Analysis of LepB94 translation products on SDS - PAGE . Translation products are visualized by the fluorescence of N - terminal Atto655 . Pausing intermediates are indicated P1 and P2 .

Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2.


Fitting of time courses ( filled circles ) of the translation of LepB mRNA constructs of increasing length ( Appendix Fig S3 ) by delay - exponential functions ( red lines ) .

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

Fitting of time courses (filled circles) of the translation of LepB mRNA constructs of increasing length (Appendix Fig S3) by delay-exponential functions (red lines).


Stopped - flow time courses of co - translational LepB insertion into SecYEG ( Atto488 donor - in at position 111 of SecY ) , as monitored by the fluorescence of the Atto655 acceptor placed at the N - terminus of the nascent peptide . Fits ( red lines ) were obtained by global fitting

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

Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting


Stopped - flow time courses of co - translational LepB insertion into SecYEG ( Atto488 donor - in at position 111 of SecY ) , as monitored by the fluorescence of the Atto655 acceptor placed at the N - terminus of the nascent peptide . Fits ( red lines ) were obtained by global

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


Intrinsic fluorescence intensities ( IFI ) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting ( red squares ) . Confidence contours ( shaded areas ) were calculated using the Fitspace

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

Intrinsic fluorescence intensities (IFI) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting (red squares). Confidence contours (shaded areas) were calculated using the Fitspace algorithm


Time course of EmrD135 translation . Translation was monitored by SDS - PAGE as in Fig 2A . Time course of EmrD ( - ) 135 translation .

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

Time course of EmrD135 translation. Translation was monitored by SDS-PAGE as in Fig 2A. Time course of EmrD(-)135 translation.


Stopped - flow time courses for translocon insertion of TM1 of EmrD ( black ) and EmrD ( - ) ( blue ) with mRNA constructs of varying chain length ( 50 to 135 aa ) . The FRET acceptor ( Atto655 ) was placed at the N - terminus of the nascent peptide , the donor ( Atto488 ) at position 111 ( donor - in ) of SecY . The results of global fitting are indicated ( red lines ) .

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

Stopped-flow time courses for translocon insertion of TM1 of EmrD (black) and EmrD(-) (blue) with mRNA constructs of varying chain length (50 to 135 aa).The FRET acceptor (Atto655) was placed at the N-terminus of the nascent peptide, the donor (Atto488) at position 111 (donor-in) of SecY. The results of global fitting are indicated (red lines).


Intrinsic fluorescence intensities ( IFIs ) calculated for EmrD ( black ) and EmrD ( - ) ( blue ) . Error margins ( shaded areas ) are calculated using the Fitspace

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

Intrinsic fluorescence intensities (IFIs) calculated for EmrD (black) and EmrD(-) (blue). Error margins (shaded areas) are calculated using the Fitspace algorithm


Stopped - flow traces for co - translational insertion of LepB94 ( red , from Fig 2 ) , EmrD135 ( black , from Fig 4E ) , and EmrD ( - ) 135 ( blue , from Fig 4E ) , rescaled according to the translation efficiency .

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

Stopped-flow traces for co-translational insertion of LepB94 (red, from Fig 2), EmrD135 (black, from Fig 4E), and EmrD(-)135 (blue, from Fig 4E), rescaled according to the translation efficiency.


A , B , C LepB ( A ) , EmrD ( B ) , and EmrD ( - ) ( C ) are translated for the times indicated and subjected to PK treatment ( Materials and Methods ) . Sensitivity to PK in the absence of SecYEG ( black circles ) or in the presence of SecYEG ( red squares ) , and co - translational translocon insertion monitored by FRET ( blue trace ) are depicted . Error bars represent standard deviations ( n ≥ 3 ) and the corresponding lines represent fits to delay - exponential functions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B, C LepB (A), EmrD (B), and EmrD(-) (C) are translated for the times indicated and subjected to PK treatment (Materials and Methods). Sensitivity to PK in the absence of SecYEG (black circles) or in the presence of SecYEG (red squares), and co-translational translocon insertion monitored by FRET (blue trace) are depicted. Error bars represent standard deviations (n≥3) and the corresponding lines represent fits to delay-exponential functions.


DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB IL - 2nil ( 32694 ) ordered by increasing fold change of tag count for TB IL - 2 compared to TB IL - 2nil . The sequence tag density is also shown in parallel at the same sites for the DNase - Seq analyses of CD4 naïve T cells ( TN ) , TB IL - 2 DMSO and TB IL - 2 Ruxolitinib and ATAC samples from in vivo derived Th2 ( Shih et al . , 2016 ) , Th1 and Th17 cells . The location of the 1000 IL - 2 pDHSs is shown to the right .

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

DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right.


Average DNase - Seq tag density profile for the 1000 IL - 2 pDHSs in both replicates of TB IL - 2 and TB IL - 2nil samples , and for naïve T cells ( TN ) .

O O O O O O O O O O O O O O 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 DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-2 and TB IL-2nil samples, and for naïve T cells (TN).


Number of IL - 2 pDHSs which are detected in in vivo purified CD4 T cell subsets ( Chen et al . , 2020 , Garg et al . , 2019 , Shih et al . , 2016 ) .

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

Number of IL-2 pDHSs which are detected in in vivo purified CD4 T cell subsets (Chen et al., 2020, Garg et al., 2019, Shih et al., 2016).


UCSC genome browser tracks with examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the Rad50 gene which controls the Th2 cytokines Il13 , Il4 and Il5 , plus IL - 2 pDHSs within the Tnfrsf9 and Cish loci . IL - 2 pDHSs are highlighted with blue boxes .

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

UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.


Jund mRNA levels in TB IL - 2 and TB IL - 2nil . Standard deviation is shown from the mean of 3 samples . P - values were calculated from the RNA - seq data using Limma with Benjamini - Hochberg correction for multiple testing .

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

Jund mRNA levels in TB IL-2 and TB IL-2nil. Standard deviation is shown from the mean of 3 samples. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.


Western blot analyses showing JUND and pSTAT5 protein levels in TB IL - 2 and TB IL - 2nil ( left panel ) with the signal calculated from 2 replicates relative to TB IL - 2 in the right panel .

Western blot analyses showing JUND and pSTAT5 protein levels in TB IL-2 and TB IL-2nil (left panel) with the signal calculated from 2 replicates relative to TB IL-2 in the right panel.


DNA motif density plots ( right ) showing the location of binding motifs for RUNX , ETS , STAT and AP - 1 transcription factors at the 32694 DHSs detected in TB IL - 2 and TB IL - 2nil , as depicted in Figure 2A , ordered according to fold enrichment of DNase - Seq tag counts in TB IL - 2 compared to TB IL - 2nil ( left ) .

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

DNA motif density plots (right) showing the location of binding motifs for RUNX, ETS, STAT and AP-1 transcription factors at the 32694 DHSs detected in TB IL-2 and TB IL-2nil, as depicted in Figure 2A, ordered according to fold enrichment of DNase-Seq tag counts in TB IL-2 compared to TB IL-2nil (left).


DNA sequence tag density plots from ChIP - Seq assays of RUNX1 , ETS1 , STAT5 and JUND in TB IL - 2 , and STAT5 and JUND in TB IL - 2nil ordered as in ( D ) .

DNA sequence tag density plots from ChIP-Seq assays of RUNX1, ETS1, STAT5 and JUND in TB IL-2, and STAT5 and JUND in TB IL-2nil ordered as in (D).


UCSC genome browser tracks at the Il13 / Il4 / Il5 Th2 cytokine locus LCR ( indicated by the grey box within the Rad50 gene ) , Gzmb and Il24 showing DNase - Seq and ChIP - Seq for STAT5 and JUND in TB IL - 2 and TB IL - 2nil . Replicate JUND and STAT5 ChIP - Seq tracks are shown for TB IL - 2 .

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UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.


DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB + IL - 2 ( A , 38645 ) and in TB + IL - 2 and TB + IL - 2nil ( B , 36067 ) , ordered by increasing fold change in tag count as indicated . The position of the 1015 IL - 2 - dependent inducible DHSs is shown alongside .

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

DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB+ IL-2 (A, 38645) and in TB+ IL-2 and TB+ IL-2nil (B, 36067), ordered by increasing fold change in tag count as indicated. The position of the 1015 IL-2-dependent inducible DHSs is shown alongside.


Average DHS signal at the 1015 IL - 2 - dependent inducible DHSs in TB IL - 2 , TB + IL - 2 , TB + IL - 2nil and naïve T cells stimulated with PMA / I ( TN + ) .

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

Average DHS signal at the 1015 IL-2-dependent inducible DHSs in TB IL-2, TB+ IL-2, TB+ IL-2nil and naïve T cells stimulated with PMA/I (TN+).


Bar graph showing the number of IL - 2 iDHSs found within 25 kb of an IL2 pDHS .

Bar graph showing the number of IL-2 iDHSs found within 25 kb of an IL2 pDHS.


Examples of IL - 2 iDHSs which are proximal to IL - 2 pDHSs at the Csf2 / Il2 and Il10 loci . IL - 2 iDHSs are highlighted in red and IL - 2 pDHSs in blue .

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

Examples of IL-2 iDHSs which are proximal to IL-2 pDHSs at the Csf2/Il2 and Il10 loci. IL-2 iDHSs are highlighted in red and IL-2 pDHSs in blue.


Hierarchical clustering of RNA - Seq data from 3 replicates as described in ( A ) .

Hierarchical clustering of RNA-Seq data from 3 replicates as described in (A).


MA plot showing the genes differentially regulated between TB IL - 2nil and TB IL - 2 . The number of genes with a fold change > 3 and adj p - value < 0 . 05 are highlighted . P - values were calculated from the RNA - seq data using Limma with Benjamini - Hochberg correction for multiple testing .

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

MA plot showing the genes differentially regulated between TB IL-2nil and TB IL-2. The number of genes with a fold change >3 and adj p-value <0.05 are highlighted. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.


log2 fold change in gene expression in TB IL - 2nil compared to TB IL - 2 for the nearest gene to the DHSs ranked according to the fold change in DNase - Seq tag count for TB IL - 2 / TB IL - 2nil . The DNase - Seq tag density plots are taken from Figure 2A for TB IL - 2 , TB IL - 2nil and TN .

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

log2 fold change in gene expression in TB IL-2nil compared to TB IL-2 for the nearest gene to the DHSs ranked according to the fold change in DNase-Seq tag count for TB IL-2/TB IL-2nil. The DNase-Seq tag density plots are taken from Figure 2A for TB IL-2, TB IL-2nil and TN.


UCSC genome browser tracks showing DNase - seq and RNA - seq at the Socs1 ( E ) and Sell loci ( F ) .

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

UCSC genome browser tracks showing DNase-seq and RNA-seq at the Socs1 (E) and Sell loci (F).


Relative mRNA expression levels in TB + IL - 2 and TB + IL - 2nil ( A ) and TB + DMSO and TB + Ruxolitinib ( B ) . The expression level in the IL - 2 / DMSO TB + sample is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - test with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . )

O O 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

Relative mRNA expression levels in TB+ IL-2 and TB+ IL-2nil (A) and TB+ DMSO and TB+ Ruxolitinib (B). The expression level in the IL-2/DMSO TB+ sample is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)


UCSC genome browser tracks showing DNase - Seq and RNA - Seq at the IL - 2 regulated genes Csf2 / Il3 and at the LCR within the Rad50 gene ( grey box ) which controls the Th2 cytokines Il13 , Il4 and Il5 . IL - 2 pDHSs are highlighted in blue and IL - 2 iDHSs in red .

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

UCSC genome browser tracks showing DNase-Seq and RNA-Seq at the IL-2 regulated genes Csf2/Il3 and at the LCR within the Rad50 gene (grey box) which controls the Th2 cytokines Il13, Il4 and Il5. IL-2 pDHSs are highlighted in blue and IL-2 iDHSs in red.


DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB IL ‑ 2nil ( data taken from Figure 2A ) , duplicate DNase - seq samples for TB IL - 7 and TB IL - 7nil are shown in parallel for the same sites .

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

DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL‑2nil (data taken from Figure 2A), duplicate DNase-seq samples for TB IL-7 and TB IL-7nil are shown in parallel for the same sites.


Average DNase - Seq tag density profile for the 1000 IL - 2 pDHSs in both replicates of TB IL - 7 and TB IL - 7nil .

O O O O 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 DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-7 and TB IL-7nil.


UCSC genome browser tracks with DNase - Seq data for TB IL - 7 and TB IL - 7nil showing examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the Rad50 gene which controls the Th2 cytokines Il13 , Il4 and Il5 , plus IL - 2 pDHSs within the Tnfrsf9 and Cish loci ( C ) IL - 2 pDHSs are highlighted with blue boxes .

UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C) IL-2 pDHSs are highlighted with blue boxes.


UCSC genome browser tracks with DNase - Seq data for TB IL - 7 and TB IL - 7nil showing examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the non - primed control regions from the CD3 gene cluster and the Tbp locus ( D ) . IL - 2 pDHSs are highlighted with blue boxes .

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

UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the non-primed control regions from the CD3 gene cluster and the Tbp locus (D). IL-2 pDHSs are highlighted with blue boxes.


Average DHS signal at the 1015 IL - 2 regulated inducible DHSs in TB + IL - 7 and TB + IL - 7nil .

Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil.


Relative mRNA expression levels in TB + IL - 7 and TB + IL - 7nil . The expression level in the TB + IL - 7 is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - test with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression levels in TB+ IL-7 and TB+ IL-7nil. The expression level in the TB+ IL-7 is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)


Flow cytometric analysis of IL - 7Rα expression by 2W1S - specific CD44hi CD4 T cells in the presence or absence of cre recombinase .

Flow cytometric analysis of IL-7Rα expression by 2W1S-specific CD44hi CD4 T cells in the presence or absence of cre recombinase.


Average number of 2W1S - specific cells recovered / mouse after sorting 3 replicate samples of Il7rf / f compared to CD4creERT2 x Il7rf / f . Errors bars indicate the standard deviation .

O O O O O O O O O O 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 number of 2W1S-specific cells recovered/mouse after sorting 3 replicate samples of Il7rf/f compared to CD4creERT2 x Il7rf/f . Errors bars indicate the standard deviation.


Average ATAC - Seq signal at the 411 IL - 2 pDHSs which contain a STAT binding motif .

Average ATAC-Seq signal at the 411 IL-2 pDHSs which contain a STAT binding motif.


UCSC genome browser tracks at loci with IL - 2 pDHSs showing replicate ATAC - Seq samples for CD4creERT2 x Il7rf / f memory T cells , Il7rf / f control memory T cells , replicate DNase - Seq samples for TB IL - 2 and TB IL - 2nil and TB IL - 2 and TB IL ‑ 2nil STAT5 ChIP - Seq samples . The grey boxes signify IL - 2 pDHSs which diminish after disruption of the Il7r gene .

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

UCSC genome browser tracks at loci with IL-2 pDHSs showing replicate ATAC-Seq samples for CD4creERT2 x Il7rf/f memory T cells, Il7rf/f control memory T cells, replicate DNase-Seq samples for TB IL-2 and TB IL-2nil and TB IL-2 and TB IL‑2nil STAT5 ChIP-Seq samples. The grey boxes signify IL-2 pDHSs which diminish after disruption of the Il7r gene.


UCSC genome browser tracks showing ChIP - Seq and DNase - Seq at the LCR within the Rad50 gene which controls the Il4 / Il13 / Il5 Th2 cytokine locus , and also Il17a , Il12rb2 and Tbx21 . The grey boxes indicate IL - 2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages .

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

UCSC genome browser tracks showing ChIP-Seq and DNase-Seq at the LCR within the Rad50 gene which controls the Il4/Il13/Il5 Th2 cytokine locus, and also Il17a, Il12rb2 and Tbx21. The grey boxes indicate IL-2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages.


mRNA expression levels of JUN family transcription factors . Normalised TPM is shown for TB IL - 2 , TB IL2nil , TB + IL - 2 and TB + IL - 2nil . Standard deviation is shown for 3 replicates .

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

mRNA expression levels of JUN family transcription factors. Normalised TPM is shown for TB IL-2, TB IL2nil, TB+ IL-2 and TB+ IL-2nil. Standard deviation is shown for 3 replicates.


Average JUNB ChIP - Seq signal at the 1000 IL - 2 pDHSs in TB IL - 2 and TB + IL - 2 ( Bevington et al . , 2016 ) .

O O O O O O O O O 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 JUNB ChIP-Seq signal at the 1000 IL-2 pDHSs in TB IL-2 and TB+ IL-2 (Bevington et al., 2016).


Average JUND ChIP - Seq signal at the 1000 IL - 2 pDHSs in Junb + / + CD4Cre ( Junb WT ) and Junbf / fCD4Cre ( Junb KO ) Th17 cells .

Average JUND ChIP-Seq signal at the 1000 IL-2 pDHSs in Junb+/+CD4Cre (Junb WT) and Junbf/fCD4Cre (Junb KO) Th17 cells.


Genome browser tracks showing open chromatin ( DNase - Seq or ATAC - Seq ) and ChIP - Seq in Th0 , Th1 and Th17 cells at the Il17a locus ( Carr et al . , 2017 , Ciofani et al . , 2012 , Vahedi et al . , 2012 ) . The grey box indicates a DHS which binds JUND .

Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Il17a locus (Carr et al., 2017, Ciofani et al., 2012, Vahedi et al., 2012). The grey box indicates a DHS which binds JUND.


Fold change in gene expression in TB + IL - 2 cells for Jund - / - compared to Jund + / + mice . Data from 2 replicates 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

Fold change in gene expression in TB+ IL-2 cells for Jund -/- compared to Jund+/+ mice. Data from 2 replicates are shown.


Genome browser tracks showing open chromatin ( DNase - Seq or ATAC - Seq ) and ChIP - Seq in Th0 , Th1 and Th17 cells at the Irf8 locus . The grey box indicates an IL - 2 pDHS which binds JUND .

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

Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Irf8 locus. The grey box indicates an IL-2 pDHS which binds JUND.


A , B . A missense mutation in SPOP gene ( Q165P , T > G ) was identified in a PCa patient . Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation ( A ) whereas the liver metastasis harbored a homozygous Q165P mutation ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. A missense mutation in SPOP gene (Q165P, T >G) was identified in a PCa patient. Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation (A) whereas the liver metastasis harbored a homozygous Q165P mutation (B).


D , E . IHC analysis of BRD4 protein expression in SPOP wild type ( WT ) and mutated ( MUT ) PCa patient samples . The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in ( D ) and the quantified IHC data are shown in ( E ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The red dot in ( E ) indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) .

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

D, E. IHC analysis of BRD4 protein expression in SPOP wild type (WT) and mutated (MUT) PCa patient samples. The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in (D) and the quantified IHC data are shown in (E). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The red dot in (E) indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).


F , G . IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples . The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in ( F ) and the quantified IHC data are shown in ( G ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The green dot indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples. The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in (F) and the quantified IHC data are shown in (G). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The green dot indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; *** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).


LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP ( A )

LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A)


LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination ( B ) .

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

LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination (B).


C . C4 - 2 cells were stably infected with the lenti - virus expressing empty vector ( EV ) or SPOP mutants and harvested for western blot analysis with the indicated antibodies .

C. C4-2 cells were stably infected with the lenti-virus expressing empty vector (EV) or SPOP mutants and harvested for western blot analysis with the indicated antibodies.


D . C4 - 2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies . T1 : tumor 1 ; T2 : tumor 2 .

O O O O O O O O O O O O O O O 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. C4-2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.


E . Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields .

E. Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields.


Co - IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant ( F )

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

Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F)


Co - IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P ( G ) .

Co-IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P (G).


A . DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis .

A. DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis.


B . MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

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

B. MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells . The survival curve showed IC50 for SPOP Q165P ( 0 . 69 µM ) and EV cells ( 1 . 08 µM ) ( C ) .

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

Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells. The survival curve showed IC50 for SPOP Q165P (0.69 µM) and EV cells (1.08 µM) (C).


DU145 cells expressing lenti - EV or lenti - HA - SPOP - Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantitative data shown in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 .

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

DU145 cells expressing lenti-EV or lenti-HA-SPOP-Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantitative data shown in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01.


F . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody . The enrichment of BRD4 at c - MYC enhancer and RAC1 promoter was analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody. The enrichment of BRD4 at c-MYC enhancer and RAC1 promoter was analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01.


G . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac - H3 antibody . The level of Ac - H3 at c - MYC enhancer and RAC1 promoter is analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac-H3 antibody. The level of Ac-H3 at c-MYC enhancer and RAC1 promoter is analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01.


B , C . SCID male mice with PDX tumors were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) , CPI - 637 ( 30 mg / kg ) , combination of JQ1 ( 50 mg / kg ) and CPI - 637 ( 30 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of drug treatment were photographed ( B ) and tumor growth are shown in ( C ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 001 .

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

B, C. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001.


PDX tumors in each treatment group were harvested for protein For western blot analysis , three tumors each group ( n = 3 ) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in ( D ) .

PDX tumors in each treatment group were harvested for protein For western blot analysis, three tumors each group (n = 3) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in (D).


PDX tumors in each treatment group were harvested for RNA extraction . For RT - qPCR , three tumors each group ( n = 3 ) were extracted RNA for RT - qPCR in ( E ) . Data are shown as means ± SEM . The P value comparing vehicle with each treatment group was calculated by unpaired two - tailed Student ' s t - test and indicated in the graph .

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

PDX tumors in each treatment group were harvested for RNA extraction. For RT-qPCR, three tumors each group (n = 3) were extracted RNA for RT-qPCR in (E). Data are shown as means ± SEM. The P value comparing vehicle with each treatment group was calculated by unpaired two-tailed Student's t-test and indicated in the graph.


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for western blot analysis with the indicated antibodies ( A ) Asterisk , SRC3 at expected molecular mass .

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for western blot analysis with the indicated antibodies (A) Asterisk, SRC3 at expected molecular mass.


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for IFC with p - AKT - S473 antibody ( B ) . E - cadherin antibody was used to indicate the cell membrane ( red ) and DAPI for nucleus ( blue ) . Scale bars : 25 µm .

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for IFC with p-AKT-S473 antibody (B). E-cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 µm.


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . The representative images of organoids after the treatment are shown in ( C )

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. The representative images of organoids after the treatment are shown in (C)


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . the quantified data of the organoid diameter are shown in ( D ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. the quantified data of the organoid diameter are shown in (D). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.


E , F . 313HR ( SPOP WT ) and 573R ( Q165P ) organoids were cultured for 5 days , followed by the treatment with the indicated inhibitors for 5 days . The organoids were stained with Caspase - 3 antibody ( green ) to detect apoptotic cells . E - cadherin antibody staining was used to define the cell membrane ( red ) and the nucleus was counterstained with DAPI ( blue ) . The representative images of cleaved Caspase - 3 IFC staining are shown in ( E ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

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

E, F. 313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with Caspase-3 antibody (green) to detect apoptotic cells. E-cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved Caspase-3 IFC staining are shown in (E). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


A . C4 - 2 PCa cells infected with virus expressing EV , SPOP Q165P or F133V were harvested for western blot analysis .

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

A. C4-2 PCa cells infected with virus expressing EV, SPOP Q165P or F133V were harvested for western blot analysis.


B MTS was performed to compare the cell growth between EV , Q165P , F133V C4 - 2 groups after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.


C . Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4 - 2 cells . The survival curve showed IC50 for EV ( 1 . 01 µM ) , SPOP Q165P ( 0 . 621 µM ) and SPOP F133V cells ( 0 . 395 µM ) .

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

C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM).


D , E . C4 - 2 cells expressing EV , Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantification data in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


F - H . A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration ( F ) . When the tumor reached 100 mm3 , mice were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of treatment were photographed ( G ) and tumor growth are shown in ( H ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 ; * * * P < 0 . 001 .

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

F - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001.


( B ) Co - immunoprecipitation of HA - tagged Btz - WT or Btz - HD with Myc - tagged GFP or eIF4AIII from transfected S2R + cells using Myc - Trap Magnetic Agarose beads . The upper Western blot is blotted with anti - Myc and the lower with anti - HA . Input samples are shown on the left and immunoprecipitates on the right . Btz - WT co - immunoprecipitates with eIF4AIII , but Btz - HD does not , although the protein is equally stable . The asterisk represents a likely cleavage product of Btz .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation of HA-tagged Btz-WT or Btz-HD with Myc-tagged GFP or eIF4AIII from transfected S2R+ cells using Myc-Trap Magnetic Agarose beads. The upper Western blot is blotted with anti-Myc and the lower with anti-HA. Input samples are shown on the left and immunoprecipitates on the right. Btz-WT co-immunoprecipitates with eIF4AIII, but Btz-HD does not, although the protein is equally stable. The asterisk represents a likely cleavage product of Btz.


( C - F ) show stage 10 egg chambers stained with anti - Vasa ( C - F ) or anti - GFP ( E ' , F ' ) . ( C ) wild type ; ( D ) germline clones homozygous for the null allele btz2 ; ( E ) btz2 germline clones rescued with Btz - WT ; ( F ) btz2 germline clones rescued with Btz - HD . Posterior is to the right . Scale bars , 80 μm . Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction - defective transgene . Wild - type Btz - GFP , but not the mutant form , is also slightly enriched at the posterior pole .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) show stage 10 egg chambers stained with anti-Vasa (C-F) or anti-GFP (E', F'). (C) wild type; (D) germline clones homozygous for the null allele btz2; (E) btz2 germline clones rescued with Btz-WT; (F) btz2 germline clones rescued with Btz-HD. Posterior is to the right. Scale bars, 80 μm. Vasa localization to the posterior pole of the oocyte is lost in btz mutants and is rescued by the wild type but not the EJC interaction-defective transgene. Wild-type Btz-GFP, but not the mutant form, is also slightly enriched at the posterior pole.


( L - N ) electron micrographs showing cross - sections of larval muscle 6 in segment A3 , with the dorsal surface to the right . Scale bars , 10 μm . ( L ) btz2 / + ; ( M ) Df ( 3R ) BSC497 / + ; ( N ) btz2 / Df ( 3R ) BSC497 . Yellow arrows indicate examples of mitochondria interspersed between muscle fibers , and red arrows indicate mitochondria at the dorsal surface . Scale bars , 2 μm .

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

(L-N) electron micrographs showing cross-sections of larval muscle 6 in segment A3, with the dorsal surface to the right. Scale bars, 10 μm. (L) btz2/+; (M) Df(3R)BSC497/+; (N) btz2/ Df(3R)BSC497. Yellow arrows indicate examples of mitochondria interspersed between muscle fibers, and red arrows indicate mitochondria at the dorsal surface. Scale bars, 2 μm.


( O ) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated . * * * , p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant by Mann - Whitney test . n = 9 ( btz / + ) , n = 11 ( Df / + , btz / Df ) , n = 6 ( btz / Df ; G14 > btz , C57 - GAL4 / + ) , n = 4 ( btz / Df ; Btz - WT , C57 > eIF4AIII RNAi ) , n = 5 ( btz / Df ; Btz - HD ) , n = 10 ( C57 > btz RNAi ) , or n = 3 ( C57 > mago RNAi ) . Error bars show 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) quantification of the ratio of the dorsal surface peak of ATP5 intensity to the second highest peak for the genotypes indicated. ***, p<0.001; **p<0.01; *p<0.05; ns, not significant by Mann-Whitney test. n=9 (btz/+), n=11 (Df/+, btz/Df), n=6 (btz/Df; G14>btz, C57-GAL4/+), n=4 (btz/Df; Btz-WT, C57>eIF4AIII RNAi), n=5 (btz/Df; Btz-HD), n=10 (C57>btz RNAi), or n=3 (C57>mago RNAi). Error bars show mean ± SEM.


( A - J ) confocal images of the NMJ on larval muscles 6 and 7 in segment A3 , stained with anti - HRP ( magenta ) to label the nerve and anti - Dlg ( green ) as a postsynaptic marker of synaptic boutons . ( A ) w1118 control ; ( B ) btz2 / + ; ( C ) btz2 / Df ( 3R ) BSC497 ; ( D ) UAS - btz / + ; da - GAL4 , btz2 / Df ( 3R ) BSC497 ; ( E ) BG380 - GAL4 / + ; UAS - btz / + ; btz2 / Df ( 3R ) BSC497 ; ( F ) 24B - GAL4 / UAS - btz ; btz2 / Df ( 3R ) BSC497 ; ( G ) BG380 - GAL4 / + ; 24B - GAL4 / UAS - btz ; btz2 / Df ( 3R ) BSC497 ; ( H ) BG380 - GAL4 / + ; UAS - btz RNAi / + ; ( I ) mhc - GAL4 / UAS - btz RNAi ; ( J ) repo - GAL4 / UAS - btz RNAi . Scale bar , 30 μm . Insets in ( A , C ) show enlargements of the boxed regions , illustrating the closer spacing of boutons in btz mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) to label the nerve and anti-Dlg (green) as a postsynaptic marker of synaptic boutons. (A) w1118 control; (B) btz2/+; (C) btz2/Df(3R)BSC497; (D) UAS-btz/+; da-GAL4, btz2/Df(3R)BSC497; (E) BG380-GAL4/+; UAS-btz/+; btz2/Df(3R)BSC497; (F) 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (G) BG380-GAL4/+; 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (H) BG380-GAL4/+; UAS-btz RNAi/+; (I) mhc-GAL4/UAS-btz RNAi; (J) repo-GAL4/UAS-btz RNAi. Scale bar, 30 μm. Insets in (A, C) show enlargements of the boxed regions, illustrating the closer spacing of boutons in btz mutants.


( K , L ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; ns , not significant by unpaired t - test with Welch ' s correction . n = 23 ( w ; da - GAL4 , btz / Df ) , n = 6 ( btz / + ) , n = 10 ( Df / + , UAS - BtzGFP / + ; btz / Df ) , n = 8 ( btz / Df ) , n = 22 ( da > btz - GFP ; btz / Df ) , n = 13 ( BG380 - GAL4 ; btz / Df ; BG380 + 24B > Btz - GFP ; btz / Df ; da - GAL4 / + ) , n = 5 ( BG380 > Btz - GFP ; btz / Df ) , n = 11 ( 24B - GAL4 ; btz / Df ; da > btz RNAi ) , n = 12 ( 24B > Btz - GFP ; btz / Df ; btz RNAi / + ) , n = 9 ( BG380 + 24B - GAL4 ; btz / Df ) , n = 15 ( BG380 - GAL4 / + ) , n = 14 ( BG380 > btz RNAi ; repo > btz RNAi ) , n = 16 ( mhc - GAL4 / + ) , n = 28 ( mhc > btz RNAi ) , or n = 21 ( repo - GAL4 / + ) . Error bars show mean ± SEM . NMJ size is reduced in btz mutants and rescued by expressing UAS - btz with da - GAL4 or with the combination of the BG380 - GAL4 and 24B - GAL4 drivers , but not with any single driver . Expressing UAS - btz RNAi with da - GAL4 or in motor neurons with BG380 - GAL4 , muscles with mhc - GAL4 or glia with repo - GAL4 also reduces NMJ size .

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

(K, L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not significant by unpaired t-test with Welch's correction. n=23 (w; da-GAL4, btz/Df), n=6 (btz/+), n=10 (Df/+, UAS-BtzGFP/+; btz/Df), n=8 (btz/Df), n=22 (da>btz-GFP; btz/Df), n=13 (BG380-GAL4; btz/Df; BG380+24B>Btz-GFP; btz/Df; da-GAL4/+), n=5 (BG380>Btz-GFP; btz/Df), n=11 (24B-GAL4; btz/Df; da>btz RNAi), n=12 (24B>Btz-GFP; btz/Df; btz RNAi/+), n=9 (BG380+24B-GAL4; btz/Df), n=15 (BG380-GAL4/+), n=14 (BG380>btz RNAi; repo>btz RNAi), n=16 (mhc-GAL4/+), n=28 (mhc>btz RNAi), or n=21 (repo-GAL4/+). Error bars show mean ± SEM. NMJ size is reduced in btz mutants and rescued by expressing UAS-btz with da-GAL4 or with the combination of the BG380-GAL4 and 24B-GAL4 drivers, but not with any single driver. Expressing UAS-btz RNAi with da-GAL4 or in motor neurons with BG380-GAL4, muscles with mhc-GAL4 or glia with repo-GAL4 also reduces NMJ size.


( I ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 by unpaired t - test or Mann - Whitney test . n = 15 ( BG380 - GAL4 ) , n = 12 ( BG380 > btz RNAi ; BG380 > mago RNAi ; BG380 > eIF4AIII RNAi ) , n = 16 ( btz / + ) , n = 13 ( btz / Df ) , or n = 10 ( Btz - WT ; btz / Df ; Btz - HD ; btz / Df ) . Error bars show mean ± SEM . NMJ size is reduced by knocking down btz in motor neurons , but slightly increased by knocking down mago or eIF4AIII . The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction - defective btz transgenes .

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

(I) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001 by unpaired t-test or Mann-Whitney test. n=15 (BG380-GAL4), n=12 (BG380>btz RNAi; BG380>mago RNAi; BG380>eIF4AIII RNAi), n=16 (btz/+), n=13 (btz/Df), or n=10 (Btz-WT; btz/Df; Btz-HD; btz/Df). Error bars show mean ± SEM. NMJ size is reduced by knocking down btz in motor neurons, but slightly increased by knocking down mago or eIF4AIII. The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction-defective btz transgenes.


( L ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; ns , not significant by unpaired t - test or Mann - Whitney test . n = 19 ( UAS - daw RNAi / + ) , n = 9 ( BG380 > daw RNAi ; btz / Df ; tub > daw ) , n = 23 ( mhc > daw RNAi ) , n = 10 ( repo > daw RNAi ; tub > daw ) , n = 12 ( btz / Df ; UAS - daw ) , or n = 14 ( btz / Df ; tub > btz ) . Error bars show mean ± SEM . Knocking down daw in motor neurons , muscles or glia reduces NMJ size . Expressing UAS - daw with tub - GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS - btz , but has no significant effect on NMJ size in a wild - type background .

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

(L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ns, not significant by unpaired t-test or Mann-Whitney test. n=19 (UAS-daw RNAi/+), n=9 (BG380>daw RNAi; btz/Df; tub>daw), n=23 (mhc>daw RNAi), n=10 (repo>daw RNAi; tub>daw), n=12 (btz/Df; UAS-daw), or n=14 (btz/Df; tub>btz). Error bars show mean ± SEM. Knocking down daw in motor neurons, muscles or glia reduces NMJ size. Expressing UAS-daw with tub-GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS-btz, but has no significant effect on NMJ size in a wild-type background.


Immunofluorescence labeling of QSOX1 and a Golgi marker ( GM130 ) in epithelial cells isolated from murine colon . QSOX1 is green , GM130 is red , and DAPI staining of nuclei is blue in the merged image . Scale bar is 10 µm .

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

Immunofluorescence labeling of QSOX1 and a Golgi marker (GM130) in epithelial cells isolated from murine colon. QSOX1 is green, GM130 is red, and DAPI staining of nuclei is blue in the merged image. Scale bar is 10 µm.


Top , average colon length of WT and QSOX1 KO mice following indicated treatments . For untreated mice , colon lengths were calculated for 3 WT and 3 KO . For treated mice , lengths were calculated from 5 mice except for the DSS - treated WT , for which 3 mice were analyzed . Error bars are standard deviation . Statistical analysis was performed using Tukey multiple comparison of means ( * * p < 0 . 01 ) . Administration of QSOX1 inhibitory antibody MAb316 . 1 ( αQSOX1 ) , did not enhance the sensitivity of WT colons to DSS ( p > 0 . 4 for MAb316 . 1 - treated WT vs . WT control antibody ( IgG ) - treated ; p > 0 . 5 for MAb316 . 1 - treated WT vs . WT without antibody treatment ) . Bottom , representative images of colons following DSS treatment . Scale bar is 1 cm .

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

Top, average colon length of WT and QSOX1 KO mice following indicated treatments. For untreated mice, colon lengths were calculated for 3 WT and 3 KO. For treated mice, lengths were calculated from 5 mice except for the DSS-treated WT, for which 3 mice were analyzed. Error bars are standard deviation. Statistical analysis was performed using Tukey multiple comparison of means (** p<0.01). Administration of QSOX1 inhibitory antibody MAb316.1 (αQSOX1), did not enhance the sensitivity of WT colons to DSS (p>0.4 for MAb316.1-treated WT vs. WT control antibody (IgG)-treated; p>0.5 for MAb316.1-treated WT vs. WT without antibody treatment). Bottom, representative images of colons following DSS treatment. Scale bar is 1 cm.


Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well - packed goblet cell granules in WT and QSOX1 KO colons . Scale bar is 5 µm .

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

Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well-packed goblet cell granules in WT and QSOX1 KO colons. Scale bar is 5 µm.


Alcian blue and PAS staining of reduced guanidine - insoluble mucins from WT and QSOX1 KO mice , separated on a 6 % polyacrylamide gel . Data information : each lane corresponds to a sample from one mouse .

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

Alcian blue and PAS staining of reduced guanidine-insoluble mucins from WT and QSOX1 KO mice, separated on a 6% polyacrylamide gel. Data information: each lane corresponds to a sample from one mouse.


Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels . Data information : each lane corresponds to a sample from one mouse .

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

Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.


Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels . Data information : each lane corresponds to a sample from one mouse .

O O O O O O 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 blood Vwf from WT and QSOX1 KO mice separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.


QSOX1 immunofluorescence ( green ) in control ( siControl ) and QSOX1 knockdown ( siQSOX1 ) MDA - MB - 231 cells . Blue is DAPI staining of nuclei . Scale bar is 20 µm . Data information : The results described in are representative of 3 experiments that were performed .

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

QSOX1 immunofluorescence (green) in control (siControl) and QSOX1 knockdown (siQSOX1) MDA-MB-231 cells. Blue is DAPI staining of nuclei. Scale bar is 20 µm. Data information: The results described in are representative of 3 experiments that were performed.


Western blot analysis of the MUC2 N - terminal region and indicated cysteine mutants in supernatants of transfected cell cultures . No differences in disulfide - mediated dimerization were observed for any MUC2 variant between siC ( siControl ) and siQ ( siQSOX1 ) . Data information : The results described in G are representative of 3 experiments that were performed .

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

Western blot analysis of the MUC2 N-terminal region and indicated cysteine mutants in supernatants of transfected cell cultures. No differences in disulfide-mediated dimerization were observed for any MUC2 variant between siC (siControl) and siQ (siQSOX1). Data information: The results described in G are representative of 3 experiments that were performed.


Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice . Lysates were treated with PEG - mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1 . As for panel A but using antibodies to St3gal1 . As for panel A but using antibodies to B3galt5 . Data information : For the experiments in panels A - C , equal amounts of total protein were applied to each gel lane , and no reducing agent was added . Experiments were repeated 3 times with fresh new lysates , and representative blots 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 colon epithelial cell lysates from WT and QSOX1 KO mice. Lysates were treated with PEG-mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1. As for panel A but using antibodies to St3gal1. As for panel A but using antibodies to B3galt5. Data information: For the experiments in panels A-C, equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.


QSOX1 oxidizes St6gal1 in vitro . MAb492 . 1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG - mal additions ( 2PEG ) , whereas protein with two pairs of free cysteines acquires four PEG - mal additions ( 4PEG ) . The change in migration per two PEG - mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs . protein QSOX1 oxidation of St6gal1 mutants . The C350 - C361 disulfide ( the redox - active disulfide present in the C139A - C403A mutant ) is oxidized rapidly , while the C142 - C406 disulfide ( present in the C350A - C361A mutant ) is oxidized slowly . Data information : Experiments were repeated 3 times with fresh new lysates , and representative blots 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

QSOX1 oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG-mal additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG-mal additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein QSOX1 oxidation of St6gal1 mutants. The C350-C361 disulfide (the redox-active disulfide present in the C139A-C403A mutant) is oxidized rapidly, while the C142-C406 disulfide (present in the C350A-C361A mutant) is oxidized slowly. Data information: Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.


( A ) Kinetics of cell numbers in Lineage - EPCR + CD150 + CD48 - ( L - ESLAM ) HSCs , Lineage - EPCR + CD150 + CD48 + ( CD150 + CD48 + ) progenitor cells , and Lineage - EPCR + CD150 - CD48 + ( CD150 - CD48 + ) progenitor cells after the administration of 5 - FU ( 250 mg / kg , i . v . ) . The small graph is an enlargement of 0 to 8 days after the administration of 5 - FU . ( * or * * ) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days , respectively .

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

(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively.


( A ) A principal component analysis ( PCA ) using the ATAC - seq data of HSCs in the steady state ( untreated ) , early phase ( 5 - FU 6d ) , or late phase ( 5 - FU 10d ) .

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

(A) A principal component analysis (PCA) using the ATAC-seq data of HSCs in the steady state (untreated), early phase (5-FU 6d), or late phase (5-FU 10d).


mRNA ( C ) in Acly in HSCs after the administration of 5 - FU .

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

mRNA (C) in Acly in HSCs after the administration of 5-FU.


protein ( D ) , and phosphorylation levels of Ser455 ( E ) in Acly in HSCs after the administration of 5 - FU .

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

protein (D), and phosphorylation levels of Ser455 (E) in Acly in HSCs after the administration of 5-FU.


The levels of intracellular Glu ( G ) in HSCs obtained from untreated or 5 - FU - treated mice .

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

The levels of intracellular Glu (G) in HSCs obtained from untreated or 5-FU-treated mice.


total acetylated lysine ( I ) in HSCs obtained from untreated or 5 - FU - treated mice .

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

total acetylated lysine (I) in HSCs obtained from untreated or 5-FU-treated mice.


( B - C ) The effects of CTPI - 2 ( B ) or BMS303141 ( BMS ) ( C ) on H3K27ac in HSCs in the early phase after the administration of 5 - FU .

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

(B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU.


( D - E ) The effects of CTPI - 2 ( D ) , BMS303141 ( BMS ) ( E ) , A - 485 ( D ) , and TV - 3664 ( E ) on HSC expansion in the early phase after the administration of 5 - FU . Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells .

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

(D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells.


Global H3K27ac ( A ) , Acly levels ( B ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU .

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

Global H3K27ac (A), Acly levels (B) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.


acetylated lysine ( C ) , and ΔΨm ( D ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU .

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

acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.


( C ) The frequencies of the CD150 + CD48 - and CD48 + fractions within the lineage - EPCR + fraction after a 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ; 100 μM ) , SB204990 ( SB ; 100 μM ) , an alternative Acly inhibitor , or CTPI - 2 ( 50 μM ) . ( D and E ) Total cell number ( D ) or CD48 + cell number ( E ) after the 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ) , SB204990 ( SB ) , or CTPI - 2 . Numbers within the graph represent the inhibitory effects of each treatment ( fold decrease by an inhibitor treatment ) .

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

(C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). (D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment).


B , C . Cryosections of samples from patients with prostate carcinoma and SCC of the lung . Samples were processed as described in the methods section . Ve = vessel , Tu = tumor . Scale 50µm . See also Fig 1D for comparative semi - quantitative analysis of a panel of samples from patients with prostate carcinoma , lung SCC and non - lung SCC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. Cryosections of samples from patients with prostate carcinoma and SCC of the lung. Samples were processed as described in the methods section. Ve=vessel, Tu=tumor. Scale 50µm. See also Fig 1D for comparative semi-quantitative analysis of a panel of samples from patients with prostate carcinoma, lung SCC and non-lung SCC.


D . Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities . For definition of the H - score reflecting binding intensity , refer to the methods section . Statistical analysis was performed using the paired , non - parametric Wilcoxon test . Sample size ( n ) and the corresponding P - values are indicated in the graphs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities. For definition of the H-score reflecting binding intensity, refer to the methods section. Statistical analysis was performed using the paired, non-parametric Wilcoxon test. Sample size (n) and the corresponding P-values are indicated in the graphs.


E , F . Binding of the Fabsc - and IgGsc - molecules to PSMA - expressing LNCaP ( E ) or 22Rv1low ( F ) cells was assessed by flow cytometry , as described in the methods section . The EC50 values were calculated using GraphPad Prism software and are presented below the figure .

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

E, F. Binding of the Fabsc- and IgGsc-molecules to PSMA-expressing LNCaP (E) or 22Rv1low (F) cells was assessed by flow cytometry, as described in the methods section. The EC50 values were calculated using GraphPad Prism software and are presented below the figure.


G - I . Binding of the Fabsc - and IgGsc - molecules to CD3 - expressing Jurkat - cells ( G ) and to CD4 T cells ( H ) and CD8 T cells ( I ) in PBMC preparations was assessed by flow cytometry . The results represent the average of three independent experiments performed with different PBMC donors . EC50 values are presented in the table below the figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I. Binding of the Fabsc- and IgGsc-molecules to CD3-expressing Jurkat-cells (G) and to CD4 T cells (H) and CD8 T cells (I) in PBMC preparations was assessed by flow cytometry. The results represent the average of three independent experiments performed with different PBMC donors. EC50 values are presented in the table below the figure.


BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP - expressing high and low amounts of PSMA , respectively . After three days , cytokine release were determined by flow cytometry as described in the methods section . Mean values and standard deviations of triplicate measurements 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 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

BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.


BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low - cells expressing high and low amounts of PSMA , respectively . After three days , cytokine release were determined by flow cytometry as described in the methods section . Mean values and standard deviations of triplicate measurements are indicated .

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BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.


B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs , and after three days cytokine release ( B ) were measured using a 3H - thymidine uptake - and a Legendplex assay , respectively . Mean values and standard deviations from triplicate samples .

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B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs, and after three days cytokine release (B) were measured using a 3H-thymidine uptake- and a Legendplex assay, respectively. Mean values and standard deviations from triplicate samples.


C - F . 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors ( 5 animals per group ) , and uptake at the indicated locations was determined by PET quantification over time ( C , D ) and at termination of the experiment after 48 hours ( E , F ) as described in the methods section . Error bars represent standard deviation of the mean value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors (5 animals per group), and uptake at the indicated locations was determined by PET quantification over time (C, D) and at termination of the experiment after 48 hours (E, F) as described in the methods section. Error bars represent standard deviation of the mean value.


B , C . 106 LNCaP cells were injected s . c . into the right flank of NSG mice ( 5 per group ) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm . BsAbs ( 2µg per dose ) were applied at day 1 , 8 and 15 ( IgGsc ) and daily at day 1 - 3 , day 8 - 10 and day 15 - 17 ( Fabsc ) , PBMC were applied at day 1 , 8 and 15 . Mean values and standard deviations of five mice per group 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. 106 LNCaP cells were injected s.c. into the right flank of NSG mice (5 per group) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm. BsAbs (2µg per dose) were applied at day 1, 8 and 15 (IgGsc) and daily at day 1-3, day 8-10 and day 15-17 (Fabsc), PBMC were applied at day 1,8 and 15. Mean values and standard deviations of five mice per group are indicated.


The IgGsc - molecule was applied in escalating doses , with a starting dose of 20µg ( patient 1 ) or 40µg ( patients 2 and 3 ) to a maximum of approx . 2 . 6mg , as indicated . PSA values were monitored and T - cell activation was assessed by flow cytometry .

The IgGsc-molecule was applied in escalating doses, with a starting dose of 20µg (patient 1) or 40µg (patients 2 and 3) to a maximum of approx. 2.6mg, as indicated. PSA values were monitored and T-cell activation was assessed by flow cytometry.


( E ) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 ( LxxAxA ) . Binding of YFP - B56α to indicated proteins was determined and quantified by LiCor .

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

(E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor.


( G ) YFP - B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins . Representative of 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

(G) YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.


( B ) IP of YFP - B56α from cells stably expressing the B56α WT , R222E , and 5A followed by immunoblotting of indicated proteins . Representative blots are shown ( top ) . hSgo1 signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) .

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

(B) IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).


( C ) Reciprocal IP of ( B ) . YFP - hSgo1 expression construct was transfected into cells stably expressing FLAG - B56α WT , R222E and 5A , followed by YFP IP and immunoblotting of indicated proteins . Representative blots are shown ( top ) . B56α signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) .

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

(C) Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).


( D ) Representative images of chromosome spreads from the indicated conditions . Scale bar , 5μm . ( E ) Quantification of ( D ) . The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 4 independent experiments and the mean and SD 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 O O O O O O O O O O O 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) Representative images of chromosome spreads from the indicated conditions. Scale bar, 5μm. (E) Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated.


( F ) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere - targeting domain ( CB ) . Representative images of chromosome spreads are shown . CB targets all the rescue constructs ( green ) to the centromere . Scale bar , 5μm . ( G ) Quantification of ( F ) . The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 3 independent experiments and the mean and SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5μm. (G) Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.


( D ) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP - hSgo1 variants . Representative immunofluorescent images are shown . Scale bar , 5μm . ( E ) B56α signal intensity was quantified . B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP - hSgo1 signal . Each circle represents an individual cell , and the average and SD 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 O O O O O O O O O O O O O O O O O O O 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) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP-hSgo1 variants. Representative immunofluorescent images are shown. Scale bar, 5μm. (E) B56α signal intensity was quantified. B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP-hSgo1 signal. Each circle represents an individual cell, and the average and SD are indicated.


( F ) Mitotic U2OS LacO Haspin CM cells expressing hSgo1 - LacI - GFP variants or LacI - GFP ( control ) were stained for PP2A - C . Scale bar , 5μm . ( G ) PP2A - C signal intensity was quantified , normalized to GFP , and then plotted . Each circle represents an individual cell , and the mean fluorescent intensity is indicated . Representative of at least 3 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 O O O O

(F) Mitotic U2OS LacO Haspin CM cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C. Scale bar, 5μm. (G) PP2A-C signal intensity was quantified, normalized to GFP, and then plotted. Each circle represents an individual cell, and the mean fluorescent intensity is indicated. Representative of at least 3 independent experiments.


( A ) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP - hSgo1 variants . All the YFP - hSgo1 rescue constructs ( green ) localize to the centromeres ( CREST , red ) . Scale bar , 5μm . ( B ) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in ( A ) and averaged for a single cell . The data are from 3 independent experiments and the mean and SD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP-hSgo1 variants. All the YFP-hSgo1 rescue constructs (green) localize to the centromeres (CREST, red). Scale bar, 5μm. (B) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in (A) and averaged for a single cell. The data are from 3 independent experiments and the mean and SD are indicated.


( C ) IP of YFP - hSgo1 from cells stably expressing hSgo1 WT , 3A , and 4A followed by immunoblotting of cohesin components , Smc1 and Smc3 . Representative blots are shown ( top ) . Signals were quantification by LiCor , normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 for Smc1 and n = 3 for Smc3 ) .

(C) IP of YFP-hSgo1 from cells stably expressing hSgo1 WT, 3A, and 4A followed by immunoblotting of cohesin components, Smc1 and Smc3. Representative blots are shown (top). Signals were quantification by LiCor, normalized to YFP and plotted (bottom). Error bars represent SD (n=4 for Smc1 and n=3 for Smc3).


( F ) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi - resistant constructs was performed . Representative still images captured during the live cell imaging showing DIC and YFP - hSgo1 WT , 3A , and 4A localization during mitosis . Time ( min ) from nuclear envelop breakdown ( NEBD ) is indicated . Scale bar , 15μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi-resistant constructs was performed. Representative still images captured during the live cell imaging showing DIC and YFP-hSgo1 WT, 3A, and 4A localization during mitosis. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15μm.


( A ) LC - MS / MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA . Signal intensities of the modifications are scaled to that for m1A , the ribonucleoside with the highest MS

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

(A) LC-MS/MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA. Signal intensities of the modifications are scaled to that for m1A, the ribonucleoside with the highest MS signal


( A ) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC - MS / MS in total tRNA extracted from parasites across the IDC time course . The average fold - change values ( relative to an arbitrary average control ) were subjected to hierarchical clustering analysis ( log2 - transformed data ) . The scale of the heat map has saturated the fold - change values of some modifications . Refer to Appendix Table S6 . ( B ) Three groups of modifications show differential regulation during the course of IDC . The majority ( 22 ) increase synchronously across the IDC ( blue ) , while 4 show asynchronous or irregular behavior ( purple ) and 2 ( orange ) are uniquely up - regulated in the ring stage . Log2Fold - change values plotted in the three graphs represent mean ± SEM ( N = 3 ) . Differences between the highest value ( denoted as peak ) and the lowest value ( denoted as trough ) were subjected to a two - tailed Student ' s t - test : NS , not

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC-MS/MS in total tRNA extracted from parasites across the IDC time course. The average fold-change values (relative to an arbitrary average control) were subjected to hierarchical clustering analysis (log2-transformed data). The scale of the heat map has saturated the fold-change values of some modifications. Refer to Appendix Table S6. (B) Three groups of modifications show differential regulation during the course of IDC. The majority (22) increase synchronously across the IDC (blue), while 4 show asynchronous or irregular behavior (purple) and 2 (orange) are uniquely up-regulated in the ring stage. Log2Fold-change values plotted in the three graphs represent mean ± SEM (N = 3). Differences between the highest value (denoted as peak) and the lowest value (denoted as trough) were subjected to a two-tailed Student's t-test: NS, not significant


iTRAQ proteomics analysis ( Appendix Fig . S2 ) resulted in 2 , 100 proteins consistently quantified at every point across the IDC . The heat map shows hierarchical clustering analysis of fold - changes ( log2 - transformed ) in the levels of the proteins relative to an arbitrary average across the IDC . The 100 most up - regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P . falciparum 3D7 . The significantly ( P < 0 . 05 ) enriched pathways among the up - regulated proteins at each time point are summarized in tables for each time

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iTRAQ proteomics analysis (Appendix Fig. S2) resulted in 2,100 proteins consistently quantified at every point across the IDC. The heat map shows hierarchical clustering analysis of fold-changes (log2-transformed) in the levels of the proteins relative to an arbitrary average across the IDC. The 100 most up-regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P. falciparum 3D7. The significantly (P < 0.05) enriched pathways among the up-regulated proteins at each time point are summarized in tables for each time point


LC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of the unmodified ( 3055 . 425 Da ) and modified ( 3143 . 43 Da ) oligos for Glu - tRNAGAA (

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LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of the unmodified (3055.425 Da) and modified (3143.43 Da) oligos for Glu-tRNAGAA (B


Collision - induced dissociation of the parent molecular ions for m / z 1046 . 477 - 3 from Glu - tRNAGA produced expected - c , - w and - y ions for the sequences CU [ mcm5s2U ]

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

Collision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCC


LC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of th unmodified ( 1835 . 20 Da ) and modified ( 1923 . 262 Da ) oligos for Gln - tRNACAA ( E )

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LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of th unmodified (1835.20 Da) and modified (1923.262 Da) oligos for Gln-tRNACAA (E)


Collision - induced dissociation of the parent molecular ions fo m / z 1923 . 262 from Gln - tRNACAA produced expected - c , - w and - y ions for the sequence CC [ mcm5s2U ]

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

Collision-induced dissociation of the parent molecular ions fo m/z 1923.262 from Gln-tRNACAA produced expected -c, -w and -y ions for the sequence CC[mcm5s2U]CCAA


Ratios of modified oligonucleotides from the anticodon stem - loop o ( H ) GluGA isoacceptors expressed as percentages of their sum

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Ratios of modified oligonucleotides from the anticodon stem-loop o (H)GluGA isoacceptors expressed as percentages of their sum total


Ratios of modified oligonucleotides from the anticodon stem - loop o ( G )

Ratios of modified oligonucleotides from the anticodon stem-loop o (G)GlnCA


Ratios of modified oligonucleotides from the anticodon stem - loop o ( I ) GlyGGA isoacceptors expressed as percentages of their sum

Ratios of modified oligonucleotides from the anticodon stem-loop o (I)GlyGGA isoacceptors expressed as percentages of their sum total