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 .

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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 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 .

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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 + 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 .

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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 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 .

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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 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 .

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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 the sorted populations , assessed by flow cytometry , is indicated above the 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 analysis 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 ) and scored for alkaline phosphatase after 6d . Error bars : standard deviation of the 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 analysis 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 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 .

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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 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

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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 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

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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 : 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 .

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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 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 .

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

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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 counts per base pair were normalised by library size and by subtracting the background ( IgG ) signal . EsrrbHi ( EH ) , EsrrbMed ( EM ) and 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 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 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 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 .

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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 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

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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 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 .

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: 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 and 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 all 60 raw files . The color coding highlights the 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 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 .

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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 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 ) .

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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 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 .

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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 weight and the amount of protein extracted by ultrasonication .

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


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

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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 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 .

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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 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 .

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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 analysis 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 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

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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 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 ) .

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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).


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 .

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

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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 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 ) .

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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 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 ) ) .

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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)).


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 ) .

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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).


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 .

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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.


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 .

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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.


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 .

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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 ) , and TH / SYNAPSIN - stained ( C ) images in the cultures terminally differentiated from mutant ( c13 , c19 ) and corrected hiPSCs ( c22 , c59 ) .

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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 levels were measured in the media conditioned in the differentiated cultures for 24 h ( D15 ) and evoked by KCl - mediated depolarization for 30 min .

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.


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

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


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

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 membrane 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 . 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 .

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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 . 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 .

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

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

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


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 .

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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 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 ) .

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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).


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 .

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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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 cells transfected with pFLAG - WT - LIN28A and 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 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

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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 the 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 the overlap between the YAP signatures obtained in Oral SCC and Breast ( YAP Signature 1 ) and CRC and 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 the overlap between YAP Signature 1 and the 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 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 ]

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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 the genes commonly upregulated in YAP signature 1 and in BCCs , well - differentiated and 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 the overlap between YAP signature 2 and the 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 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 [

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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 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

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

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 ) 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 .

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

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

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

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 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 .

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

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 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 .

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 ±

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

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

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

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

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

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

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


( 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 ) .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

(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 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 ) .

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

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

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

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


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 ) .

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

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 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 .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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

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 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 ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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

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 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 ) .

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

(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 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 .

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

(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 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 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

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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O B-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 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-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 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 .

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

(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 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 ) .

O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 ) .

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

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 . 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 .

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

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

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O 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) 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 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 .

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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 and NLRP3 knockout THP - 1 cells . 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 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 .

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(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 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 .

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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 protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains .

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


( 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 .

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

(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.


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 .

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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.


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 . Levels of viral M1 mRNA ( L ) , Mx1 mRNA ( M ) and IP10 mRNA ( N ) expression were detected by RT q ‑ PCR . The mean ± standard deviation for cells from four patients are shown . Individiual patients are color coded .

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

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. Levels of viral M1 mRNA (L), Mx1 mRNA (M) and IP10 mRNA (N) expression were detected by RT q‑PCR. The mean ± standard deviation for cells from four patients are shown. Individiual patients are color coded.


( A - D ) THP - 1 cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Flow cytometry results for annexin V single positive ( A and C ) and annexin V and zombie dye double positive ( B and D ) cells . Percentage of positive cells are depicted at 12 hpi or 24hpi .

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(A-D) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Flow cytometry results for annexin V single positive (A and C) and annexin V and zombie dye double positive (B and D) cells. Percentage of positive cells are depicted at 12 hpi or 24hpi.


( E and F ) THP - 1 cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . LDH release measured from supernatants of mock treated or infected cells at 12 hpi ( E ) or 24hpi ( F ) .

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(E and F) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi (E) or 24hpi (F).


G THP - 1 Caspase 1 - / - cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi . n = 3 for 12hpi , n = 5 for 24hpi . Medians are indicated , statistical analysis was performed by one - way ANOVA .

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G THP-1 Caspase 1 -/- cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi. n=3 for 12hpi, n=5 for 24hpi. Medians are indicated, statistical analysis was performed by one-way ANOVA.


( A ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Detection of the full - length gasdermin D . Equal loading was confirmed by beta actin . One representative blot of five independent repeats is shown . ( B ) Quantification of band intensity for the gasdermin D full - length isoform . The mean ± standard deviation five independent experiments is shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , The asterisk ( * ) marks a significant difference when compared to the mock group * * p < 0 . 01 .

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(A) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of the full-length gasdermin D. Equal loading was confirmed by beta actin. One representative blot of five independent repeats is shown. (B) Quantification of band intensity for the gasdermin D full-length isoform. The mean ± standard deviation five independent experiments is shown. Statistical analysis was performed by the paired two‑tailed Student's t test., The asterisk (*) marks a significant difference when compared to the mock group ** p < 0.01.


( C ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Detection of cleaved gasdermin D N - terminal and C - terminal isoforms by immunoblot in supernatants ; one representative experiment of three independent repeats is shown . ( D ) Quantification of band intensity for N - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , * p < 0 . 05 . ( E ) Quantification of band intensity for C - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , * p < 0 . 05 .

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(C) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of cleaved gasdermin D N-terminal and C-terminal isoforms by immunoblot in supernatants; one representative experiment of three independent repeats is shown. (D) Quantification of band intensity for N-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (E) Quantification of band intensity for C-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05.


( F ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Pro - caspase - 1 and active caspase - 1 were detected by immunoblot in cell lysates ( cell , upper panels ) and supernatants ( sup , lower panels ) from the same experiment . One representative experiment of three independent repeats is shown . ( G ) Quantification of band intensity for active caspase - 1 band . Five independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . * p < 0 . 05 . ( H ) Levels of total pro - caspase - 1 were quantified on pellets and respective supernatants . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test .

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

(F) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Pro-caspase-1 and active caspase-1 were detected by immunoblot in cell lysates (cell, upper panels) and supernatants (sup, lower panels) from the same experiment. One representative experiment of three independent repeats is shown. (G) Quantification of band intensity for active caspase-1 band. Five independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. * p < 0.05. (H) Levels of total pro-caspase-1 were quantified on pellets and respective supernatants. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test.