microscopy . Oct4 negative , also identified by immunostaining , were excluded from . ( B ) red vertical horizontal lines indicate negative thresholds defined by ESCs differentiatied for 3 days in absence of LIF presence of retinoic acid . red trendline represents moving average of data distribution at day 3 . Representative of two independent experiments each including at least 1000 nuclei . ( C ) Histogram plots of data presented in B .

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B,C: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured in N2B27/LIF/2i and released in GMEMβ/FCS/LIF for 1, 2 or 3 days. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. (B) The red vertical and horizontal lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid. The red trendline represents the moving average of the data distribution at day 3. Representative of two independent experiments each including at least 1000 nuclei. (C) Histogram plots of the data presented in B.


: Sorted SSEA - 1 + Esrrb - GFPhigh / Nanog - mCherryhigh NER ESCs were replated in GMEMβ / FCS / LIF SSEA - 1 + were analysed daily for Esrrb - GFP Nanog - mCherry expression . Wild - type E14Tg2a set negative thresholds .

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: Sorted SSEA-1+ and Esrrb-GFPhigh/Nanog-mCherryhigh NER ESCs were replated in GMEMβ/FCS/LIF and SSEA-1+ cells were analysed daily for Esrrb-GFP and Nanog-mCherry expression. Wild-type E14Tg2a set the negative thresholds.


E : SSEA - 1 + TNG E - tdT ESCs from sorted populations used above ( panel A , day 0 ) were replated at clonal density in GMEMβ / LIF / FCS or N2B27 / LIF / 2i stained for alkaline phosphatase activity after 6 days . Error bars : standard deviation of number of colonies observed in two independent experiments .

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E: SSEA-1+ TNG E-tdT ESCs from the sorted populations used above (panel A, day 0) were replated at clonal density in GMEMβ/LIF/FCS or N2B27/LIF/2i and stained for alkaline phosphatase activity after 6 days. Error bars: standard deviation of the number of colonies observed in two independent experiments.


A : Flow cytometric of GFP expression in SSEA - 1 + E - GFPd1 ESCs cultured in N2B27 / LIF / 2i or GMEMβ / LIF / FCS compared to wild - type E14Tg2a ESCs .

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A: Flow cytometric analysis of GFP expression in SSEA-1+ E-GFPd1 ESCs cultured in N2B27/LIF/2i or GMEMβ/LIF/FCS compared to wild-type E14Tg2a ESCs.


B : SSEA - 1 + E - GFPd1 ESCs sorted into EsrrbHi , EsrrbMed or EsrrbNeg subpopulations . Purity of sorted populations , assessed by flow cytometry , is indicated above sorting gates .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

B: SSEA-1+ E-GFPd1 ESCs sorted into EsrrbHi, EsrrbMed or EsrrbNeg subpopulations. Purity of the sorted populations, assessed by flow cytometry, is indicated above the sorting gates.


C : Quantitative Esrrb mRNA expression of populations sorted Error bars : standard deviation of gene expression values measured in four independent experiments .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

C: Quantitative Esrrb mRNA expression analysis of populations sorted Error bars: standard deviation of gene expression values measured in four independent experiments.


D : Populations sorted were replated at clonal density in GMEMβ / LIF / FCS ( top ) or N2B27 / LIF / 2i ( bottom ) scored for alkaline phosphatase after 6d . Error bars : standard deviation of number of colonies observed in two independent experiments .

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D: Populations sorted were replated at clonal density in GMEMβ/LIF/FCS (top) or N2B27/LIF/2i (bottom) and scored for alkaline phosphatase after 6d. Error bars: standard deviation of the number of colonies observed in two independent experiments.


E : Quantitative mRNA expression of populations sorted Error bars : standard deviation of gene expression values measured in four independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

E: Quantitative mRNA expression analysis of populations sorted Error bars: standard deviation of gene expression values measured in four independent experiments.


F : Microarray of populations sorted represented as a ordered by expression change between EsrrbHi EsrrbNeg . Expression were measured from three independent experiments genes expressed in at least one sample are shown ( n = 14 , 553 ) . central plot summarises average fold change per population compared to EsrrbHi ( log2 scale ) with colour coding indicated below heat map . Previously identified Nanog Esrrb targets are indicated on right .

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F: Microarray analysis of populations sorted represented as a heatmap ordered by expression change between EsrrbHi and EsrrbNeg. Expression levels were measured from three independent experiments and all genes expressed in at least one sample are shown (n = 14,553). The central line plot summarises the average fold change per cell population compared to EsrrbHi cells (log2 scale) with the colour coding indicated below the heat map. Previously identified Nanog and Esrrb targets are indicated on the right.


G : Pearson correlation coefficients of gene expression profiles of EsrrbH ( H ) , EsrrbM ( M ) , Esrrb - ( Neg ) E - GFPd1 ESCs derived ( late bud stage ) TNG E - 2a - tdT EpiSCs ( EpiSC ) compared to those of naïve ESC

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G: Pearson correlation coefficients of the gene expression profiles of EsrrbH (H), EsrrbM (M), Esrrb- (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) compared to those of naïve ESC and EpiLC


H : Pearson correlation coefficients in gene expression obtained comparing EsrrbHi ( H ) , EsrrbMed ( M ) , EsrrbNeg ( Neg ) E - GFPd1 ESCs derived ( late bud stage ) TNG E - 2a - tdT EpiSCs ( EpiSC ) to epiblast / ectoderm from dissected at successive developmental stages ( CAV , cavity ; PS , prestreak ; ES , early - streak ; MS , midstreak ; LMS , late midstreak ; LS , late streak ; OB / EB , no bud / early bud ; LB , late bud ) . This was restricted to top 500 differentially expressed gene identified at successive developmental stages sorting by Hotelling T²

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD 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-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O

H: Pearson correlation coefficients in gene expression obtained comparing EsrrbHi (H), EsrrbMed (M), EsrrbNeg (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) to epiblast/ectoderm cells from embryos dissected at successive developmental stages (CAV, cavity; PS, prestreak; ES, early-streak; MS, midstreak; LMS, late midstreak; LS, late streak; OB/EB, no bud/early bud; LB, late bud). This analysis was restricted to the top 500 differentially expressed gene identified at successive developmental stages sorting by Hotelling T² score


A : of methylated CpG dinucleotides profiled across Esrrb Nanog enhancer Nanog promoter in sorted SSEA - 1 + / EsrrbHi , EsrrbMed or EsrrbNeg E - GFPd1 ESCs TNG E - 2a - tdT EpiSC . CpG methylation was assessed by measuring protection from digestion of HpaII , AciI , Hin6I or TaqI restriction sites ( indicated by vertical lines in gene structure maps derived from mouse reference genome ( mm9 ) expressed relative to TSS in ESCs Values represent total CpG methylation ( 5mC + 5hmC ) . Error bars : standard deviation of measures in four independent experiments .

O O O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

A: Percentage of methylated CpG dinucleotides profiled across the Esrrb and Nanog enhancer and the Nanog promoter in sorted SSEA-1+ / EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs and TNG E-2a-tdT EpiSC. CpG methylation was assessed by measuring protection from digestion of the HpaII, AciI, Hin6I or TaqI restriction sites (indicated by vertical lines in the gene structure maps derived from the mouse reference genome (mm9) and expressed relative to the TSS in ESCs Values represent total CpG methylation levels (5mC + 5hmC). Error bars: standard deviation of the measures in four independent experiments.


B : Methylated CpG dinucleotides across Esrrb enhancer were assessed SSEA - 1 + / EsrrbHi E - GFPd1 ESCs were sorted placed back in culture overnight before resorting methylation relative to Esrrb - E - GFPd1 ESCs . Error bars : standard deviation of measures in three independent experiments .

O O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O

B: Methylated CpG dinucleotides across the Esrrb enhancer were assessed SSEA-1+ / EsrrbHi E-GFPd1 ESCs were sorted and placed back in culture overnight before resorting and methylation analysis relative to Esrrb- E-GFPd1 ESCs. Error bars: standard deviation of the measures in three independent experiments.


C : sorted E - GFPd1 ESCs populations were assessed by immunoblot for Esrrb Nanog ( left ) or Oct4 ( right ) by - detection ( Licor ) .

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C: The sorted E-GFPd1 ESCs populations were assessed by immunoblot for Esrrb and Nanog (left) or Oct4 (right) by fluorescence-based detection (Licor).


D : Quantitative ChIP - PCR of OCT4 , NANOG ESRRB binding histone modifications at Esrrb Nanog enhancers Nanog promoter in sorted populations Error bars : standard deviation of measures in two ( NANOG , ESRRB , H3K27ac ) or three ( OCT4 , H3K4me3 , H3K4me1 ) independent ChIP experiments , each performed on pooled from at least three independently sorted samples . diagrams at bottom show approximate position of regions analysed for transcription factor binding or histone modifications ( in color or black ) , along with relative control genomic locations ( grey ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O 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-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

D: Quantitative ChIP-PCR analysis of OCT4, NANOG and ESRRB binding and histone modifications at the Esrrb and Nanog enhancers and the Nanog promoter in the sorted populations Error bars: standard deviation of the measures in two (NANOG, ESRRB, H3K27ac) or three (OCT4, H3K4me3, H3K4me1) independent ChIP experiments, each performed on pooled chromatin from at least three independently sorted samples. The diagrams at the bottom show the approximate position of the regions analysed for transcription factor binding or histone modifications (in color or black), along with the relative control genomic locations (grey).


Average binding profile of ( B ) OCT4 in binding peaks where binding in EsrrbNeg ESCs was kept , lost or gained relative to EsrrbHi E - GFPd1 ESCs . Read per base pair were normalised by library by subtracting background ( IgG ) signal . EsrrbHi ( EH ) , EsrrbMed ( EM ) EsrrbNeg ( EN )

O B-EXP_ASSAY O O O O O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O

Average binding profile of (B) OCT4 in binding peaks where binding in EsrrbNeg ESCs was kept, lost or gained relative to EsrrbHi E-GFPd1 ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal. EsrrbHi (EH), EsrrbMed (EM) and EsrrbNeg (EN)


Average binding profile of ( D ) NANOG in binding peaks where binding in EsrrbNeg ESCs was kept , lost or gained relative to EsrrbHi E - GFPd1 ESCs . Read per base pair were normalised by library by subtracting background ( IgG ) signal . EsrrbHi ( EH ) , EsrrbMed ( EM ) EsrrbNeg ( EN )

Average binding profile of (D) NANOG in binding peaks where binding in EsrrbNeg ESCs was kept, lost or gained relative to EsrrbHi E-GFPd1 ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal. EsrrbHi (EH), EsrrbMed (EM) and EsrrbNeg (EN)


G : Boxplots showing fold change in expression of genes proximal to Class I elements , Class II elements or both Class I Class II elements in EsrrbHi ( EH ) , EsrrbMed ( EM ) or EsrrbNeg ( EN ) E - GFPd1 ESCs EpiSC ( Epi ) . Boxes span inter - quartile range ( IQR ) from first to third quartile . indicates median . Whiskers extend up to 1 . 5 IQR , outliers are plotted .

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G: Boxplots showing the fold change in expression levels of genes proximal to Class I elements, Class II elements or both Class I and Class II elements in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) E-GFPd1 ESCs and EpiSC (Epi). Boxes span the inter-quartile range (IQR) from the first to the third quartile. The line indicates the median. Whiskers extend up to 1.5 IQR, and outliers are plotted.


H , I : Binding profiles of OCT4 NANOG in proximity of two genes representative of ( H ) Class I ( Tet2 ) or ( I ) Class II ( Sall1 ) in EsrrbHi , EsrrbMed or EsrrbNeg E - GFPd1 ESCs . relative occupancies of NANOG OCT4 at Tet2 or Sall1 ( positions highlighted by hatched boxes ) alongside expression of Tet2 Sall1 mRNAs in EsrrbHi ( EH ) , EsrrbMed ( EM ) or EsrrbNeg ( EN ) GFPd1 ESCs EpiSC ( Epi ) is shown at bottom . Oct4 binding data from

O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O

H, I: Binding profiles of OCT4 and NANOG in the proximity of two genes representative of (H) Class I (Tet2) or (I) Class II (Sall1) in EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs. The relative occupancies of NANOG and OCT4 at Tet2 or Sall1 (positions highlighted by hatched boxes) alongside expression levels of Tet2 and Sall1 mRNAs in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) GFPd1 ESCs and EpiSC (Epi) is shown at the bottom. Oct4 binding data from EpiSC


: Average binding profile of ESRRB , KLF4 , NANOG , OCT4 SOX2 to Class I ( green ) , Class II ( orange ) or Nanog Oct4 bound ( grey dashed ) elements in ESCs . Read per base pair were normalised by library by subtracting background ( IgG ) signal .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

: Average binding profile of ESRRB, KLF4, NANOG, OCT4 and SOX2 to Class I (green), Class II (orange) or all Nanog and Oct4 bound (grey dashed line) elements in ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal.


B : Average FAIRE profile or ChIP - Seq profiles for H3K27ac , OCT4 , Ep300 or OTX2 in ESCs or EpiLC for Class I Class II elements .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O

B: Average FAIRE profile or ChIP-Seq profiles for H3K27ac, OCT4, Ep300 or OTX2 in ESCs or EpiLC for Class I and Class II elements.


B ) Box - whisker plots of log2 - transformed peptide intensities across 60 raw files . color coding highlights samples plate of origin .

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B) Box-whisker plots of log2-transformed peptide intensities across all 60 raw files. The color coding highlights the samples plate of origin.


C ) Cumulative frequency curve [ % ] of observed coefficient of variation ( CV ) of that were identified quantified with a minimum of three valid values within each day . Here , ten raw files of each day are evaluated individually . resulting median average CV for each day are shown .

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C) Cumulative frequency curve [%] of the observed coefficient of variation (CV) of proteins that were identified and quantified with a minimum of three valid values within each day. Here, the ten raw files of each day are evaluated individually. The resulting median and average CV for each day are shown.


D ) Pearson correlation of 60 raw files additional sixteen manually prepared HeLa SP3 samples . displayed data are filtered for 75 % data completeness ( Table 1 ) . Please note narrow scaling ( 1 - 0 . 94 ) .

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D) Pearson correlation heatmap of all 60 raw files and additional sixteen manually prepared HeLa SP3 samples. The displayed data are filtered for 75% data completeness (Table 1). Please note the narrow scaling (1-0.94).


C ) Volcano plot showing differential expression using Limma moderated t - statistics for comparison of lepidic samples against other samples . passing significance thresholds of - log10 p - value < 0 . 05 ( Benjamini - hochberg adjusted ) an absolute log2 fold change of > 1 are highlighted in orange .

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C) Volcano plot showing differential expression analysis using Limma moderated t-statistics for the comparison of lepidic samples against all other samples. Proteins passing significance thresholds of -log10 p-value < 0.05 (Benjamini-hochberg adjusted) and an absolute log2 fold change of >1 are highlighted in orange.


B ) Correlation between mg wet tissue amount of extracted by ultrasonication .

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B) Correlation between mg wet tissue weight and the amount of protein extracted by ultrasonication.


C ) Pearson correlation of peptide intensities across five replicates of each sample type ( heart , liver , kidney , HeLa ) with corresponding average .

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C) Pearson correlation heatmap of peptide intensities across five replicates of each sample type (heart, liver, kidney, HeLa cells) with the corresponding average.


D ) relative number of identified quantified across five replicates of each sample type . average number of identified standard deviation across five replicates is shown on top .

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D) The relative number of identified and quantified proteins across the five replicates of each sample type. The average number of identified proteins and the standard deviation across five replicates is shown on top.


E ) Cumulative frequency curve [ % ] of observed coefficient of variation ( CV ) of that were identified quantified in at least three out of five replicates in each sample type . resulting median average CV for each sample type are shown .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Cumulative frequency curve [%] of the observed coefficient of variation (CV) of proteins that were identified and quantified in at least three out of five replicates in each sample type. The resulting median and average CV for each sample type are shown.


Defects in midbrain - type dopamine ( mDA ) neuron - specific developmental factor expressions in ventral midbrain ( VM ) of Lin28a / b cKO . Embryonic VM sections from 3 WT cKO each were immunoflorescent stained for midbrain - specific markers in parallel mean fluoresecent intensities ( MFI ) were measured . n = 20 for each group . * * * p < 0 . 0001 , t - . Reduced mDA neurons in embryonic VM ( E12 . 5 ) adult SN at 2 - 7 months ( young ) at 14 - 18 months ( old ) of Lin28a / b cKO mice . * p < 0 . 05 , * * p < 0 . 005 , n = 4 each wild - type ( WT ) cKO , 9 ( WT ) , 8 ( cKO ) in adult mice . Enhanced DA transporter ( DAT ) + TH + fiber intensities of Lin28a / b cKO adult mice ( : 4 - 7 months ) . Striatal sections of WT cKO mice ( 3 for each group ) were subjected to immunofluorescent against DAT TH in parallel , MFI was measured from 101 microscopic fields of dorsolateral striatum .

O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-CELL_TYPE I-CELL_TYPE O B-ORGANISM B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O 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-ORGANISM O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O

Defects in midbrain-type dopamine (mDA) neuron-specific developmental factor expressions in the ventral midbrain (VM) of Lin28a/b cKO embryos. Embryonic VM sections from 3 WT and cKO animals each were immunoflorescent stained for the midbrain-specific markers in parallel and mean fluoresecent intensities (MFI) were measured. n = 20 cells for each group. *** p<0.0001, t-test. Reduced mDA neurons in the embryonic VM (E12.5) and adult SN at 2-7 months (young age) and at 14-18 months (old age) of the Lin28a/b cKO mice. *p<0.05, **p<0.005, n=4 each wild-type (WT) and cKO embryos, and 9 (WT), 8 (cKO) in adult mice. Enhanced DA transporter (DAT)+ and TH+ fiber intensities of Lin28a/b cKO adult mice (age: 4-7 months). Striatal sections of WT and cKO mice (3 animals for each group) were subjected to immunofluorescent staining against DAT and TH in parallel, and MFI was measured from 101 microscopic fields of dorsolateral striatum.


DA uptakes assessed by nanoScanPET / MRI using [ 18F ] FP - CIT . n = 9 ( WT ) , 8 ( cKO ) . * p < 0 . 05 , * * * p < 0 . 001 .

B-SMALL_MOLECULE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DA uptakes assessed by nanoScanPET/MRI analysis using [18F]FP-CIT. n=9 (WT), 8 (cKO). *p<0.05, ***p<0.001.


Enhanced susceptibility of Lin28a / b cKO mice to Parkinsonian toxin MPTP . WT cKO mice were injected with MPTP ( 20mg / kg , i . p ) for 5 days behaviors of were assessed for 1 month after MPTP

O O O B-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE

Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. WT and cKO mice were injected with MPTP (20mg/kg, i.p) for 5 days and behaviors of the animals were assessed for 1 month after the MPTP injection


Enhanced susceptibility of Lin28a / b cKO mice to Parkinsonian toxin MPTP . mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH + mDA neuron at SN ( L ) DAT MFI at striatum ( M ) .

O O O B-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM B-TISSUE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O B-GENEPROD O O B-TISSUE O O O O

Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. The mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH+ mDA neuron counts at SN (L) and DAT MFI at striatum (M).


growth population doubling ( PDLs ) during passaging . In ( B ) , dots represent PDLs , boxes bars represent mean ± SE of five PDL values of + / + + / m lines . * Significantly different from control with p < 0 . 05 , ANOVA . Phase - contrast ( left ) , alkaline phosphatase - stained ( middle ) , immunocytochemical stained with pluripotent markers ( right ) . were taken at passage P8 ( + / + , + / m ) or P6 ( m / m ) . Scale bars , 100 µm .

B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell growth and population doubling levels (PDLs) during passaging. In (B), dots represent individual PDLs, boxes and bars represent mean ± SE of five PDL values of +/+ and +/m lines. *Significantly different from control with p<0.05, ANOVA. Phase-contrast (left), alkaline phosphatase-stained (middle), and immunocytochemical images stained with pluripotent cell markers (right). Images were taken at cell passage P8 (+/+, +/m) or P6 (m/m). Scale bars, 100 µm.


Unsupervised hierarchical clustering of gene expression ( ratios of mutant / WT ) categorized in " neurodegenerative disorders " ( ' Parkinson ' s disease ' , ' Alzheimer ' s disease ' , ' Huntington ' s disease ' ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE I-DISEASE O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O

Unsupervised hierarchical clustering heatmap of the gene expression (ratios of mutant/WT) categorized in "neurodegenerative disorders" ('Parkinson's disease', 'Alzheimer's disease', and 'Huntington's disease').


Neural differentiation of hESCs was induced on a feeder layer of MS5 stromal . Representative colonies differentiated from WT ( + / + , c42 ) homozygous mutant ( m / m , c38 ) hESCs are shown ( left ) . Neural induction efficiencies were estimated by % neuroepithelial colonies out of total colonies ( n = 5 ~ 7 dishes for each group ) . Neuroepithelial colonies were identified by neuroepithelial structure ( neural rosettes ) by of markers specific for primitive neuroepithelial ( PLZF , N - CADHERIN , SOX2 ) rostral embryonic brain ( OTX2 ) . Scale bars , 100 µm . Ventral midbrain ( VM ) patterning was assessed during 24 days of + / + + / m hESC differentiation ( chemically defined protocol ) . Each dot represents mRNA expression detected by qPCR ; boxes bars represent means SEs of + / + + / m lines . gene expression of + / + + / m at day 24 are compared in last graph ( n = 4 , * p < 0 . 05 or * * p < 0 . 001 , t - ) .

B-CELL_TYPE O O B-CELL_TYPE O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-ORGANISM B-TISSUE O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neural differentiation of hESCs was induced on a feeder layer of MS5 stromal cells. Representative colonies differentiated from the WT (+/+, c42) and homozygous mutant (m/m, c38) hESCs are shown (left). Neural induction efficiencies were estimated by % neuroepithelial colonies out of total colonies (n=5~7 dishes for each group). Neuroepithelial cell colonies were identified by neuroepithelial cell structure (neural rosettes) and by staining of markers specific for primitive neuroepithelial cells (PLZF, N-CADHERIN, and SOX2) and rostral embryonic brain (OTX2). Scale bars, 100 µm. Ventral midbrain (VM) patterning was assessed during 24 days of +/+ and +/m hESC differentiation (chemically defined protocol). Each dot represents mRNA expression detected by qPCR; boxes and bars represent means and SEs of +/+ and +/m lines. The gene expression levels of +/+ and +/m at day 24 are compared in the last graph (n=4, *p<0.05 or **p<0.001, t-test).


Neural ( NSC ) expansion estimated by growth curve PDL during NSC passages ( n = 12 , * p < 0 . 001 , t - ) . NSCs were derived from neuroepithelial colonies ( shown in ( A ) ) .

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Neural stem cell (NSC) expansion estimated by cell growth curve and PDL during NSC passages (n=12, *p<0.001, t-test). NSCs were derived from neuroepithelial cell colonies (shown in (A)).