( d ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status . Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate ( and HUWE1 mutation itself ) is not due to silencing of MLH1 ( Mann - Whitney , p = 0 . 0007 , n ≥ 11 ) .

O O O O O O O O O O O O O O B-ORGANISM B-DISEASE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-DISEASE O B-GENEPROD O O O O O O O O O B-GENEPROD 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

(d) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status. Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate (and HUWE1 mutation itself) is not due to silencing of MLH1 (Mann-Whitney, p = 0.0007, n ≥ 11).


( e ) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels . Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy . ( f ) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels . Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy .

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

(e) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy. (f) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy.


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Log Rank , p < 0 . 001 , n ≥ 15 ) .

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

(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Log Rank, p < 0.001, n ≥ 15).


( c ) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) . ( d ) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) .

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

(c) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset). (d) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset).


( a ) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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

(a) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice . Deletion of one copy of Mcl1 led to a significant increase in survival of these animals ( Log Rank , p = 0 . 0052 , n = 9 vs 10 ) .

O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O B-GENEPROD 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) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice. Deletion of one copy of Mcl1 led to a significant increase in survival of these animals (Log Rank, p = 0.0052, n = 9 vs 10).


( c ) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice . Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion ( Mann - Whitney , p = 0 . 0281 , n = 8 v 8 ) .

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

(c) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice. Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion (Mann-Whitney, p=0.0281, n = 8 v 8).


( A ) Ookinetes sandwiched between two hydrogel surfaces .

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(A) Ookinetes sandwiched between two hydrogel surfaces.


( B ) Percentage of ookinetes moving for more than one parasite length within the time of observation ( 5 - 10 min ) if sandwiched between soft , medium ( med ) or stiff hydrogel or glass . Bars represent the mean and error bars the standard deviation of at least three independent experiments . ( C ) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B .

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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O

(B) Percentage of ookinetes moving for more than one parasite length within the time of observation (5-10 min) if sandwiched between soft, medium (med) or stiff hydrogel or glass. Bars represent the mean and error bars the standard deviation of at least three independent experiments. (C) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B.


( D ) Percentage of motile ookinetes after 20 - 26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in ( B ) . Shown is the average ± S . D . from at least two independent experiments . ( E ) Speed of motile ookinetes after 20 - 26 hours of incubation between hydrogels . Speed on glass was not analyzed ( n . a . ) due to the low fraction of motile ookinetes at this timepoint .

O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-TISSUE O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O

(D) Percentage of motile ookinetes after 20-26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in (B). Shown is the average ± S.D. from at least two independent experiments. (E) Speed of motile ookinetes after 20-26 hours of incubation between hydrogels. Speed on glass was not analyzed (n.a.) due to the low fraction of motile ookinetes at this timepoint.


( F ) Migration patterns of ookinetes . Image : Overlay of tracked migration paths ( black ) and DIC image showing ookinetes at the end of the recorded image sequence . Graph : Quantitative analysis of the indicated different migration patterns on soft , medium ( med ) and stiff hydrogels .

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

(F) Migration patterns of ookinetes. Image: Overlay of tracked migration paths (black) and DIC image showing ookinetes at the end of the recorded image sequence. Graph: Quantitative analysis of the indicated different migration patterns on soft, medium (med) and stiff hydrogels.


( G ) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints .

O O O O O B-EXP_ASSAY O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O

(G) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints.


( A ) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel .

O O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-TISSUE O O O O B-SMALL_MOLECULE O

(A) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel.


( B ) Overlay of sporozoite tracks ( black ) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence . Scale bar , 20 µm .

O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(B) Overlay of sporozoite tracks (black) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence. Scale bar, 20 µm.


( C ) Speed of WT sporozoites moving in the skin in vivo ( Douglas et al , 2018b ) and in soft hydrogels with small or large pores .

O O O B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(C) Speed of WT sporozoites moving in the skin in vivo (Douglas et al, 2018b) and in soft hydrogels with small or large pores.


( D , E ) Speed of indicated mutant and control ( Ctrl ) parasites in hydrogels with small ( D ) and large ( E ) pores .

O O O O O B-EXP_ASSAY 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

(D, E) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores.


( F , G ) Speed of drug - treated and control parasites in hydrogels with small ( F ) and large ( G ) pores . Data information : Note the faster speed in gels with larger pores . Data from at least two independent experiments . Numbers above bars indicate the number of sporozoites analyzed . Significance determined by Mann - Whitney test . Box - and - whisker plots ( C - G ) depict the 25 % quantile , median , 75 % quantile and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Outliers beyond this range are shown as black dots .

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

(F, G) Speed of drug-treated and control parasites in hydrogels with small (F) and large (G) pores. Data information: Note the faster speed in gels with larger pores. Data from at least two independent experiments. Numbers above bars indicate the number of sporozoites analyzed. Significance determined by Mann-Whitney test. Box-and-whisker plots (C-G) depict the 25% quantile, median, 75% quantile and nearest observations within 1.5 times the interquartile range (whiskers). Outliers beyond this range are shown as black dots.


( A ) Percentage of motile sporozoites on endothelial cells ( LSECs ) versus glass . Shown is average ± S . D . from three independent experiments . Significance determined using an unpaired t - test .

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

(A) Percentage of motile sporozoites on endothelial cells (LSECs) versus glass. Shown is average ± S.D. from three independent experiments. Significance determined using an unpaired t-test.


( B ) Left : Merged fluorescence images showing sporozoites on cultured HFF cells . Actin filaments of HFF cells were stained with phalloidin ( shown in red ) , nuclei were stained with Hoechst ( blue ) and sporozoites expressed GFP ( green ) . Scale bar , 50 µm . Right : Maximum projection of sporozoites moving for 3 min on cultured HFF cells . Scale bar , 50 µm .

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

(B) Left: Merged fluorescence images showing sporozoites on cultured HFF cells. Actin filaments of HFF cells were stained with phalloidin (shown in red), nuclei were stained with Hoechst (blue) and sporozoites expressed GFP (green). Scale bar, 50 µm. Right: Maximum projection of sporozoites moving for 3 min on cultured HFF cells. Scale bar, 50 µm.


( C ) Percentage of motile sporozoites on confluent layers of endothelial cells ( HUVEC ) , fibroblasts ( HFF ) and on glass . Shown is average ± S . D . from two independent experiments . No significant differences as determined by One - way analysis of variance with Bonferroni ' s Multiple Comparison test .

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

(C) Percentage of motile sporozoites on confluent layers of endothelial cells (HUVEC), fibroblasts (HFF) and on glass. Shown is average ± S.D. from two independent experiments. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


( D ) Sporozoite on planar uncoated hydrogels .

(D) Sporozoite on planar uncoated hydrogels.


( E ) Overlay of sporozoite tracks ( black ) and DIC image showing sporozoites on a hydrogel . Scale bar , 50 µm . ( F ) Percentage of motile sporozoites on soft , medium ( med ) or stiff hydrogel or glass 30 min after activation with BSA . Shown is the average ± S . D . from at least three independent experiments . Significance determined by One - way analysis of variance with Bonferroni ' s Multiple Comparison test .

O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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

(E) Overlay of sporozoite tracks (black) and DIC image showing sporozoites on a hydrogel. Scale bar, 50 µm.(F) Percentage of motile sporozoites on soft, medium (med) or stiff hydrogel or glass 30 min after activation with BSA. Shown is the average ± S.D. from at least three independent experiments. Significance determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


( G ) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA . All sporozoites moving for more than one parasite length within the time of observation were analyzed , even if they stopped moving or detached during imaging . Significance determined by Kruskal - Wallis test with Dunn ' s Multiple Comparison test .

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

(G) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA. All sporozoites moving for more than one parasite length within the time of observation were analyzed, even if they stopped moving or detached during imaging. Significance determined by Kruskal-Wallis test with Dunn's Multiple Comparison test.


( H ) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses . Note that not all sporozoites analyzed in ( G ) complied with that requirement . To be able to analyze a high number of sporozoites , movies were taken 10 to 45 min after activation with BSA . Speed of sporozoites on soft hydrogels was not analyzed ( n . a . ) due to the low fraction of motile sporozoites on these hydrogels . No significant differences as determined by One - way analysis of variance with Bonferroni ' s Multiple Comparison test .

O O O B-EXP_ASSAY O B-TISSUE O O 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 B-TISSUE O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(H) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses. Note that not all sporozoites analyzed in (G) complied with that requirement. To be able to analyze a high number of sporozoites, movies were taken 10 to 45 min after activation with BSA. Speed of sporozoites on soft hydrogels was not analyzed (n.a.) due to the low fraction of motile sporozoites on these hydrogels. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


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 .

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

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

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 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 .

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

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 core protein ( αDG - Core ) , β - dystroglycan ( βDG ) and laminin - 2 is similar to control ( Scale bar , 100 μm ) .

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

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 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 .

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

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 , and PII . 5 ) . Note normal basement membrane compactation ( arrowheads ) . Original magnification : 26 , 700 .

O O O O O O O B-TISSUE 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 B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O

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 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 .

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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 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 .

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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 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 .

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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 patients .

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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 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 ) .

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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 + 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 .

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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 " 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 .

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

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 ) , 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 ) .

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

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 , 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 ) .

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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 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 ) .

O O O O O B-GENEPROD O O O O O O B-CELL_TYPE 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 O O O O O 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 B-ORGANISM O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O 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 O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O B-CELL_TYPE O 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 O B-ORGANISM O B-GENEPROD O O B-CELL_TYPE 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 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 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 .

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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.


B α - dystroglycan glycosylation of infected immortalized myoblasts from patient - LV - NICD - GFP at differentiation day 5 ( D5 ) showed a striking reduction , in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression . α - dystroglycan in myoblasts from patient - LV - GFP showed a reduction of glycosylation compared to control - LV - GFP at D5 , similar to the results obtained in the adult muscle from patients ( Fig . 2 ) .

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B α-dystroglycan glycosylation of infected immortalized myoblasts from patient-LV-NICD-GFP at differentiation day 5 (D5) showed a striking reduction, in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression. α-dystroglycan in myoblasts from patient-LV-GFP showed a reduction of glycosylation compared to control-LV-GFP at D5, similar to the results obtained in the adult muscle from patients (Fig. 2).


C - D C2C12 myogenic cells showed a reduced level of glycosylated α - dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium . This indicates that reducing Notch signaling can affect α - dystroglycan glycosylation in wild - type C2C12 myoblasts cells , which do not harbor any known mutations in α - dystroglycan glycosyltransferases or Poglut1 .

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C-D C2C12 myogenic cells showed a reduced level of glycosylated α-dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium. This indicates that reducing Notch signaling can affect α-dystroglycan glycosylation in wild-type C2C12 myoblasts cells, which do not harbor any known mutations in α-dystroglycan glycosyltransferases or Poglut1.


E - F qRT - PCR experiments show that upon DAPT or LY3039478 treatment , a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation .

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E-F qRT-PCR experiments show that upon DAPT or LY3039478 treatment, a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation.


K - P Realtime qRT - PCR revealed the expression profiles of four zebrafish chs genes in different tissues ( K ) , different gut sections ( L ) , and different developmental stages ( M - P ) . One representative example of three repeat experiments is shown . Data were the mean ± SEM of n = 3 technical replicates . n . s . no significant , no label P < 0 . 05 by student ' s t - test .

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K-P Realtime qRT-PCR revealed the expression profiles of four zebrafish chs genes in different tissues (K), different gut sections (L), and different developmental stages (M-P). One representative example of three repeat experiments is shown. Data were the mean ± SEM of n=3 technical replicates. n.s. no significant, no label P < 0.05 by student's t-test.


F - M Compared with wild - types ( F - I ) , mutants ( J - M ) completely lost the CM ( red ) in the gut of 7dpf larva ( F , J ) , as well as in the intestinal bulb ( G , K ) , middle intestine ( H , L ) and posterior intestine ( I , M ) of adults . Red , SNAP - CBD stain ; blue , DAPI stain ; WT , wild - type ; MU , chs1 - / - mutant .

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F-M Compared with wild-types (F-I), mutants (J-M) completely lost the CM (red) in the gut of 7dpf larva (F, J), as well as in the intestinal bulb (G, K), middle intestine (H, L) and posterior intestine (I, M) of adults. Red, SNAP-CBD stain; blue, DAPI stain; WT, wild-type; MU, chs1-/- mutant.


A , B The survival rates of wild - types and mutants in the first eight days ( A ) and the first six months ( B ) . Two independent batches ( 1 and 2 ) were conducted for each experiment . n , the initial numbers of zebrafish . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by long - rank ( Mantel - Cox ) test . Please see Table EV1 for data on another three batches of independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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 The survival rates of wild-types and mutants in the first eight days (A) and the first six months (B). Two independent batches (1 and 2) were conducted for each experiment. n, the initial numbers of zebrafish. * P < 0.05, ** P < 0.01, *** P < 0.001 by long-rank (Mantel-Cox) test. Please see Table EV1 for data on another three batches of independent experiments.


E - J 6 / 7 dpf larva mutants were fed with 6 - μm polystyrene beads . The beads jammed in the intestinal bulb and middle intestine ( E , F , H , I ) . In some cases , the beads escaped the bead cord and stuck between villi ( white triangles ) ( E , G , I , J ) . Blue rectangle indicates the intestinal bulb . Right panels were magnification of the red rectangle area in left panels .

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E-J 6/7 dpf larva mutants were fed with 6-μm polystyrene beads. The beads jammed in the intestinal bulb and middle intestine (E, F, H, I). In some cases, the beads escaped the bead cord and stuck between villi (white triangles) (E, G, I, J). Blue rectangle indicates the intestinal bulb. Right panels were magnification of the red rectangle area in left panels.


B Comparison of 3 - month - old wild - types and mutants in the pairwise microbiota compositional dissimilarity ( Jaccard distance ) between the digesta ( niche1 ) and the epithelia ( niche2 ) . This metric represents the radial gradient of the gut bacterial composition . The comparison was done on four taxonomy levels . Data of n = 14 individuals were represented in box ( interquartile range ) and whisker ( min to max ) plots . Individual data points were plotted as black dots . * P < 0 . 05 , * * P < 0 . 01 by the conservative , nonparametric Kruskal - Wallis test . Please see Appendix Fig S10 for another way of data presentation , and Dataset EV1 for the details of the sample information and raw statistics . Note that each 16S - rRNA library contains only one sample from one animal .

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

B Comparison of 3-month-old wild-types and mutants in the pairwise microbiota compositional dissimilarity (Jaccard distance) between the digesta (niche1) and the epithelia (niche2). This metric represents the radial gradient of the gut bacterial composition. The comparison was done on four taxonomy levels. Data of n=14 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05, ** P < 0.01 by the conservative, nonparametric Kruskal-Wallis test. Please see Appendix Fig S10 for another way of data presentation, and Dataset EV1 for the details of the sample information and raw statistics. Note that each 16S-rRNA library contains only one sample from one animal.


Comparison of the bacterial community richness of the intestine samples ( S3 - S6 ; including both the digesta and the epithelia ) between six 3 - month - old wild - types and six 3 - month - old mutants . Three alpha diversity metrics were used : Ace ( abundance - based coverage estimator ) , Chao1 Data of n = 6 individuals were represented in box ( interquartile range ) and whisker ( min to max ) plots . Individual data points were plotted as black dots . * P < 0 . 05 by Kruskal - Wallis test .

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Comparison of the bacterial community richness of the intestine samples (S3-S6; including both the digesta and the epithelia) between six 3-month-old wild-types and six 3-month-old mutants. Three alpha diversity metrics were used: Ace (abundance-based coverage estimator), Chao1 Data of n=6 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05 by Kruskal-Wallis test.


E Immunohistochemical detection of BrdU - labeled cells in the intestine bulb ( section S2 ) of two 3 - month - old adult wild - types and two 3 - month - old adult mutants to measure the rate of cell proliferation ( new cells were stained bright green ) and migration . Compared with wild - types ( WT ) , the mutants had more green spots along the villi 2 hours after treatment , and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments . The violin plots ( interquartile range with mean line ) show the relative numbers of green puncta from tip to base of villi , which were calculated from 3 - 5 villi from the raw version of these images . Please see Appendix Fig S19 for the images of more animals and time points .

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

E Immunohistochemical detection of BrdU-labeled cells in the intestine bulb (section S2) of two 3-month-old adult wild-types and two 3-month-old adult mutants to measure the rate of cell proliferation (new cells were stained bright green) and migration. Compared with wild-types (WT), the mutants had more green spots along the villi 2 hours after treatment, and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments. The violin plots (interquartile range with mean line) show the relative numbers of green puncta from tip to base of villi, which were calculated from 3-5 villi from the raw version of these images. Please see Appendix Fig S19 for the images of more animals and time points.


Comparison of the body length , weight and external appearance of five wild - types and five mutants after the alternative treatments of two sets of antibiotic mixtures . The treatments were started with 8 - week - old fish and continued for six weeks . The measurements and photoshoots were conducted five weeks later . Data of n = 5 are represented in box ( min to max with mean line ) plots . Individual data points for each animal were plotted as grey dots . n . s . , no significant , * * P < 0 . 01 by student ' s t - test . The raw statistics was listed in Table EV3 .

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

Comparison of the body length, weight and external appearance of five wild-types and five mutants after the alternative treatments of two sets of antibiotic mixtures. The treatments were started with 8-week-old fish and continued for six weeks. The measurements and photoshoots were conducted five weeks later. Data of n=5 are represented in box (min to max with mean line) plots. Individual data points for each animal were plotted as grey dots. n.s., no significant, ** P < 0.01 by student's t-test. The raw statistics was listed in Table EV3.


( C - D ) Snapshots from confocal live imaging movies of a representative embryo ( N = 7 ) during cellularisation co - expressing opto - Notch , Mito - Zdk , MCP : : GFP and sim - MS2 . Continuous photo - activation from the onset of cellularisation with a stack size of z = 25μm ( λ = 488 nm ) was alternated with mCherry excitation ( λ = 561 nm ) at 1 min intervals . Single confocal z - slices of the mCherry channel are shown to record opto - Notch localization before , and 10 minutes after sustained photo - activation . Magnified insets show opto - Notch bound to the mitochondrial anchor in the dark ( C ) and in the nucleus upon photo - activation ( D ) . Scale bar , 10 μm .

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

(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.


( F - H ) Maximum intensity z - projections ( z = 25 μm ) of the same embryo simultaneously photoactivated and imaged in the GFP channel ( λ = 488 nm ) to record sim expression at a time resolution of 30 s . Shown are the first cycle of photo - activation , which approximates the dark state ( F ) , 15 min ( G ) , and 35 min ( H ) after continuous photo - activation . Magnified insets show that sim is not expressed in the dark at this stage ( F ) , but it is expressed in lateral ectodermal cells after 15 min of photo - activation ( G ) , and appeared to be shut off in most nuclei after 35 mins ( H ) . Scale bar , 10 μm .

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(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.


The number of sim - expressing nuclei as counted by automated spot detection over time ( A ) Continuous opto - Notch activation initially induces sim expression across a large number of nuclei . Over time , the majority of nuclei adapt to the input and terminate expression .

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The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression.


( D - E ) Quantification of nuclear localisation of mCherry : : NICD during opto - Notch photo - activation . Raw data was bleaching - corrected with an exponential fit and shown is the nuclear / total ratio , which further normalises bleaching and embryo - to - embryo variability . NICD displays persistent nuclear enrichment during continuous activation ( D ) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol ( E ) .

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(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).


( D - E ) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant ( Hairless H2 ) and under overexpression of Twist ( Twist OE ) The observed outcomes are most consistent with the predictions from the state - dependent inactivation model .

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(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE) The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.


( 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 .

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

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(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 the lung and liver of a Pax5Jak2 / + tumor mouse . Infiltrating and blasting tumor cells are indicated by an arrow .

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(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 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 .

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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 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 ) .

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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 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

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

(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 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 .

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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 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 .

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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 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 ) .

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

(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 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 .

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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 , 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

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

(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 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 .

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

(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 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 .

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-DISEASE I-DISEASE I-DISEASE O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-SMALL_MOLECULE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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) 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 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 .

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

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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 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 .

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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 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 .

O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O B-ORGANISM B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O B-EXP_ASSAY 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 B-GENEPROD 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 O B-GENEPROD 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 B-EXP_ASSAY O O O O

(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 , 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

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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) 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 ) 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 .

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

(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 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 .

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

(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 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 )

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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 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 .

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

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.


( A , Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + and Nlrp3A350V / wt CreT - mice were incubated with LPS ( 3 hr ) before culture media were analysed for secreted IL - 1β ( A ) Cytokine values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA and P - values were corrected for multiple comparisons ( Bonferroni ) . * * , p ≤ 0 . 01 .

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

(A, Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS (3 hr) before culture media were analysed for secreted IL-1β (A) Cytokine values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA and P-values were corrected for multiple comparisons (Bonferroni). **, p ≤ 0.01.


B ) . Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + and Nlrp3A350V / wt CreT - mice were incubated with LPS and cell lysates and culture media were immunoblotted for β - actin , caspase - 1 and IL - 1β ( B ) .

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B). Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS and cell lysates and culture media were immunoblotted for β-actin, caspase-1 and IL-1β (B).


( C ) Representative pictures of Nlrp3A350V / wt MRP8 - CreTg and Nlrp3A350V / wt MRP8 - Cre - mice on days 4 and 8 after birth . Left side in each picture : Nlrp3A350V / wt MRP8 - Cre - mice ; right side in each picture : Nlrp3A350V / wt MRP8 - CreTg . Abscesses ( day 4 ) and scaling erythema ( day 8 ) are observed in Nlrp3A350V / wt MRP8 - CreTg mice .

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(C) Representative pictures of Nlrp3A350V/wt MRP8-CreTg and Nlrp3A350V/wt MRP8-Cre- mice on days 4 and 8 after birth. Left side in each picture: Nlrp3A350V/wt MRP8-Cre- mice; right side in each picture: Nlrp3A350V/wt MRP8-CreTg. Abscesses (day 4) and scaling erythema (day 8) are observed in Nlrp3A350V/wt MRP8-CreTg mice.


( D ) Survival curves of Nlrp3A350V / wt MRP8 - CreTg ( n = 22 ) and littermate Nlrp3A350V / wt MRP8 - Cre - ( n = 21 ) mice . Statistical significance was analysed by the log - rank Mantel - Cox test . * * * * , p ≤ 0 . 0001 .

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

(D) Survival curves of Nlrp3A350V/wt MRP8-CreTg (n=22) and littermate Nlrp3A350V/wt MRP8-Cre- (n=21) mice. Statistical significance was analysed by the log-rank Mantel-Cox test. ****, p ≤0.0001.