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 .

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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 .

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 .

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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 .

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 .

: 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 .

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 .

: 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 .

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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 .

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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 .

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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

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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 .

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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 .

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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 ) .

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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 )

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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

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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 .

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 .

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 O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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

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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 ) .

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 .

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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 ' ) .

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 - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)).


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . General survival ( calcein - AM ) / ( EthD - 1 ) . ( B ) caspase - 3 + apoptotic . ( C ) senescence ( β - gal ) . ( D ) damage estimated by γH2AX . with > 4 γH2AX foci in nucleus were counted . ( E ) Mitochondrial ROS ( MitoSox ) . ( F ) Mitochondrial potential ( MitoID ) .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID).


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Seahorse . Terminal differentiation of neural ( NSCs ) was induced oxygen consumption rate ( OCR ) extracellular acidification rate ( ECAR ) were analyzed on NSCs at differentiation day 4 . p < 0 . 001 * , n = 7 ( WT ) , 10 ( mutant ) , t - .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Seahorse test. Terminal differentiation of neural stem cells (NSCs) was induced and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed on the NSCs at differentiation day 4. p<0.001*, n=7 (WT), 10 (mutant), t-test.


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Western blot analyses for ER stress marker .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Western blot analyses for ER stress marker proteins.


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Resistance of mDA neurons against mitochondrial toxins . Differentiated mDA neurons were exposed to toxins indicated for 1 h , viable TH + mDA neurons were counted . Data are expressed as % TH + relative to untreated . Three cultures were analyzed for each . N = 3 independent experiments , * Significance at p < 0 . 05 . Scale bars , 100 µm .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Resistance of mDA neurons against mitochondrial toxins. Differentiated mDA neurons were exposed to the toxins indicated for 1 h, and viable TH+ mDA neurons were counted. Data are expressed as % TH+ cells relative to untreated. Three cultures were analyzed for each cell line. N=3 independent experiments, *Significance at p<0.05. Scale bars, 100 µm.


Representative in vitro phase - contrast ( A ) , TH / DAPI or TH ( insets ) ( B ) , TH / SYNAPSIN - stained ( C ) in cultures terminally differentiated from mutant ( c13 , c19 ) corrected hiPSCs ( c22 , c59 ) .

Representative in vitro phase-contrast (A), TH/DAPI or TH (insets) (B), and TH/SYNAPSIN-stained (C) images in the cultures terminally differentiated from mutant (c13, c19) and corrected hiPSCs (c22, c59).


DA neuron differentiation of DA neurons were assessed by DA neuronal yields


DA neuron morphological of DA neurons were assessed by TH + fiber

DA neuron morphological of DA neurons were assessed by TH+ fiber lengths


DA neuron , synaptical of DA neurons were assessed by synaptic puncta densities


DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA were measured in media conditioned in differentiated cultures for 24 h ( D15 ) evoked by KCl - mediated depolarization for 30 min .

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DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA levels were measured in the media conditioned in the differentiated cultures for 24 h (D15) and evoked by KCl-mediated depolarization for 30 min.


breaks estimated by γH2AX . with > 4 γH2AX foci in nucleus were counted .

DNA breaks estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted.


SA - β - gal was performed for senescence .

SA-β-gal staining was performed for cellular senescence analysis.


Mitochondrial ROS ( MitoSox ) were analyzed after terminal differentiation .

Mitochondrial ROS (MitoSox) were analyzed after terminal differentiation.


mitochondrial potential ( MitoID ) were analyzed after terminal differentiation .

mitochondrial membrane potential (MitoID) were analyzed after terminal differentiation.


Accumulation of α - synuclein oligomers analyzed by western blotting . mutant ( c13 ) corrected ( c22 ) neural ( NSCs ) were transduced with α - synuclein - expressing lentiviruses . At 6 days after differentiation , were extracted fractionated to Triton X - 100 ( 1 % ) - soluble insoluble fractions . Shown is representative WB image of three independent experiments . α - Synuclein oligomers are indicated with a bracket , while monomer bands are marked with an arrowhead .

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Accumulation of α-synuclein oligomers analyzed by western blotting. The mutant (c13) and corrected (c22) neural stem cells (NSCs) were transduced with α-synuclein-expressing lentiviruses. At 6 days after differentiation, cells were extracted and fractionated to Triton X-100 (1%)-soluble and insoluble fractions. Shown is the representative WB image of three independent experiments. α-Synuclein oligomers are indicated with a bracket, while monomer bands are marked with an arrowhead.


Amphetamine - induced rotation scores . net rotation values of transplanted with differentiated mutant ( n = 7 ) corrected ( n = 6 ) pt - hiPSCs are depicted . Significant differences from mutant - transplanted at each post - transplantation time point are at * p < 0 . 05 or * * p < 0 . 001 . engraftments measured by integrated intensities in grafted with DiR - labeled pt corrected hiPSCs . * p < 0 . 05 .

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Amphetamine-induced rotation scores. The ipsilateral net rotation values of individual animals transplanted with differentiated mutant (n=7) and corrected (n=6) pt-hiPSCs are depicted. Significant differences from the mutant cell-transplanted at each post-transplantation time point are at *p<0.05 or **p<0.001. Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05.


Overviews of hNCAM + / TH + grafts at 8 post - transplantation . Insets , TH + DA neuron in grafts shown with high magnification .

Overviews of hNCAM+/TH+ cell grafts at 8 weeks post-transplantation. Insets, TH+ DA neuron morphology in grafts shown with high magnification.


Co - expression of mDA neuron - specific FOXA2 , LMX1A , NURR1 , DAT in TH + DA generated by corrected pt - hiPSC transplantation .

Co-expression of the mDA neuron-specific FOXA2, LMX1A, NURR1, DAT in TH+ DA cells generated by the corrected pt-hiPSC transplantation.


engraftments measured by integrated intensities in grafted with DiR - labeled pt corrected hiPSCs . * p < 0 . 05 . Total number of TH + in graft ( TH + numbers / mm3 ) per .

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Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05. Total number of TH+ cells in the graft (TH+ numbers/mm3) per animal.


LIN28A stability assays . HEK293 were transfected with pFLAG - WT - LIN28A pFLAG - LIN28A R192G . samples were prepared during 24 h of cycloheximide ( 50 μg / ml ) treatment . Representative western blot data for LIN28A level changes normalized to β - actin ( upper ) or with normalization on initial ( 0 h ) LIN28A ( lower ) . Band intensities were quantified using ImageJ , normalized values are depicted ( lower graph ) ( n = 5 ) .

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

LIN28A protein stability assays. HEK293 cells were transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G. Protein samples were prepared during 24 h of cycloheximide (50 μg/ml) treatment. Representative western blot data for LIN28A protein level changes normalized to β-actin protein levels (upper) or with normalization based on initial (0 h) LIN28A protein levels (lower). Band intensities were quantified using ImageJ, and normalized values are depicted (lower graph) (n=5).


proteasome inhibitor MG132 treatment increases LIN28A ( R192G ) level . After serum starvation for 3 hours , HEK293T expressing LIN28A ( R912G ) were treated with or without MG132 ( 0 . 1 - 10 uM ) for 30 minutes , then followed by WB analyses .

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The proteasome inhibitor MG132 treatment increases LIN28A(R192G) protein level. After serum starvation for 3 hours, HEK293T cells expressing LIN28A(R912G) were treated with or without MG132 (0.1-10 uM) for 30 minutes, and then followed by WB analyses.


LC - MS / MS . Immunoprecipitated FLAG - LIN28A WT was loaded on an SDS gel ( left upper ) . Bold red sequences indicate peptides matching LIN28A ( left lower ) . Right , LC - MS / MS spectra of WT LIN28A .

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LC-MS/MS analysis. Immunoprecipitated FLAG-LIN28A WT was loaded on an SDS gel (left upper). Bold red sequences indicate peptides matching LIN28A (left lower). Right, LC-MS/MS spectra of WT LIN28A.


localization of WT LIN28A LIN28A ( R192G ) . HeLa transfected with pFLAG - WT - LIN28 pFLAG - LIN28A ( R192G ) were stained with FLAG antibody counterstained with DAPI .

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Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI.


LET - 7 microRNA regulatory activity of LIN28A . HeLa were transfected with plasmids expressing FLAG - tagged WT - LIN28A , LIN28AR192G , or mock pFLAG vector ( control ) , Let - 7a , b , c were quantified by RT - qPCR . level of was normalized to that of U6 snRNA . p < 0 . 05 , n = 26 from 6 independent experiments , ns = not significant , ANOVA .

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LET-7 microRNA regulatory activity of LIN28A. HeLa cells were transfected with plasmids expressing FLAG-tagged WT- LIN28A, LIN28AR192G, or mock pFLAG vector (control), and Let-7a, b, and c levels were quantified by RT-qPCR. The level of RNA was normalized to that of U6 snRNA. p<0.05, n=26 from 6 independent experiments, ns = not significant, ANOVA.


Affinity of LIN28A binding to its cognate mRNA 3 ′ UTR region . Upper : schematic for pmirGLO - LIN28A used in this , a bicistronic Firefly / Renilla luciferase ( FLuc / RLuc ) plasmid containing part of LIN28A 3 ′ UTR containing putative LIN28A binding sites ( black dots ) Wilbert et al . , 2012 ( ) . Lower : HeLa were transiently co - transfected with pmirGLO - LIN28A 3 ′ UTR along with pFLAG - WT - LIN28A , pFLAG - LIN28A R192G , or mock pFLAG control . LIN28A - mRNA binding activities were determined by relative luciferase activity of FLuc normalized to those of RLuc . * p < 0 . 05 , n = 18 from 6 independent experiments .

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Affinity of LIN28A protein binding to its cognate mRNA 3′UTR region. Upper: schematic for pmirGLO-LIN28A used in this assay, a bicistronic Firefly/Renilla luciferase (FLuc/RLuc) plasmid containing the part of the LIN28A 3′UTR containing the putative LIN28A protein binding sites (black dots)Wilbert et al., 2012(). Lower: HeLa cells were transiently co-transfected with pmirGLO-LIN28A 3′UTR along with pFLAG-WT-LIN28A, pFLAG-LIN28A R192G, or mock pFLAG control. LIN28A protein-mRNA binding activities were determined by relative luciferase activity levels of FLuc normalized to those of RLuc. *p<0.05, n=18 from 6 independent experiments.


LIN28A - RHA co - IP assays in HEK293 transfected with pFLAG - WT - LIN28A pFLAG - LIN28A R192G .

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LIN28A-RHA co-IP assays in HEK293 cells transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G.


GSEA showing enrichment of BCC [ 28 ] , well - differentiated EMT containing SCC [ 33 ] for YAP gene signatures obtained from mouse hepatocellular carcinoma [ 40 ] human breast cancer [ 39 ] , human oral squamous carcinoma [ 42 ] human colorectal cancer [ 41 ] . Well - diff SCC : well - differentiated SCC ; EMT SCC : EMT - containing SCC

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GSEA analysis showing enrichment of BCC [28], well-differentiated and EMT containing SCC [33] for YAP gene signatures obtained from mouse hepatocellular carcinoma [40]human breast cancer cell line [39], human oral squamous cell carcinoma [42] and human colorectal cancer [41]. Well-diff SCC: well-differentiated SCC; EMT SCC: EMT-containing SCC


( A ) Venn diagram showing overlap between four published YAP signatures [ 39 - 42 ]

(A)Venn diagram showing the overlap between four published YAP signatures [39-42]


( B ) Venn diagram showing overlap between YAP signatures obtained in Oral SCC Breast ( YAP Signature 1 ) CRC HCC ( YAP Signature 2 ) . (

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(B) Venn diagram showing the overlap between the YAP signatures obtained in Oral SCC and Breast (YAP Signature 1) and CRC and HCC (YAP Signature 2) .(C


. ( C ) Venn diagram showing overlap between YAP Signature 1 genes upregulated by more than 1 . 5 fold in BCC compared to IFE [

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.(C) Venn diagram showing the overlap between YAP Signature 1 and the genes upregulated by more than 1.5 fold in BCC compared to IFE[28


( D ) Venn diagram showing overlap between YAP Signature 1 genes upregulated by more than 1 . 5 fold in well - differentiated SCC versus IFE in EMT containing SCC versus HF [ 33 ]

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(D) Venn diagram showing the overlap between YAP Signature 1 and the genes upregulated by more than 1.5 fold in well-differentiated SCC versus IFE and in EMT containing SCC versus HF[33]


( E ) Venn diagram showing genes commonly upregulated in YAP signature 1 in BCCs , well - differentiated EMT - containing

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(E) Venn diagram showing the genes commonly upregulated in YAP signature 1 and in BCCs, well-differentiated and EMT-containing SCCs


( F ) Venn diagram showing overlap between YAP signature 2 genes upregulated by more than 1 . 5 fold in BCC compared to IFE [ 28 ]

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(F) Venn diagram showing the overlap between YAP signature 2 and the genes upregulated by more than 1.5 fold in BCC compared to IFE [28]


( G ) Venn diagram showing overlap between YAP signature 2 genes upregulated by more than 1 . 5 fold in well - differentiated SCC versus IFE in EMT containing SCC versus HF [

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(G) Venn diagram showing the overlap between YAP signature 2 and the genes upregulated by more than 1.5 fold in well-differentiated SCC versus IFE and in EMT containing SCC versus HF [33


( H ) Venn diagram showing genes commonly upregulated YAP Signature 2 in BCCs , well - differentiated EMT - containing SCCs . HCC : hepatocellular carcinoma ; CRC : colorectal cancer ; Well - diff SCC : well - differentiated SCC ; EMT SCC : EMT containing SCC

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(H) Venn diagram showing the genes commonly upregulated the YAP Signature 2 and in BCCs, well-differentiated and EMT-containing SCCs. HCC: hepatocellular carcinoma; CRC: colorectal cancer; Well-diff SCC: well-differentiated SCC; EMT SCC: EMT containing SCC


A . Anterior lateral of two with ELP6 variants showing dysmorphic features at 20 ( 2 ) 19 ( 3 ) years of .

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A. Anterior and lateral photographs of two patients with ELP6 variants showing dysmorphic features at 20 (Patient 2) and 19 (Patient 3) years of age.


B . Mid - sagittal ( I ) , coronal ( II ) transversal ( III ) MRI scans of 1 with newly identified ELP4 variants 2 3 with ELP6 variants . Atrophy of cerebellum with prominence of cerebellar folia is marked by circles . Arrows indicate thin corpus callosum , red arrowheads point to enlarged extra - axial CSF spaces black arrowheads to cerebral sulci in keeping with cerebral cerebellar volume loss .

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B. Mid-sagittal (I), coronal (II) and transversal (III) MRI scans of Patient 1 with newly identified ELP4 variants and Patient 2 and Patient 3 with ELP6 variants. Atrophy of the cerebellum with prominence of the cerebellar folia is marked by circles. Arrows indicate thin corpus callosum, red arrowheads point to enlarged extra-axial CSF spaces and black arrowheads to cerebral sulci in keeping with cerebral and cerebellar volume loss.


C . EEG of 3 . Left panel : 1 - 2 Hz left fronto - temporal spike multi - spike slow wave discharges ( Fp1 - F7 T3 - T5 ) spread to homologous same hemisphere opposite hemisphere , predominantly seen on bipolar montage EEG . Right panel : Spike - multi - spike slow wave activities together with fast rhythms originating from bilateral fronto - temporal - frontocentral regions indicate electrographic seizure activity . Described changes are marked by circles .

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C. EEG of Patient 3. Left panel: 1-2 Hz left fronto-temporal spike and multi-spike slow wave discharges (Fp1-F7 and T3-T5) spread to the homologous same hemisphere and opposite hemisphere, predominantly seen on bipolar montage EEG. Right panel: Spike-and multi-spike slow wave activities together with fast rhythms originating from bilateral fronto-temporal-frontocentral regions indicate electrographic seizure activity. Described changes are marked by circles.


C . Pull - down experiments of assembled mElp123456 complexes carrying - derived mutations . mElp123WT subcomplexes were immobilized on Dynabeads ( strep ) via Elp3 incubated with Elp456 variants . inputs pull downs were loaded on SDS - PAGE gels visualized by Coomassie . molecular mass marker is indicated on left ( M ) .

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C. Pull-down experiments of the assembled mElp123456 complexes carrying patients-derived mutations. The mElp123WT subcomplexes were immobilized on the Dynabeads (strep) via Elp3 protein and incubated with the Elp456 variants. The inputs and pull downs were loaded on the SDS-PAGE gels and visualized by Coomassie staining. The molecular mass marker is indicated on the left (M).


D . Co - immunoprecipitation analyses of Elongator complexes in - derived fibroblast . Whole lysates ( left ) eluates after immune - precipitation ( right ) are shown for control fibroblasts ( ctrl ) cultured fibroblasts ( Elp4Y91C / L296I ( 1 ) ; Elp6L118W / L118W ( 2 ) , Elp6L118W / L118W ( 3 ) . Used antibodies for immobilization ( ELP1 ) detection ( ELP1 , ELP3 , ELP4 , ELP5 α - tubulin ) are indicated . n = 4 independent experiments .

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D. Co-immunoprecipitation analyses of Elongator complexes in patient-derived fibroblast cell. Whole cell lysates (left) and eluates after immune-precipitation (right) are shown for control fibroblasts (ctrl) and cultured patient fibroblasts (Elp4Y91C/L296I (patient 1); Elp6L118W/L118W (patient 2), Elp6L118W/L118W (patient 3). Used antibodies for immobilization (ELP1) and detection (ELP1, ELP3, ELP4 , ELP5 and α-tubulin) are indicated. n = 4 independent experiments.


A . High - performance liquid chromatography ( HPLC ) coupled to mass spectrometry ( MS ) used to quantify Elongator - dependent tRNA modification 5 - carbamoylmethyluridine ( ncm5U ) in human fibroblasts . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . B . HPLC - MS of Elongator - dependent tRNA modification 5 - methoxy - carbonylmethyluridine ( mcm5U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . C . HPLC - MS of Elongator - dependent tRNA modification 5 - methoxycarbonylmethyl - 2 - thiouridine ( mcm5s2U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) used to quantify the Elongator-dependent tRNA modification 5-carbamoylmethyluridine (ncm5U) in human fibroblasts. Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. B. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxy-carbonylmethyluridine (mcm5U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. C. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.


D . HPLC - MS of Elongator - independent tRNA modification 1 - methyladenosine ( m1A ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . E . HPLC - MS of Elongator - independent tRNA modification 7 - methylguanosine ( m7G ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± .

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

D. HPLC-MS quantification of Elongator-independent tRNA modification 1-methyladenosine (m1A). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. E. HPLC-MS quantification of Elongator-independent tRNA modification 7-methylguanosine (m7G). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.


F . Differential expression ( DE ) of peptides in fibroblasts obtained from individuals with ELP4 ELP6 variants relative to control ( n = 3 technical repeats per genotype ) . Down - ( red ) upregulated ( blue ) peptides are depicted on volcano plots .

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F. Differential expression (DE) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control (n = 3 technical repeats per genotype). Down- (red) and upregulated (blue) peptides are depicted on volcano plots.


A . Representative of Golgi - Cox - stained brain sections in hippocampal of adult ( 2 - months - old ) wild - type mutant mice . Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction subsequent analyses . Scale bar : 100 μm . Abbreviations : DG - dentate gyrus , CA1 - cornu ammonis 1 , CA2 - cornu ammonis

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A. Representative images of Golgi-Cox-stained brain sections in the hippocampal area of adult (2-months-old) wild-type and mutant mice. Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses. Scale bar: 100 μm. Abbreviations: DG - dentate gyrus, CA1 - cornu ammonis 1, CA2 - cornu ammonis 2


C . of total dendritic from dendritic tree reconstructions shown in B . n = 15 ; 5 neurons per 3 per genotype . D . Sholl of complexity of basal dendritic arbors . n = 15 ; 5 neurons per 3 per genotype . Data information : Statistical : unpaired two - t - ( α = 0 . 05 ) with Welch ' s correction . Holm - Sidak correction was applied to adjust for multiple comparisons ( D ) . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ± ( with error bars in D removed when smaller than corresponding data point ) .

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C. Quantification of total dendritic length from dendritic tree reconstructions shown in B. n = 15; 5 neurons per animal and 3 animals per genotype. D. Sholl analysis of the complexity of basal dendritic arbors. n = 15; 5 neurons per animal and 3 animals per genotype. Data information: Statistical analysis: unpaired two-tailed t-test (α = 0.05) with Welch's correction. Holm-Sidak correction was applied to adjust for multiple comparisons (D). Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM (with error bars in D removed when smaller than the corresponding data point).


E . Representative of dendritic spines on basal dendrites of cortical neurons . of spine density per 10 μm on secondary dendrites is shown . n = 15 ; 5 neurons per 3 per genotype . Scale bar : 10 μm . Data information : Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ±

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E. Representative images of dendritic spines on basal dendrites of the cortical neurons. Quantification of spine density per 10 μm on secondary dendrites is shown. n = 15; 5 neurons per animal and 3 animals per genotype. Scale bar: 10 μm. Data information: Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM


( A - D ) Relative growth of ( top ) or western blot of YAP in ( bottom ) indicated prostate cancer lines expressing control , YAP - WT , or YAP - 5SA constructs through lentiviral infection . Of note , LNCaP C4 - 2 express full - AR while 22RV1 R1 - D567 express AR splicing variants V7 V567ES , respectively . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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(A-D) Relative growth of (top) or western blot analysis of YAP in (bottom) the indicated prostate cancer cell lines expressing control, YAP-WT, or YAP-5SA constructs through lentiviral infection. Of note, LNCaP and C4-2 express full-length AR while 22RV1 and R1-D567 express AR splicing variants V7 and V567ES, respectively. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( M ) Western blot of LATS1 , LATS2 , YAP , phosphorylated YAP at S109 ( pS109 ) S127 ( pS127 ) in C4 - 2 treated with two independent LATS1 / 2 siRNAs .

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(M) Western blot analysis of LATS1, LATS2, YAP, phosphorylated YAP at S109 (pS109) and S127 (pS127) in C4-2 cells treated with two independent LATS1/2 siRNAs.


( Q , Western blot of LATS1 , LATS2 , YAP in ( Q ) C4 - 2 treated with indicated siRNA .

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(Q, Western blot analysis of LATS1, LATS2, and YAP in (Q) C4-2 cells treated with the indicated siRNA.


( E ) Relative mRNA of AR indicated AR target genes ( left ) western blot of AR ( right ) in C4 - 2 treated with increasing concentrations of XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

(E) Relative mRNA levels of AR and the indicated AR target genes (left) and western blot analysis of AR (right) in C4-2 cells treated with increasing concentrations of XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( G ) Relative mRNA of indicated AR or YAP target genes in C4 - 2 treated with control ( siNC ) or YAP siRNA ( siYAP _ 1 or siYAP _ 2 ) for 24 hours with or without 2 μM XMU - MP - 1 for 12 hours . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

(G) Relative mRNA levels of the indicated AR or YAP target genes in C4-2 cells treated with control (siNC) or YAP siRNA (siYAP_1 or siYAP_2) for 24 hours and with or without 2 μM XMU-MP-1 for 12 hours. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Relative mRNA of indicated AR or YAP target genes ( J , in C4 - 2 ( J , expressing control ( NC ) , YAP - WT or YAP - 5SA constructs through lentiviral infection . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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Relative mRNA levels of the indicated AR or YAP target genes (J, in C4-2 (J, cells expressing control (NC), YAP-WT or YAP-5SA constructs through lentiviral infection. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( A ) Western blot of AR ( left ) of AR ( right ) in C4 - 2 treated CHX for indicated time .

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(A) Western blot analysis of AR (left) and quantification of AR (right) in C4-2 cells treated CHX for the indicated time.


Western blot of AR YAP ( I , in C4 - 2 stably expressing Tet - O - YAP - 5SA treated with 0 . 2 μg / ml doxycycline ( Dox ) for indicated periods of time . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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Western blot analysis of AR and YAP (I, in C4-2 cells stably expressing Tet-O-YAP-5SA and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Western blot of AR YAP L ) in C4 - 2 stably expressing Tet - O - YAP - 5SAS94A treated with 0 . 2 μg / ml doxycycline ( Dox ) for indicated periods of time .

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Western blot analysis of AR and YAP L) in C4-2 cells stably expressing Tet-O-YAP-5SAS94A and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time.


relative mRNA of TEAD1 TEAD4 indicated AR target genes ( B ) C4 - 2 treated with two independent TEAD1 / 3 / 4 siRNA or control siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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relative mRNA levels of TEAD1 and TEAD4 and the indicated AR target genes (B) C4-2 cells treated with two independent TEAD1/3/4 siRNA or control siRNA. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( D ) Relative mRNA of indicated AR target genes ( left ) western blot of TEAD ( right ) in C4 - 2 expression control or TEAD4 constructs treated with vehicle or XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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(D) Relative mRNA levels of the indicated AR target genes (left) and western blot analysis of TEAD (right) in C4-2 cells expression the control or TEAD4 constructs and treated with vehicle or XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( H - J ) HEK - 293T transfected with indicated constructs . lysates were immunoprecipitated with indicate antibodies . Immunoprecipitates lysates ( input ) were analyzed by western blot with indicated antibodies . ( K ) In vitro binding using GST or GST - TEAD4 purified from Flag - AR immunopurified from HEK293 .

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(H-J) HEK-293T cells transfected with the indicated constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies. (K) In vitro binding assay using GST or GST-TEAD4 purified from bacteria and Flag-AR immunopurified from HEK293 cells.


HEK - 293T transfected with indicated AR , TEAD constructs . lysates were immunoprecipitated with indicate antibodies . Immunoprecipitates lysates ( input ) were analyzed by western blot with indicated antibodies .

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HEK-293T cells transfected with the indicated AR, TEAD constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.


( O - P ) ChIP - qPCR ( O ) RT - PCR analyses show that exogenous expression of wild type ( TEAD4 - WT ) but not binding deficient form ( TEAD4m ) of TEAD4 rescued AR binding to promoter / enhancer regions of KLK2 KLK3 ( O ) as well as expression of these genes ( P ) in C4 - 2 treated with TEAD1 / 3 / 4 siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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(O-P) ChIP-qPCR (O) and RT-PCR analyses show that exogenous expression of wild type (TEAD4-WT) but not the DNA binding deficient form (TEAD4m) of TEAD4 rescued AR binding to the promoter/enhancer regions of KLK2 and KLK3 (O) as well as the expression of these genes (P) in C4-2 cells treated with TEAD1/3/4 siRNA. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( D ) YAP - 5SA but not YAP - 5SAS94A inhibited AR / TEAD interaction . C4 - 2 stably expressing Tet - O - GFP , Tet - O - YAP - 5SA or Tet - O - YAP - 5SAS94A were treated with 0 . 2 μg / ml doxycycline ( Dox ) for 10 hours , followed by CoIP western blot analyses with indicated antibodies .

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(D) YAP-5SA but not YAP-5SAS94A inhibited AR/TEAD interaction. C4-2 cells stably expressing Tet-O-GFP, Tet-O-YAP-5SA or Tet-O-YAP-5SAS94A were treated with 0.2 μg/ml doxycycline (Dox) for 10 hours, followed by CoIP and western blot analyses with the indicated antibodies.


( G - H ) ChIP - qPCR of AR ( G ) TEAD ( H ) binding to promoter / enhancer regions of KLK2 , KLK3 CTGF in Tet - O - YAP - 5SA expressing C4 - 2 treated with 0 . 2 μg / ml doxycycline ( Dox ) for indicated time . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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(G-H) ChIP-qPCR analysis of AR (G) and TEAD (H) binding to the promoter/enhancer regions of KLK2, KLK3 and CTGF in Tet-O-YAP-5SA expressing C4-2 cells treated with 0.2 μg/ml doxycycline (Dox) for the indicated time. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, **P<0.01, ***P<0.001 (Student's t-test).


Western blot of YAP TEAD ( left ) mRNA of indicated genes ( right ) in C4 - 2 treated with siYAP / TAZ for 18 ( A ) , 36 ( B ) , hours . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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Western blot analysis of YAP and TEAD (left) and mRNA levels of the indicated genes (right) in C4-2 cells treated with siYAP/TAZ for 18 (A), 36 (B), hours. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( A - F ) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4 - 2 or 22RV1 tumors that stably express Tet - O - YAP - 5SA . NOD scid gamma ( NSG ) mice bearing C4 - 2 stably expressing a Tet - O - EGFP construct were used as control . NOD scid gamma ( NSG ) mice bearing indicated tumors were injected i . p . with PBS containing Dox ( 20mg / kg ) or PBS daily for indicated time . growth curve ( A , D ) , photograph of samples ( B , E ) , of ( C , F ) at end of treatment were shown . XMU - MP - 1 inhibited AR positive prostate cancer growth in xenografts bearing C4 - 2 ( G - I ) , tumors . NOD scid gamma ( NSG ) mice bearing indicated tumors were treated daily with XUM - MP - 1 at indicated concentrations or with vehicle for indicated periods of time . growth curve ( G , J , of samples ( H , K , of ( I at end of treatment were shown . Data information : Data are ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . n = 7 mice for each group .

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(A-F) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4-2 or 22RV1 tumors that stably express Tet-O-YAP-5SA. Male NOD scid gamma (NSG) mice bearing C4-2 tumor cells stably expressing a Tet-O-EGFP construct were used as control. Male NOD scid gamma (NSG) mice bearing the indicated tumors were injected i.p. with PBS containing Dox (20mg/kg) or PBS daily for the indicated time. Tumor growth curve (A, D), photograph of tumor samples (B, E), and quantification of tumor weight (C, F) at the end of treatment were shown. XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing C4-2 (G-I), tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 at the indicated concentrations or with vehicle for the indicated periods of time. Tumor growth curve (G, J, images of tumor samples (H, K, and quantification of tumor weight (I at the end of treatment were shown. Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.


XMU - MP - 1 inhibited AR positive prostate cancer growth in xenografts bearing 22RV1 tumors . NOD scid gamma ( NSG ) mice bearing indicated tumors were treated daily with XUM - MP - 1 or with vehicle for indicated periods of time . growth curve J , Data information : Data are ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . n = 7 mice for each group .

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

XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing 22RV1 tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 or with vehicle for the indicated periods of time. Tumor growth curve J, Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.


THP - 1 derived macrophages were infected with wildtype ( Wt ) ΔF2 H5N1 ( VN ) at MOI 10 . Viral expression for PB1 - F2 ( A ) was detected by immunoblotting at 24 h post - infection ( hpi ) Equal loading was confirmed by detection of beta actin .

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THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for PB1-F2 (A) was detected by immunoblotting at 24 h post-infection (hpi) Equal loading was confirmed by detection of beta actin.


B THP - 1 derived macrophages were infected with wildtype ( Wt ) ΔF2 H5N1 ( VN ) at MOI 10 . Viral expression for NP ( B ) was detected by immunoblotting at indicated time points , respectively . Equal loading was confirmed by detection of beta actin .

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B THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for NP (B) was detected by immunoblotting at indicated time points, respectively. Equal loading was confirmed by detection of beta actin.


( C ) THP - 1 derived macrophages were infected with wildtype ( Wt ) ΔF2 H5N1 ( VN ) at MOI 10 . of viral M1 mRNA expression were detected by RT q ‑ PCR at 16 hpi . mean ± standard deviation of three independent biological samples is shown . Statistical was performed by students t - .

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(C) THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Levels of viral M1 mRNA expression were detected by RT q‑PCR at 16 hpi. The mean ± standard deviation of three independent biological samples is shown. Statistical analysis was performed by students t-test.


( D ) THP - 1 derived macrophages were infected with VN PR8 Wt ΔF2 strains for 24 h at MOI 10 . of IL - 1β were quantified by ELISA from supernatants . mean ± standard deviation of seven independent experiments is shown . Statistical was performed by one - way ANOVA , p - values are indicated .

(D) THP-1 derived macrophages were infected with VN and PR8 Wt and ΔF2 strains for 24 h at MOI 10. Levels of IL-1β were quantified by ELISA from the supernatants. The mean ± standard deviation of seven independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( E ) THP - 1 control knockout for NLRP3 were mock treated or infected with VN Wt ΔF2 strains for 24 h at MOI 10 IL - 1β were assessed by ELISA from supernatants . mean ± standard deviation of five independent experiments is shown . Statistical was performed by one - way ANOVA , p - values are indicated .

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(E) THP-1 control and knockout cells for NLRP3 were mock treated or infected with the VN Wt and ΔF2 strains for 24 h at MOI 10 and IL-1β levels were assessed by ELISA from the supernatants. The mean ± standard deviation of five independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( F ) Immunoblot detection for NLRP3 on control NLRP3 knockout THP - 1 . Equal loading was confirmed by specific blotting against beta actin . Representative blot of three independent repeats is shown .

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(F) Immunoblot detection for NLRP3 on control and NLRP3 knockout THP-1 cells. Equal loading was confirmed by specific blotting against beta actin. Representative blot of three independent repeats is shown.


( G ) Human macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt ΔF2 for 5 h at MOI 10 . of IL - 1β were measured by ELISA in technical replicates . mean of two independent experiments is shown . Statistical was performed by one - way ANOVA , p - values are indicated .

(G) Human primary macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt and ΔF2 for 5 h at MOI 10. Levels of IL-1β were measured by ELISA in technical replicates. The mean of two independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( H ) THP - 1 derived macrophages were infected with a human H3N2 IAV ( Wyo ) Wt ΔF2 for 24 h at MOI 4 . of IL - 1β were measured by ELISA from supernatants . mean ± standard deviation of four independent experiments is shown . Statistical was performed by one - way ANOVA , p - values are indicated .

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(H) THP-1 derived macrophages were infected with a human H3N2 IAV strain (Wyo) Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation of four independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( I ) Detection of viral matrix M1 by immunoblotting in infected with H3N2 Wt ΔF2 strains .

(I) Detection of viral matrix protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains.


( J ) Human macrophages isolated differentiated from PBMCs from three healthy donors were infected with H3N2 Wt ΔF2 for 24 h at MOI 4 . of IL - 1β were measured by ELISA . mean ± standard deviation of three independent experiments is shown . Statistical was performed by one - way ANOVA , p - values are indicated .

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(J) Human primary macrophages isolated and differentiated from PBMCs from three healthy donors were infected with H3N2 Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


human alveolar macrophages from BALF of five pediatric ( are color coded ) were infected with human H3N2 ( Wyo ) Wt ΔF2 for 16 h at MOI 1 . ( K ) of IL - 1β were measured by ELISA from supernatants . mean ± standard deviation is shown .

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Primary human alveolar macrophages from BALF of five pediatric patients (patients are color coded) were infected with the human H3N2 strain (Wyo) Wt and ΔF2 for 16 h at MOI 1. (K) Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation is shown.