( A ) GNS was localized by microscopy in indicated strains grown in medium containing glucose or galactose were inverted to visualize possible defects . First column shows DIC image of . Scale bar , 5 µm . ( B ) of AP - 3 defect from ( A ) . Linear plots were laid over grown in medium containing glucose ( D ) or galactose ( G ) , AP - 3 defect was calculated by dividing GFP - intensities on vacuolar by sum of of vacuolar , plasma signal ( n ≥ 15 ) . Bars show mean vacuolar signal over total signal ± standard deviation .

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

(A) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects. First column shows DIC image of cells. Scale bar, 5 µm. (B) Quantification of the AP-3 defect from (A). Linear intensity plots were laid over cells grown in medium containing glucose (D) or galactose (G), the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 15 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.


( C ) Effect of different truncations deletions on AP - 3 targeting to mitochondria . Indicated strains were grown at 30 ° C in YPG kept in logarithmic phase . Localization of Apl5 or Apl6 Vps41 was analysed by microscopy . First column shows DIC image of . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( D ) of ( C ) . ( - ) Control corresponds to Vps41 ( ΔPEST ) - GFP - Fis1 Apl6 - mCherry ( CUY12558 ) ( + ) corresponds to Vps41 ( S - D ) - GFP - Fis1 Apl6 - mCherry ( CUY12560 ) . Apl5 - / Apl6 - dots co - localizing with GFP signal were counted plotted in of total number of Apl5 - / Apl6 - dots ( n ≥ 63 ) . Bars show mean of co - localization ± standard deviation .

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

(C) Effect of different truncations and deletions on AP-3 targeting to mitochondria. Indicated strains were grown at 30°C in YPG and kept in logarithmic phase. Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC image of cells. Scale bar, 5 µm. Scale bar in inset, 2 µm. (D) Quantification of (C). (-) Control corresponds to Vps41(ΔPEST)-GFP-Fis1 Apl6-mCherry (CUY12558) and (+) corresponds to Vps41(S-D)-GFP-Fis1 Apl6-mCherry (CUY12560). Apl5- /Apl6-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5- /Apl6-dots (n ≥ 63). Bars show the mean percentage of co-localization ± standard deviation.


( C ) Volcano plots of in close proximity to Apl5 - TurboID versus control , from a label - free proteomics of streptavidin - biotin pulldowns . logarithmic ratios of intensities are plotted against negative logarithmic P values of two Student ' s t - , equal variance , performed from n = 3 independent experiments . red dashed ( significance , 0 . 05 ) separates specifically identified ( top right portion of plot ) from background . Selected top hits are indicated with black dots ,

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots,


( E ) Volcano plots of in close proximity to Apl5 - TurboID versus control in a GAL1pr - Vps41 - GFP - Fis1 background , from a label - free proteomics of streptavidin - biotin pulldowns with grown in YPG . logarithmic ratios of intensities are plotted against negative logarithmic P values of two Student ' s t - , equal variance , performed from n = 3 independent experiments . red dashed ( significance , 0 . 001 ) separates specifically identified ( top right portion of plot ) from background . Selected top hits are indicated with black

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots


( B ) AP - 3 sorting defects in GAP deletions . AP - 3 reporter was localized relative to FM4 - 64 stained vacuoles in indicated strains . First column shows DIC image of . Scale bar , 5 µm .

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

(B) AP-3 sorting defects in GAP deletions. The AP-3 reporter was localized relative to FM4-64 stained vacuoles in the indicated strains. First column shows the DIC image of cells. Scale bar, 5 µm.


( D ) Localization of AP - 3 in ArfGAP deletion strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in indicated strains . First column shows DIC image of . Scale bar , 5 µm . ( E ) of Apl5 - dots in ( D ) . Apl5 - positve dots were counted averaged to number of dots per . Box boundaries indicate 25 % 75 % of dataset . middle indicates median small square mean .

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

(D) Localization of AP-3 in ArfGAP deletion strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. First column shows DIC image of cells. Scale bar, 5 µm. (E) Quantification of Apl5-dots in (D). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


( F ) Co - localization of C - terminally GFP - tagged Apl5 C - terminally mCherry - tagged Sec7 in wt age2Δ background . were grown at 30 ° C kept in logarithmic phase . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( G ) of co - localization from ( F ) normalized per . Number of co - localizing dots was determined normalized to number of co - localization events per . Bars show mean number of co - localizing dots per ± standard deviation ( n ≥ 60 ) .

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

(F) Co-localization of C-terminally GFP-tagged Apl5 and C-terminally mCherry-tagged Sec7 in wt and age2Δ background. Cells were grown at 30°C and kept in logarithmic phase. Scale bar, 5 µm. Scale bar in inset, 2 µm. (G) Quantification of co-localization from (F) normalized per cell. Number of co-localizing dots was determined and normalized to the number of co-localization events per cell. Bars show the mean number of co-localizing dots per cell ± standard deviation (n ≥ 60).


( A , B ) AP - 3 sorting defects in strains overexpressing Gcs1 ( A ) or Age2 ( B ) . AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in indicated strains . Scale bar , 5 µm .

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

(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.


( C ) Localization of AP - 3 in ArfGAP overexpression strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in indicated strains . Scale bar , 5 µm . ( D ) of Apl5 dots in ( C ) . Apl5 - positve dots were counted averaged to number of dots per . Box boundaries indicate 25 % 75 % of dataset . middle indicates median small square mean .

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

(C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


( E , F ) AP - 3 sorting defects in GAP - dead mutants of Gcs1 ( E ) Age2 ( F ) . AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in indicated strains . Scale bar , 5 µm .

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

(E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.


( G ) Localization of AP - 3 in GAP - dead mutants of Gcs1 Age2 . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in indicated strains . Scale bar , 5 µm . ( H ) of Apl5 dots in ( G ) . Apl5 - positve dots were counted averaged to number of dots per . Box boundaries indicate 25 % 75 % of dataset . middle indicates median small square mean .

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

(G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


B . of cycle arrest ( in G2 ) in response to DSB induction assayed by flow cytometry .

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

B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.


C . Rad53 phosphorylation ( Rad53 - P ) in response to DSB assayed by western blotting .

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C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.


D . survival upon DSB induction . Average ± SD ( n = 4 ) is shown for each genotype .

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D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.


O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.


D . Dynamics of ( TG1 - 3 ) n addition monitored by qPCR through a time - course experiment ( asynchronous populations ) . Y axis shows a fold increase in de novo telomere - specific PCR product relative to background at 0 h normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 ; NK4112 , NK4113 ; NK4114 , NK4115 ; NK3292 est2 ∆ , NK3293 est2 ∆ ; NK4232 , NK4233 .

O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.


F . Comparative of ssDNA / dsDNA at de novo telomere addition loci in SRS2 srs2Δ during a time - course experiment ( synchronised populations ) . Y axis shows a fold increase in de novo telomere - specific PCR product relative to background at 0 h normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Top set of error bars represents SD in relative increase of de novo telomere - specific PCR product ( as in panel D ) while lower set of error bars corresponds to quantifications of ss / dsDNA fractions . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.


B . Southern blot of re - synthesis of resected during BIR in SRS2 srs2Δ corresponding to data quantifications in C . was digested with EcoRI BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon hybridised to mixture of 4 probes ( three RS probes a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in repair ) . A representative image of one of three repeats is shown . C . Re - synthesis of resected on chr . VIIL in SRS2 srs2Δ ( solid dashed lines , respectively ) at of 15 . 2 ( RS15 . 2 ) , 6 . 8 ( RS6 . 8 ) 2 . 6 ( RS2 . 6 ) kb away from homology region . Average ± SD ( n = 3 ) is shown for each time point .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


D . Southern blot of BIR - dependent synthesis in SRS2 srs2Δ corresponding to data quantifications in D . was digested with EcoRI BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon hybridised to mixture of 4 probes ( three BIR probes a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in repair ) . A representative image of one of three repeats is shown . C - control NK3980 . E . BIR - dependent synthesis in SRS2 srs2Δ ( solid dashed lines , respectively ) at of 6 ( BIR6 ) , 36 ( BIR36 ) 77 ( BIR77 ) kb away from homology region . Average ± SD ( n = 3 ) is shown for each time point .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


B . Frequency of DSB repair via SSA in SRS2 srs2Δ with without functional HR in on system shown in panel A . Average ± SD ( n = 4 ) is shown for each genotype . Strains used in B D : NK4691 - 4693 ; NK4805 - 4808 ; NK5081 - 5084 ; NK5085 - 5091 .

O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.


D . Fragment L formation in SRS2 srs2Δ in presence absence of Rad51 . Also see Figure EV3 for blot . Average ± SD ( n = 4 ) is shown for each time point .

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

D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.


( D ) Absolute numbers of indicated types were determined by flow - cytometric of bone marrow from 3 - week - old Pax5 + / + Pax5Jak2 / + mice . Average numbers are shown with were statistically analyzed by multiple t - ( unpaired two - with Holm - Šídák ' s correction ) ; ns , not significant ( P > 0 . 05 ) . One of 3 independent experiments is shown .

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

(D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown.


( F ) Kaplan - Meier survival of Pax5Jak2 / + ( black ) control Pax5 + / + ( grey ) mice . n , number of mice analyzed . A P value of < 0 . 0001 was determined for survival curves by statistical with log - rank ( Mantel - Cox ) .

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

(F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test.


( H ) Eosin - hematoxylin - stained sections of lung liver of a Pax5Jak2 / + mouse . Infiltrating blasting are indicated by an arrow .

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

(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.


( I ) Flow - cytometric of lymph node from a control Pax5 + / + mouse a 10 - week - old Pax5Jak2 / + mouse . ( J ) Flow - cytometric of B220lowCD19 + B from bone marrow of a 4 - week - old Pax5Jak2 / + mouse .

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(I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.


( D - G ) GSEA of 327 repressed ( D ) or 330 activated ( F ) Pax5 target genes identified in pro - B as compared with ranked log2 - fold gene expression changes in Pax5Jak2 / + ( PJ ) B - ALLs versus control ( Ctrl ) Pax5 + / - Cdkn2ab + / - B - ALLs ( left ) . NES , normalized enrichment score . expression of five genes that are upregulated in Pax5Jak2 / + B - ( upper row ) Pax5 - / - ( KO ) pro - B ( lower row ) is shown in ( E ) . Likewise , expression of five genes that are upregulated in control B - ( upper row ) Pax5 + / + ( WT ) pro - B ( lower row ) is shown in ( G ) . TPM , transcripts per million . Mean TPM values with are shown for following - seq experiments : 3 ( WT pro - B ) , 2 ( KO pro - B ) , 2 ( Ctrl B - ) , 4 ( PJ B - ) .

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(D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL).


( H ) Expression of Ptprc ( CD45 ) gene from exon 3 to exon 8 , as determined by - seq of Pax5Jak2 / + Pax5 + / - Cdkn2ab + / - B - . alternatively spliced exons 4 ( A ) , 5 ( B ) 6 ( C ) are indicated in orange in respective exon - intron structure of Ptprc gene . Ptprc transcripts of B220 + Pax5 + / - Cdkn2ab + / - B - contain three exons , thus giving rise to expression of CD45 isoform RABC ( known as B220 ) . In contrast , reads at exon 4 are barely detectable reads at exon 6 are strongly reduced in mRNA of Pax5Jak2 / + B - , which likely gives rise to CD45 isoforms RBC

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(H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB


( D ) Flow - cytometric of bone marrow from 4 - week - old Pax5ihCd2 / + Pax5Jak2 / ihCd2 mice . expression of human ( h ) CD2 from Pax5ihCd2 allele is shown for CD19 + B220 + ( grey ) CD19 + B220low ( black ) B .

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(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.


( G ) Kaplan - Meier survival of Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + ( black ) Cd79a - Cre Pax5LSL - Jak2 / + ( grey ) mice . Statistical of survival curves was performed with log - rank ( Mantel - Cox ) ; * * * * P < 0 . 0001 . n , number of mice analyzed .

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(G) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.


( H ) Flow - cytometric of B220 CD19 expression in B - from lymph node of a Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + mouse ( black ; left ) . Pax5 expression in these B - ( black ) control Pax5 + / + lymph node B ( grey filled ) was determined by intracellular Pax5 ( right ) .

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(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).


( B , Flow - cytometric of bone marrow spleen from Pax5Jak2 - KD / + ( black ; n = 11 ) control Pax5 + / + ( grey ; n = 7 ) mice at of 6 - 8 . frequencies of indicated B types are shown for each organ ( B ) . data are presented as mean percentages with were statistically analyzed by multiple t - ( unpaired two - with Holm - Šídák ' s correction ) ; ns ( P > 0 . 5 ) , * P < 0 . 05 , * * P < 0 . 01 .

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(B, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01.


( D , progression in mice , transplanted with Pax5Jak2 - Luc / + , in presence or absence of JAK1 / 2 inhibitor ruxolitinib . At day 14 after transfer , transplanted mice were treated twice daily with ruxolitinib or vehicle , mass was monitored by bioluminescence measurements . of three representative mice with or without ruxolitinib treatment are

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(D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown


( F ) Kaplan - Meier survival of transplanted mice upon prolonged treatment with ruxolitinib ( black ) vehicle ( grey ) . Statistical of survival curves ( F ) was performed with log - rank ( Mantel - Cox ) ; * * P < 0 . 01 .

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(F) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.


( B ) Genome - wide binding of Pax5 - Jak2 in in vitro cultured pro - B from Pax5Jak2 / + Rosa26BirA / + mice at of 3 ( expressing Pax5 ) ex vivo Pax5Jak2 / + Rosa26BirA / + B - tumors ( lacking Pax5 ) , as determined by Bio - ChIP - seq - binding pattern of full - Pax5 was determined by Bio - ChIP - seq of ex vivo sorted Pax5Bio / Bio pro - B , which carried a C ‐ terminal biotin acceptor together with an IRES ‐ BirA gene insertion in 3 ' untranslated region of Pax5 Two independent Bio - ChIP - seq experiments were performed for each type . Representative binding patterns of Pax5 Pax5 - Jak2 in three B types are shown for a selected genomic region , with horizontal bars indicating Pax5 or Pax5 - Jak2 peaks that were identified by MACS peak calling ( left ) . number of Pax5 ( ) Pax5 - Jak2 ( grey or black ) peaks , which were defined by stringent MACS peak calling with a P value of < 10 - 10 in three B types , are shown to right .

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(B) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.


( F , G ) Flow - cytometric of bone marrow spleen from Pax5Prd * - Jak2 / + ( black ; n = 6 ) control Pax5 + / + ( grey ; n = 5 ) mice at of 6 - 7 . frequency of indicated B types in each organ is indicated in ( F ) , flow - cytometric of CD19 B220 expression on bone marrow B is shown in ( G ) . Statistical data are shown as mean percentages with were analyzed by multiple t - ( unpaired two - with Holm - Šídák ' s correction ) : ns > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 . Each dot corresponds to one mouse .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse.


( H ) Kaplan - Meier survival of Pax5Prd * - Jak2 / + ( black ) Pax5 + / + ( grey ) mice . A P value of < 0 . 0001 was determined for survival curves by statistical with log - rank ( Mantel - Cox ) . n , number of mice analyzed .

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(H) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed.


( C ) Specificity of anti - H3Y41ph antibody , as shown by immunoblot . One phosphorylated ( pY41 ) or non - phosphorylated peptide was coupled to ubiquitin ( Ubi , 8 . 5 kDa ) , followed by separation of conjugates on SDS - PAGE immunoblot detection with anti - H3Y41ph antibody present in 8B2 - C1 supernatant . Following treatment with calf intestinal alkaline phosphatase ( CIP ) , dephosphorylated Ubi - pY41 peptide conjugate could no longer be detected with anti - H3Y41ph antibody .

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(C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.


( D ) Immunoblot of whole - extracts prepared from Pax5Jak2 / + control Pax5Etv6 / + Cdkn2ab + / - B - as well as from human HEL , TMD8 K1106 lines . Phosphorylated ( p ) STAT5 was detected with an anti - STAT5 ( pY694 ) antibody pY41 - peptide with purified anti H3Y41ph antibody One to five pY41 - peptides were coupled to ubiquitin , which was added in range of 50 ng per well . Gapdh histone H3 were analyzed as loading control . An unspecific is denoted by an asterisk . One representative of 5 immunoblot experiments is shown .

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(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.


( A , progression in Il7 + / + ( grey ) , Il7 + / - ( dashed ) Il7 - / - ( black ) mice transplanted with Pax5Jak2 - Luc / + , as determined by bioluminescence measurements at indicated days after transfer . of three transplanted mice for each Il7 genotype are shown at indicated days after

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(A, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer


( C ) Kaplan - Meier survival of Il7 + / + , Il7 + / - Il7 - / - mice transplanted with Pax5Jak2 - Luc / + tumors . Statistical of survival curves was performed with log - rank ( Mantel - Cox ) ; ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

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(C) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01.


( E ) of median ( MFI ) values of p - STAT5 determined by intracellular of splenic FO B from Pax5 + / + ( grey ) , Pax5Jak2 / + ( black ) Pax5Jak2 - KD / + ( ) mice at of 3 - 10 Statistical data are presented as mean values with were analyzed by two - unpaired Student ' s t - : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

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(E) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( F ) Flow cytometric of p - STAT5 in pro - B ( B220 + CD19 + Kit + IgM - ) leukemic B220low B of Pax5Jak2 / + mice ( black ) as well as in control pro - B of Pax5 + / + ( WT ) mice ( grey ) at of 4 - 5 . A representative flow cytometric of bone marrow of a Pax5Jak2 / + mouse ( left ) representative intracellular p - STAT5 stainings ( middle ) are shown . p - STAT5 in pro - B leukemic B220low B ( right ) are quantified as MFI values relative to that of Pax5 + / + pro - B . Statistical data are presented as mean values with were analyzed by two - unpaired Student ' s t - : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

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(F) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( I ) Upregulation of genes Cxcr5 , Syndig1l Sema4a in Pax5Jak2 / + ( PJ ) B - . Mean TPM values with are shown for following - seq experiments : 2 ( Ctrl B - ) , 4 ( PJ B - )

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(I) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL)


K ) Identification of Cxcr5 , Syndig1l Sema4a as activated STAT5 target genes . ChIP - seq identified STAT5 - binding regions at three loci ( K ) . Horizontal bars below ChIP - seq track indicate STAT5 - binding regions identified by MACS peak calling . RPM , reads per million .

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K) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.


MPK4 ‐ HA is detected in PAT1 ‐ Myc immunoprecipitates from double transgenic Arabidopsis . Immunoblots of input anti ‐ Myc IPs probed with anti ‐ HA anti ‐ Myc antibodies . Left panel , input ; right panel , anti ‐ Myc IP .

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MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP.


Yeast pat1Δ mutants are unable to grow at 37 ° C while wild ‐ type ( B4742 ) grows at 30 ° C ( left panel ) 37 ° C ( right ) . Growth at 37 ° C is restored in pat1Δ expressing AtPAT1 . Serial dilutions of each yeast were plated on YPAD agar plates grown at indicated temperatures .

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Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.


Co ‐ IP between PAT1 ‐ HA LSM1 ‐ GFP . were transiently co ‐ expressed in N . benthamiana tissue harvested 3 days post ‐ infiltration . Immunoblots of GFP IPs ( top panel ) inputs ( 20 μg each , bottom panel ) . Immunoblots were cut in half probed with anti ‐ HA antibodies anti ‐ GFP antibodies .

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Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.


Agarose gel electrophoresis of 5 ′ RACE PCR to detect capped transcripts of EIF4A1 , UGT87A2 EXPL1 in 14 ‐ day ‐ old Col ‐ 0 pat1 ‐ 1 .

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Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.


MPK4 MPK6 were immunoprecipitated from extracts of Col ‐ 0 seedlings treated with 200 nM flg22 for 10 min . For negative control ( − ) , extracts were incubated with agarose beads without antibodies . IPs were incubated with His6 ‐ PAT1 , His6 ‐ PAT1 S208A or MBP for 60 min at 37 ° C before boiling SDS ‐ PAGE . Autoradiogram ( top panel ) , Coomassie ‐ stained gel for loading control ( bottom ) .

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MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).


pat1 mutants have a serrated leaf , semi ‐ dwarf phenotypePlants photographed at 4 of growth in short day conditions , genotypes as indicated . Col ‐ 0 eds1 ‐ 2 pat1 ‐ 1 pat1 ‐ 1 / eds1 ‐ 2 , respectively , were placed in middle of same pots before pictures were taken .

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pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.


PR gene expression is elevated in mpk4 ‐ 2 pat1 ‐ 1 mutants . Four ‐ week ‐ old were used for extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) PR2 ( black ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of mean is indicated by errors bars ( n = 3 ) . Statistical significance between mean values was determined by ANOVA followed by Fisher ' s LSD , P ‐ values are only shown for mean values significantly different from Col ‐ 0 .

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PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0.


pat1 is more resistant to colonization by P . syringae pv . tomato DC3000 . were syringe ‐ infiltrated samples taken 3 days post ‐ infiltration . Data are shown as log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of mean is indicated by errors bars ( n = 4 ) . Statistical significance between mean values was determined by ANOVA followed by Fisher ′ s LSD .

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pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.


Confocalmicroscopy with root elongation zones . Five ‐ day ‐ old Col ‐ 0 / PAT1 ‐ GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min ( second panel ) followed by treatment with 100 μg / ml cycloheximide for 60 min ( bottom panel ) . scale bar corresponds to 10 μm .

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Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.


Confocalmicroscopy with 5 ‐ day ‐ old VCS ‐ GFP seedlings treated as in ( A ) . scale bar corresponds to 10 μm .

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Confocalmicroscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm.


PAT1 is induced by PAMP treatment . Immunoblot detection of equal amounts of from Col ‐ 0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water ( 180 min after infiltration ) . Immunoblots were probed with anti ‐ PAT1 antibodies . Negative control pat1 ‐ 1 was loaded for comparison . Coomassie brilliant blue loading control is indicated by CBB .

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PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB.


Anti ‐ GFP immunoblotting of extracted from Col ‐ 0 / PAT1 ‐ GFP seedlings treated over a time course with flg22 ( top panel ) Coomassie brilliant blue ( CBB ) ‐ stained PVDF loading control is shown ( bottom panel ) . No PAT1 ‐ GFP was detected in negative control Col ‐ 0 .

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Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.


Elevated PR gene expression in pat1 mutants is suppressed by summ2 ‐ 8 . Four ‐ week ‐ old were used for extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) PR2 ( black bars ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of mean is indicated by errors bars ( n = 3 ) .

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Elevated PR gene expression in pat1 mutants is suppressed by summ2‐8. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3).


pat1 resistance to P . syringae pv . tomato DC3000 is suppressed by summ2 ‐ 8 . were syringe ‐ infiltrated samples taken 3 days post ‐ infiltration . Data are log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of mean is indicated by errors bars ( n = 4 ) .

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pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2‐8. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).


14 ‐ day ‐ old seedlings grown on ms plates were used for extraction . Next , 1 μg of from each sample was treated with XRN1 ( NEB ) or mock treated before RT ‐ qPCR . Data were normalized to ACT2 , transcript were compared between XRN1 mock ‐ treated for each genotype . Standard error of mean is indicated by error bars ( n = 4 ) . Statistical significance between mean values was determined by ANOVA followed by Fisher ' s LSD .

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14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test.


Phosphorylation of PAT1 peptide SSFVSYPPPGSISPDQR , which include Ser208 , in Col ‐ 0 mkk1 / 1 ‐ summ2 before after flg22 treatment . phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment . ratios were obtained using quantitative iTRAQ mass spectrometry .

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Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col‐0 and mkk1/1‐summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.


were transiently co ‐ expressed ( PAT1 ‐ GFP + MPK4 ‐ HA or SUMM2 ‐ HA ) in N . benthamiana tissue harvested 2 days post ‐ infiltration . Anti ‐ HA immunoblots of inputs ( 20 μg each ) are shown in bottom panel . Immunoblots of GFP IPs were probed with anti ‐ HA antibodies , then stripped probed with anti ‐ GFP antibodies . Molecular weights are shown in kDa on right .

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Proteins were transiently co‐expressed (PAT1‐GFP + MPK4‐HA or SUMM2‐HA) in N. benthamiana and tissue harvested 2 days post‐infiltration. Anti‐HA immunoblots of inputs (20 μg each) are shown in the bottom panel. Immunoblots of GFP IPs were probed with anti‐HA antibodies, then stripped and probed with anti‐GFP antibodies. Molecular weights are shown in kDa on the right.


Confocalimages of yellow complementation . PAT1 ‐ Nc was transiently co ‐ expressed with either SUMM2 ‐ Nn or At4g12010 in N . benthamiana . Confocalmicroscopy on leaf disks was conducted 2 days post ‐ infiltration . Merged pictures show overlay of brightfield , chlorophyll eYFP . scale bar corresponds to 25 μm .

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Confocalimages of yellow fluorescent protein complementation. PAT1‐Nc was transiently co‐expressed with either SUMM2‐Nn or At4g12010 in N. benthamiana. Confocalmicroscopy on leaf disks was conducted 2 days post‐infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. The scale bar corresponds to 25 μm.


A , B Bottom panels : gene annotation track ( mm10 ) ; activated gene is marked in green location of TALE - VP64 target is shown by vertical dashed green . Middle panels : DamID tracks of NL interactions in control ( " control " , blue ) expressing TALE - VP64 ( " experimental " , red ) . n indicates number of independent biological replicates that were combined . Noise was suppressed by a running mean filter of indicated window . Shading between lines corresponds to color of sample with highest value . Arrowheads on right - side mark 5th 95th percentiles of genome - wide DamID values . Top panels : domainograms ; for every window of indicated ( vertical axis ) centered on a genomic position ( horizontal axis ) pixel shade indicates ranking of change in DamID score ( experimental minus control ) in this window compared to genome - wide changes in DamID scores across possible windows of same . Blue : DamID score is highest in control samples ; red : DamID score is highest in experimental samples ( color key on right of panel ( a ) ) . In ( a ) activation of Sox6 was experimental perturbation , activation of Nrp1 ( which is located on a different chromosome ) served as control ; in ( b ) activation of Nrp1 was experimental condition activation of Sox6 served as control .

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A, B Bottom panels: gene annotation track (mm10); the activated gene is marked in green and the location of the TALE-VP64 target sequence is shown by the vertical dashed green line. Middle panels: DamID tracks of NL interactions in control cells ("control", blue line) and cells expressing TALE-VP64 ("experimental", red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Arrowheads on the right-hand side mark the 5th and 95th percentiles of genome-wide DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis) the pixel shade indicates the ranking of the change in DamID score (experimental minus control) in this window compared to the genome-wide changes in DamID scores across all possible windows of the same size. Blue: DamID score is highest in control samples; red: DamID score is highest in experimental samples (color key on the right of panel (a)). In (a) activation of Sox6 was the experimental perturbation, activation of Nrp1 (which is located on a different chromosome) served as control; in (b) activation of Nrp1 was the experimental condition and activation of Sox6 served as control.


( a - b ) Plots as in Figure 1 , showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 ( a ) SOX6 ( b ) . Control were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes . Vertical green dotted lines mark position of sgRNA target .

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(a-b) Plots as in Figure 1, showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 (a) and SOX6 (b). Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes. Vertical green dotted lines mark the position of the sgRNA target sequence.


( c ) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa . Genomic regions are aligned by respective sgRNA target positions , oriented so that activated genes are transcribed from left to right .

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(c) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa. Genomic regions are aligned by the respective sgRNA target positions, and oriented so that the activated genes are all transcribed from left to right.


Average DamID values plotted against average expression of eight genes activated by CRISPRa ( red ; n = 2 ) or in control that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes ( blue ; n ranging from 19 to 27 ) .

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Average DamID values plotted against average expression levels of eight genes activated by CRISPRa (red; n = 2) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes (blue; n ranging from 19 to 27).


A - C CRISPRa activation of ADAM22 ( a ) , ABCB1 ( b ) PTN ( c ) in human RPE - 1 . Top panels visualize DamID data similar to Figure 2a - b . Middle panels show maps of replication timing at same resolution in same plotting style as panels , except that different colors are used as indicated . Bottom panels show gene track , with activated gene highlighted in green .

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A-C CRISPRa activation of ADAM22 (a), ABCB1 (b) and PTN (c) in human RPE-1 cells. Top panels visualize DamID data similar to Figure 2a-b. Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels, except that different colors are used as indicated. Bottom panels show gene track, with activated gene highlighted in green.


Changes in NL interactions after CRISPRa activation of MLK4 in human RPE - 1 . Visualization of DamID data as in Figure 2a - b .

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Changes in NL interactions after CRISPRa activation of MLK4 in human RPE-1 cells. Visualization of DamID data as in Figure 2a-b.


( a ) DamID domainograms of NL interactions after activation of CCSER1 , GRID2 , or both . data are from human RPE - 1 .

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(a) DamID domainograms of NL interactions after activation of CCSER1, GRID2, or both. All data are from human RPE-1 cells.


( b ) DamID domainograms after activation of ADAM22 , ABCB1 , or genes ABCB4 , ABCB1 , ADAM22 RUNC3B simultaneously . data are from human RPE - 1 . DamID data of activation of ADAM22 ABCB1 alone are same as in Figure 4a , b .

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(b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously. All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b.


( a ) DamID profiles of 129Sv allele of Morc1 locus with deletions of promoters of Morc , Morc1 DppA2 ( deletions marked by yellow vertical boxes ) , in control . ( b ) Same as ( a ) , but for non - mutated Cast alleles .

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(a) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc, Morc1 and DppA2 (deletions marked by yellow vertical boxes), and in control cells. (b) Same as (a), but for the non-mutated Cast alleles.


( c - d ) Same design as ( a - b ) , but with only a single mono - allelic deletion of Morc promoter ( vertical yellow box ) .

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(c-d) Same design as (a-b), but with only a single mono-allelic deletion of the Morc promoter (vertical yellow box).


( e - f ) Effect of PAS insertion on NL interactions of Cobl gene locus . Same design as ( a - b ) but with insertion of a PAS ( located at red triangle vertical dotted ) that truncates 129Sv allele of Cobl transcription unit . 129Sv allele is shown in ( e ) , non - mutated Cast allele in ( f ) . Clones with Morc1 locus mutations ( each assayed in four independent biological replicates ) served collectively as control in ( e - f )

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(e-f) Effect of PAS insertion on NL interactions of Cobl gene locus. Same design as (a-b) but with insertion of a PAS (located at red triangle and vertical dotted line) that truncates the 129Sv allele of the Cobl transcription unit. 129Sv allele is shown in (e), non-mutated Cast allele in (f). Clones with Morc1 locus mutations (each assayed in four independent biological replicates) served collectively as control in (e-f)


( c ) Average DamID profiles across 17 transgene integration sites inside LADs . Blue curve shows alleles without integrations , red curve shows corresponding alleles with integrations . Shading between lines shows which curve has highest value .

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(c) Average DamID profiles across 17 transgene integration sites inside LADs. Blue curve shows alleles without integrations, red curve shows the corresponding alleles with integrations. Shading between the lines shows which curve has the highest value.


( d ) Relative expression of barcoded transgenes in LADs iLADs .

(d) Relative expression levels of individual barcoded transgenes in LADs and iLADs.


( e ) Estimated expression of integrated transgenes in LADs ( vertical orange lines ; median value indicated by arrowhead ) , compared to distribution of expression of active endogenous genes ( black curve ) .

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(e) Estimated expression levels of integrated transgenes in LADs (vertical orange lines; median value indicated by arrowhead), compared to the distribution of expression levels of all active endogenous genes (black curve).


A family pedigree , where circles denote members , squares members , solid symbols affected members , symbols asymptomatic members with normal physical exam ; dots indicate heterozygous carriers double denotes a consanguineous marriage . pictures show scapular winging which is a consistent clinical sign in affected individuals .

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A The family pedigree, where circles denote female members, squares male members, solid symbols affected members, and white symbols asymptomatic members with normal physical exam; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage. The pictures show scapular winging which is a consistent clinical sign in affected individuals.


B Hematoxylin eosin ( H & amp ; amp ; E ) of skeletal muscle from II . 1 shows histological features of moderate to severe dystrophic pattern . ( Scale bar , 50 μm ) .

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B Hematoxylin and eosin staining (H&amp;E) of skeletal muscle from patient II.1 shows histological features of moderate to severe dystrophic pattern. (Scale bar, 50 μm).


C T1 - weighted MRI axial at thigh calf show that fatty degeneration is more prominent in thigh , equally affecting posterior anterior compartment , with relative sparing of rectus femoris , sartorius gracilismuscles until late stages ( 4 , 10 11 , respectively ) . Strikingly , fatty tissue is located in internal parts of almost affected in thigh ( 1 , 2 , 3 , 5 - 9 ) , while external regions are spared . At calf level , only gastrocnemius medialis muscle ( 12 ) shows this pattern , while soleus ( 13 ) is diffusely involved . II . 2 ( PII . 2 ) shows late - stage thigh with an unusual involvement of tibialis posteriormuscle ( 14 ) in lower leg .

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C T1-weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles, equally affecting posterior and anterior compartment, with relative sparing of the rectus femoris, sartorius and gracilismuscles until late stages (4, 10 and 11, respectively). Strikingly, the fatty tissue is located in the internal parts of almost all the affected muscles in thigh (1, 2, 3, 5-9), while the external regions are spared. At calf level, only the gastrocnemius medialis muscle (12) shows this pattern, while the soleus (13) is diffusely involved. Patient II.2 (PII.2) shows late-stage thigh muscles with an unusual involvement of the tibialis posteriormuscle (14) in the lower leg.


A Muscle sections show variable labeling using an antibody against glycosylated α - dystroglycan ( αDG - IIH6 ) , whereas labeling using antibodies against α - dystroglycan ( αDG - ) , β - dystroglycan ( βDG ) laminin - 2 is similar to control ( Scale bar , 100 μm ) .

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A Muscle sections show variable labeling using an antibody against glycosylated α-dystroglycan (αDG-IIH6), whereas labeling using antibodies against α-dystroglycan core protein (αDG-Core), β-dystroglycan (βDG) and laminin-2 is similar to control (Scale bar, 100 μm).


B Western blots ligand - overlay ( O / L ) of wheat - germ agglutinin - enriched muscle fibroblasts lysates from PII . 2 , PII . 5 , healthy sibling ( HII . 3 ) healthy controls ( Ctr , Ctr1 , Ctr2 ) ( Muscle : 250μg / lane , Fibroblasts : 800μg / lane ) . In muscle , expression of αDG - IIH6 is reduced but αDG - shows similar expression as controls , with a slight reduction in molecular ; laminin overlay detected some binding activity but was diminished compared to controls , whereas agrin - binding activity showed no difference with controls . In fibroblasts , both αDG - IIH6 αDG - expression laminin - binding activity were normal , suggesting that unlike other muscular dystrophies , α - dystroglycan glycosylation defect is muscle - specific in POGLUT1 .

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B Western blots and ligand-overlay (O/L) of wheat-germ agglutinin-enriched muscle and fibroblasts lysates from PII.2, PII.5, the healthy sibling (HII.3) and healthy controls (Ctr, Ctr1, and Ctr2) (Muscle: 250μg protein/lane, Fibroblasts: 800μg/lane). In muscle, expression of αDG-IIH6 is reduced but the αDG-Core shows similar expression as controls, with a slight reduction in molecular weight; laminin overlay assay detected some binding activity but was diminished compared to controls, whereas agrin-binding activity showed no difference with controls. In fibroblasts, both αDG-IIH6 and αDG-Core expression and laminin-binding activity were normal, suggesting that unlike other muscular dystrophies, α-dystroglycan glycosylation defect is muscle-specific in POGLUT1 patients.


C No ultrastructural alterations are observed in muscle by electron microscopy ( 6 - 7 fields were captured from PII . 1 , PII . 3 , PII . 5 ) . Note normal basement compactation ( arrowheads ) . Original magnification : 26 , 700 .

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C No ultrastructural alterations are observed in muscle by electron microscopy (6-7 fields were captured from PII.1, PII.3, and PII.5). Note normal basement membrane compactation (arrowheads). Original magnification: 26,700.


A O - glucosyltransferase activity of wild - type ( WT ) or D233E mutant POGLUT1 toward human factor IX EGF repeat ( hFIX - EGF ) . Wild - type shows higher O - glucosyltransferase activity than D233E , this activity is dependent on of acceptor substrate hFIX - EGF repeat ( left ) on of donor substrate UDP - glucose ( UDP - Glc ) ( right ) . Values indicate mean ± from three independent assays .

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A Protein O-glucosyltransferase activity of wild-type (WT) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat (hFIX-EGF). Wild-type shows higher O-glucosyltransferase activity than D233E, and this activity is dependent on the concentration of the acceptor substrate hFIX-EGF repeat (left) and on the concentration of donor substrate UDP-glucose (UDP-Glc) (right). Values indicate mean ± SEM from three independent assays.


B O - glucosyltransferase activity towards five different single EGF repeats from mouse Notch1 . Wild - type POGLUT1 shows higher activity toward EGF repeats tested than POGLUT1D233E . Y - axis shows normalized activity relative to wild - type POGLUT1 . EGF repeats were at 10 μM . Values indicate mean ± from three independent assays .

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B O-glucosyltransferase activity towards five different single EGF repeats from mouse Notch1. Wild-type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E. The Y-axis shows the normalized activity relative to wild-type POGLUT1. EGF repeats were at 10 μM. Values indicate mean ± SEM from three independent assays.


C Elution profiles of POGLUT1 reaction products on reverse phase HPLC . hFIX - EGF repeat was incubated with wild - type or D233E mutant POGLUT1 donor substrate , UDP - Glc or UDP - xylose ( UDP - Xyl ) , at 37ºC overnight . Values on top of peaks indicate measured masses . Addition of Glc ( 162 Da ) or xylose ( 132 Da ) to hFIX - EGF ( 5696 . 2 Da ) by wild - type POGLUT1 caused a shift to an earlier retention time ( left ) . Similarly , products exhibited a similar shift after incubation with POGLUT1D233E ( right ) . These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX - EGF repeats thus has residual enzymatic activity .

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C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC. hFIX-EGF repeat was incubated with wild-type or D233E mutant POGLUT1 and donor substrate, UDP-Glc or UDP-xylose (UDP-Xyl), at 37ºC overnight. Values on top of the peaks indicate the measured masses. Addition of Glc (162 Da) or xylose (132 Da) to hFIX-EGF (5696.2 Da) by wild-type POGLUT1 caused a shift to an earlier retention time (left). Similarly, the products exhibited a similar shift after incubation with POGLUT1D233E (right). These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX-EGF repeats and thus has residual enzymatic activity.


A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain ( NICD ) in .

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A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain (NICD) in patients.


B - C qRT - PCR on extracted from skeletal - muscle shows that expression of HES1 ( B ) PAX7 ( C ) was significantly lower in D233E compared to healthy controls . Mean ± , Student ´ s t - ( B ) Mann - Whitney U ( C ) .

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B-C qRT-PCR on RNA extracted from skeletal-muscle shows that expression of HES1 (B) and PAX7 (C) was significantly lower in D233E patients compared to healthy controls. Mean ± SEM, Student´s t-test (B) and Mann-Whitney U test (C).


D PAX7 + in skeletal muscle sections , demonstrating that satellite are less abundant in D233E than in controls ( n = 3 with 10 - 15 fields analyzed per muscle ) . Mean ± ; Mann - Whitney U ; Scale bar , 50 μm .

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D PAX7+ cells in skeletal muscle sections, demonstrating that satellite cells are less abundant in D233E patients than in controls (n = 3 muscles with 10-15 fields analyzed per muscle). Mean ± SEM; Mann-Whitney U test; Scale bar, 50 μm.


A - A " of Drosophila indirect flight at 25 % pupal development with 22C10 antibody ( myotube marker ) anti - Twist antibody ( myoblast marker ) indicates that in wild - type , indirect flight are formed from 6 myotubes , a large number of Twist + myoblasts are present . B - B " rumi79 / 79 mutants raised at 18C show no obvious defects in myoblast number or myotube . C - D " rumi79 / 79 mutants exhibit decreased number of myoblasts at 25 ° - 30 ° C either an incomplete set of short myotubes when raised at 25C or aberrant of myotubes when raised at 30C . E - E " rumi79 / 79 overexpressing FLAG - tagged POGLUT1WT in muscle compartment using Mef2 - GAL4 raised at 25C during pupal stage show a rescue of rumi79 / 79 muscle phenotype . F - G " Overexpression of FLAG - tagged POGLUT1D233E only partially rescues rumi79 / 79 muscle phenotype , with some variation in myotube number of restored Twist + ( compare G F ) . Note that number of Twist + restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT . Scale bar in A ' is 50 µm applies to A - G " . H of myotube lengths in rumi79 / 79 mutants with or without POGLUT1 expression indicates a weak rescue of by POGLUT1D233E compared to POGLUT1WT . Mean ± standard deviation is shown . differences in myotube lengths are statistically significant ( n = 22 - 54 ) . Mean ± standard deviation is shown ; One - way Anova with Bonferroni ´ s multiple comparisons .

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A-A" Staining of Drosophila indirect flight muscles at 25% pupal development with 22C10 antibody (myotube marker) and anti-Twist antibody (myoblast marker) indicates that in wild-type animals, indirect flight muscles are formed from 6 myotubes, and a large number of Twist+myoblasts are present.B-B" rumi79/79 mutants raised at 18C show no obvious defects in myoblast number or myotube morphology.C-D" rumi79/79 mutants exhibit decreased number of myoblasts at 25°-30°C and either an incomplete set of short myotubes when raised at 25C or aberrant morphology of myotubes when raised at 30C.E-E" rumi79/79 animals overexpressing FLAG-tagged POGLUT1WT in the muscle compartment using Mef2-GAL4 and raised at 25C during the pupal stage show a rescue of the rumi79/79 muscle phenotype.F-G" Overexpression of FLAG-tagged POGLUT1D233E only partially rescues the rumi79/79 muscle phenotype, with some variation in the myotube length and the number of restored Twist+cells (compare G and F). Note that the number of Twist+cells restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT. Scale bar in A' is 50 µm and applies to A-G".H Quantification of myotube lengths in rumi79/79 mutants with or without POGLUT1 expression indicates a weak rescue of the phenotypes by POGLUT1D233E compared to POGLUT1WT. Mean ± standard deviation is shown. The differences in myotube lengths are statistically significant (n = 22-54). Mean ± standard deviation is shown; One-way Anova with Bonferroni´s multiple comparisons test.


A - F Proliferation assays examined proliferating ( PAX7 + MyoD + , arrows ) , self - renewing ( PAX7 + MyoD − , open arrows ) , differentiating ( PAX7 − MyoD + , arrowheads ) from D233E ( n = 2 ) , healthy controls ( n = 3 ) disease controls ( n = 3 ) at 1 , 4 , 8 days growing in proliferation medium . ( A ) shows representative from day 8 . number of patientmyoblasts is lower than that of controls at three time points , indicating a slower proliferation rate in patientmyoblasts ( B ) , accordingly , of proliferating ( C ) is smaller in . ( D ) of self - renewing is small in ´ s culture , reflecting a poor capacity for maintaining pool of quiescent SCs . In addition , of PAX7 + ( E ) is smaller in ´ s culture , similarly to what was found in adult D233E muscle ( shown in Fig 5D ) . ( F ) of differentiating is higher in . We examine proliferation capturing 7 - 12 randomly fields per day condition ( mean ± is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons ; Scale bar , 50 µm ) .

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A-F Proliferation assays examined proliferating (PAX7+MyoD+, arrows), self-renewing (PAX7+ MyoD−, open arrows), and differentiating (PAX7− MyoD+, arrowheads) cells from D233E patients (n = 2), healthy controls (n = 3) and disease controls (n = 3) at 1, 4, and 8 days growing in proliferation medium. (A) shows representative images from day 8. The number of patientmyoblasts is lower than that of controls at all three time points, indicating a slower proliferation rate in patientmyoblasts (B), and accordingly, the percentage of proliferating cells (C) is smaller in the patient. (D) The percentage of self-renewing cells is small in patient´s culture, reflecting a poor capacity for maintaining the pool of quiescent SCs. In addition, the percentage of PAX7+ cells (E) is smaller in patient´s culture, similarly to what was found in adult D233E muscle (shown in Fig 5D). (F) The percentage of differentiating cells is higher in the patient. We examine proliferation capturing 7-12 randomly fields per day and condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).


G - I When myoblasts started to be confluent , proliferation medium was replaced with differentiation medium , myoblasts started to fuse yielding myotubes , which were analyzed after four days of differentiation . ( H ) fusion index , which measures of nuclei in myotubes with respect to total number of nuclei in myogenic , was higher in D233E cultures than in controls . ( I ) Besides , myogenin showed increased expression in D233E myoblast cultures compared to controls . data support that differentiation process is facilitated in ´ s muscle . Data was collected from 3 - 10 randomly chosen areas per condition ( mean ± is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons ( H ) One - way Anova with Bonferroni ´ s multiple comparisons ( I ) ; Scale bar , 50 µm ) .

O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 When myoblasts started to be confluent, the proliferation medium was replaced with differentiation medium, and the myoblasts started to fuse yielding myotubes, which were analyzed after four days of differentiation. (H) The fusion index, which measures the percentage of nuclei in myotubes with respect to the total number of nuclei in myogenic cells, was higher in D233E cultures than in controls. (I) Besides, myogenin showed increased expression in D233E myoblast cultures compared to controls. The data support that differentiation process is facilitated in patient´s muscle. Data was collected from 3-10 randomly chosen areas per condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test (H) and One-way Anova with Bonferroni´s multiple comparisons test (I); Scale bar, 50 µm).


A - C We overexpressed GFP - tagged NICD in immortalized myoblasts to rescue features displayed by immortalized D233E myoblasts . ( B ) We analyzed proliferation at 1 , 3 5 days , proliferation rate in - LV - NICD - GFP reached level observed in control - LV - GFP , which were higher than in - LV - GFP . ( C ) However , of PAX7 + in - LV - NICD - GFP showed same reduced value as in - LV - GFP , which were significantly lower than in control - LV - GFP . majority of control myoblasts were proliferating ( PAX7 + MyoD + , arrows ) , with no quiescent few differentiating detected . Data were collected from n = 2 independent assays ( 5 - 8 per condition ) . Mean ± is shown ; One - way Anova with Tukey multiple comparisions ( B ) Kruskal - Wallis with Dunn ´ s multiple comparisons ( C ) ; Scale bar , 50 µm ) .

O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C We overexpressed GFP-tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts. (B) We analyzed the proliferation at 1, 3 and 5 days, and proliferation rate in patient-LV-NICD-GFP reached the level observed in control-LV-GFP, which were higher than in patient-LV-GFP. (C) However, the percentage of PAX7+cells in patient-LV-NICD-GFP showed the same reduced value as in patient-LV-GFP, which were significantly lower than in control-LV-GFP. The majority of control myoblasts were proliferating cells (PAX7+ MyoD+, arrows), with no quiescent cells and few differentiating cells detected. Data were collected from n = 2 independent assays (5-8 images per condition). Mean ± SEM is shown; One-way Anova with Tukey multiple comparisions test (B) and Kruskal-Wallis with Dunn´s multiple comparisons test (C); Scale bar, 50 µm).


D - F As previously described in ' s myoblasts , - LV - GFPmyoblasts showed a striking facilitated differentiation compared to control - LV - GFP - myoblasts . This phenotype fully disappeared in - LV - GFP - NICD myoblasts , as of fusion index ( E ) , myogenin expression ( F ) demonstrated . data support a pathogenic role for Notch inhibition on altered differentiation in our . Data were collected from n = 3 independent assays ( 13 - 17 per condition ) . Mean ± is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons ; Scale bar , 50 µm ) .

O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F As previously described in patient's primary myoblasts, patient-LV-GFPmyoblasts showed a striking facilitated differentiation compared to control-LV-GFP-myoblasts. This phenotype fully disappeared in patient-LV-GFP-NICD myoblasts, as quantification of the fusion index (E), and myogenin expression (F) demonstrated. The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients. Data were collected from n = 3 independent assays (13-17 images per condition). Mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).


A Immortalized myoblasts from a healthy control showed a progressive pattern of α - dystroglycan glycosylation during five days of differentiation ( D0 - D5 ) , as has been previously described by Goddeeris et al . Immortalized myoblasts from displayed an irregular pattern of glycosylation , with a final level of glycosylated α - dystroglycan that was lower than control .

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

A Immortalized primary myoblasts from a healthy control showed a progressive pattern of α-dystroglycan glycosylation during five days of differentiation (D0-D5), as has been previously described by Goddeeris et al. Immortalized primary myoblasts from patients displayed an irregular pattern of glycosylation, with a final level of glycosylated α-dystroglycan that was lower than control.