Figure 5A . Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3 , HA - Ubiquitin , TRIM50 or its truncation mutants . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . B . SDD - AGE analysis of NLRP3 oligomerization in NLRP3 together with TRIM50 or its coiled - coil domain - deleted mutants ( ΔC - C1 and ΔC - C2 ) transfected HEK293T cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . C . SDD - AGE analysis of the oligomerization of TRIM50 in TRIM50 or its coiled - coil domain - deleted mutants ( ΔC - C1 and ΔC - C2 ) transfected HEK293T cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . D . SDD - AGE analysis of the oligomerization of NLRP3 and TRIM50 in HEK293T cells overexpressing NLRP3 together with TRIM50 or its coiled - coil domain - deleted mutants . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results . E . Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 coiled - coil domain - deleted mutants , followed with the cycloheximide ( CHX ) treatment for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA ( E ) . Data are representative of three biological replicates with similar results . ( mean ± SD in E ) . F . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in HEK293T cells transfected with His - NLRP3 and TRIM50 or TRIM50 coiled - coil domain - deleted mutants . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

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Figure 5A. Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3, HA-Ubiquitin, TRIM50 or its truncation mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.B. SDD-AGE analysis of NLRP3 oligomerization in NLRP3 together with TRIM50 or its coiled-coil domain-deleted mutants (ΔC-C1 and ΔC-C2) transfected HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.C. SDD-AGE analysis of the oligomerization of TRIM50 in TRIM50 or its coiled-coil domain-deleted mutants (ΔC-C1 and ΔC-C2) transfected HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.D. SDD-AGE analysis of the oligomerization of NLRP3 and TRIM50 in HEK293T cells overexpressing NLRP3 together with TRIM50 or its coiled-coil domain-deleted mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.E. Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 coiled-coil domain-deleted mutants, followed with the cycloheximide (CHX) treatment for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA(E). Data are representative of three biological replicates with similar results. (mean ± SD in E).F. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate(soluble) in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50 coiled-coil domain-deleted mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


Figure 6A . Survival status of WT and Trim50 - / - mice after intraperitoneal injection with LPS ( n = 10 mice / group ) . Data information : P - values are shown , two - way ANOVA ( A ) Data are representative of three biological replicates with similar results . ( mean ± SDB . ELISA analysis of IL - 1β , TNF - α and IL - 6 in serum of WT or Trim50 - / - mice after i . p . injection with LPS for 6h ( n = 4 - 6 mice / group ) . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDC . Histopathological analysis of the lung tissues by hematoxylin and eosin ( H & E ) staining . The quantified lung injury was depicted by defined clinical parameters in ALI score ( n = 6 mice / group ) . Scale bars : 50μm . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDD . Flow cytometry analysis of the neutrophils ( left ) and Ly6C + monocytes ( right ) in peritoneum of WT or Trim50 - / - mice after i . p . injection with Alum . ( n = 6 mice / group ) . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDE . WT or Trim50 - / - mice were i . p . injected with FA for 36h , followed with immunoblot analysis of NLRP3 , pro - IL - 1β , Caspase1 p20 , IL - 1β in the kidney tissue samples . Data information : P - values are shown , two - way ANOVA ( A ) or two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ± SDF . H & E staining of kidney tissue sections . Scale bars : 20μm . G . Periodic - acid - Schiff ( PAS ) staining of kidney tissue sections . Scale bars : 20μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 6A. Survival status of WT and Trim50-/- mice after intraperitoneal injection with LPS (n=10 mice/group). Data information: P-values are shown, two-way ANOVA (A) Data are representative of three biological replicates with similar results. (mean ± SDB. ELISA analysis of IL-1β, TNF-α and IL-6 in serum of WT or Trim50-/- mice after i.p. injection with LPS for 6h (n=4-6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDC. Histopathological analysis of the lung tissues by hematoxylin and eosin (H&E) staining. The quantified lung injury was depicted by defined clinical parameters in ALI score (n=6 mice/group). Scale bars: 50μm. Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDD. Flow cytometry analysis of the neutrophils (left) and Ly6C+ monocytes (right) in peritoneum of WT or Trim50-/- mice after i.p. injection with Alum. (n=6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDE. WT or Trim50-/- mice were i.p. injected with FA for 36h, followed with immunoblot analysis of NLRP3, pro-IL-1β, Caspase1 p20, IL-1β in the kidney tissue samples. Data information: P-values are shown, two-way ANOVA (A) or two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SDF. H&E staining of kidney tissue sections. Scale bars: 20μm.G. Periodic-acid-Schiff (PAS) staining of kidney tissue sections. Scale bars: 20μm.


Figure 1 ( A ) Coordinates for injection in normal NHPs of the pilot group , study group 1 and study group 2 were calculated on the bases of pre - surgery MRI scans . ( C ) The brain was cut in 6 mm - thick slices using an adjustable brain matrix , obtaining 9 - 10 slices / brain . ( D ) Each brain slice was divided along the midline and each part subdivided in blocks . The cervical spinal cord was cut in 4 blocks .

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Figure 1(A) Coordinates for injection in normal NHPs of the pilot group, study group 1 and study group 2 were calculated on the bases of pre-surgery MRI scans.(C) The brain was cut in 6 mm-thick slices using an adjustable brain matrix, obtaining 9-10 slices/brain. (D) Each brain slice was divided along the midline and each part subdivided in blocks. The cervical spinal cord was cut in 4 blocks.


Figure 2 ( A ) Representative confocal images showing GFP + cells ( green ) expressing markers of neurons ( NeuN ) , astrocytes ( GFAP ) and oligodendrocytes ( CNPase ) close to the posterior injection site of NHP P2 . Arrows indicate co - localization of IF signals . ( B ) Representative confocal images showing neurons ( NeuN ) overexpressing ARSA ( green ) close to the posterior injection site of NHP S2 . 2 . Arrows indicate co - localization of IF signals . Note the granular perinuclear ARSA staining , likely representing protein localized in the Golgi / lysosomal vesicles . ( C ) Confocal z - stack images showing the presence of ARSA - positive cells ( green ) co - stained with oligodendrocyte markers CNPase and APC ( arrows ) . ( D ) Undetectable ARSA immunoreactivity in tissues of untreated animals . In all pictures nuclei are counterstained with ToPro ( blue ) . Scale bars : A , 80 µm ; B , D 60µm , C ,

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Figure 2(A) Representative confocal images showing GFP+ cells (green) expressing markers of neurons (NeuN), astrocytes (GFAP) and oligodendrocytes (CNPase) close to the posterior injection site of NHP P2. Arrows indicate co-localization of IF signals.(B) Representative confocal images showing neurons (NeuN) overexpressing ARSA (green) close to the posterior injection site of NHP S2.2. Arrows indicate co-localization of IF signals. Note the granular perinuclear ARSA staining, likely representing protein localized in the Golgi/lysosomal vesicles.(C) Confocal z-stack images showing the presence of ARSA-positive cells (green) co-stained with oligodendrocyte markers CNPase and APC (arrows). (D) Undetectable ARSA immunoreactivity in tissues of untreated animals. In all pictures nuclei are counterstained with ToPro (blue). Scale bars: A, 80 µm; B, D 60µm, C, 80µm


Figure 3Vector copy number ( VCN ) cartography shows integrated LV genome ( assessed by qPCR ) along the rostro - caudal axis ( slices 1 - 10 ) in LV . hARSA - injected S1 . 1 and S2 . 2 NHP in a side - by - side comparison with ARSA mRNA expression ( assessed by qPCR analyses using probe and primers annealing to sequences conserved in both the human and Macaca fascicularis and expressed as fold increase to region - matched blocks of the contralateral hemisphere ) . Grading of colors for VCN ranged from white ( VCN < 0 . 001 ; corresponding to CT > 37 ) to dark orange ( VCN = 1 - 3 ) . The highest VCN is found in close correspondence to the injection sites , as confirmed by comparison with post - surgery MR images ( yellow and orange circles indicate viral suspension close to the injection sites ) . Grading of colors for ARSA expression ranges from white ( < 1 . 5 fold the physiological level ) to dark purple ( 100 - fold the physiological level ) .

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Figure 3Vector copy number (VCN) cartography shows integrated LV genome (assessed by qPCR) along the rostro-caudal axis (slices 1-10) in LV.hARSA-injected S1.1 and S2.2 NHP in a side-by-side comparison with ARSA mRNA expression (assessed by qPCR analyses using probe and primers annealing to sequences conserved in both the human and Macaca fascicularis and expressed as fold increase to region-matched blocks of the contralateral hemisphere). Grading of colors for VCN ranged from white (VCN<0.001; corresponding to CT>37) to dark orange (VCN=1-3). The highest VCN is found in close correspondence to the injection sites, as confirmed by comparison with post-surgery MR images (yellow and orange circles indicate viral suspension close to the injection sites). Grading of colors for ARSA expression ranges from white (<1.5 fold the physiological level) to dark purple (100-fold the physiological level).


Figure 4 ( A ) ARSA overexpression detected by IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2 . 3 . ( B ) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV . GFP - injected NHP P1 . Scale bar , 100 µm . Insets show ARSA - expressing cells at higher magnification . Note the perinuclear accumulation of ARSA protein . ( C ) Quantification of immunopositive signal in brain slices 3 to 6 ( n = 8 blocks / slice ) . Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 ( physiological ) . Two - way ANOVA and Bonferroni post - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs physiological .

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Figure 4(A) ARSA overexpression detected by IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2.3.(B) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV.GFP-injected NHP P1. Scale bar, 100 µm. Insets show ARSA-expressing cells at higher magnification. Note the perinuclear accumulation of ARSA protein.(C) Quantification of immunopositive signal in brain slices 3 to 6 (n= 8 blocks/slice). Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 (physiological). Two-way ANOVA and Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 vs physiological.


Figure 5 ( A ) Schematic representation of ARSA activity in the brain of NHP S1 . 1 and S2 . 2 along the rostro - caudal axis ( slices 2 - 9 ) . Data are expressed as fold increase to ARSA activity detected in matched blocks of UT and LV . GFP - injected NHPs ( physiological levels ) . Grading of colors ranges from light yellow ( < 1 . 2 fold ) to dark green ( ≥ 2 fold ) . ( B - C ) Enzymatic activity measured in single brain slices of NHPs of study group 1 ( B ) and study group 2 ( C ) ( filled grey bars , injected hemisphere ; striped grey bars , contralateral hemisphere ) , and in UT and LV . GFP - injected NHPs ( white bars ; physiological levels ) . Arrows on x axis indicate the injection sites . n = 2 - 12 tissue blocks / slice . Number of tissue blocks analyzed per animal : 51 ( UT / LV . GFP ) , 98 - 122 ( S1 . 1 and S2 . 2 ) , 12 - 22 ( S1 . 2 , S1 . 3 , S2 . 2 , S2 . 3 ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison tests , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs matched slices of UT / LV . GFP . ( D ) Summary of ARSA activity in the injected and contralateral hemispheres of NHPs in study group 1 and study group 2 . Number of tissue blocks analysed : 51 ( UT / LV . GFP ) , 105 ( study group 1 , injected hemisphere ) , 52 ( study group 1 , contralateral hemisphere ) ; 103 ( study group 2 , injected hemisphere ) , 72 ( study group 2 , contralateral hemisphere ) . One - way ANOVA and Tukey ' s multiple comparison tests , * * * p < 0 . 001 versus UT / LV . GFP , § p < 0 . 05 versus study group 1 . ( E ) ARSA activity in the spinal cord of individual LV . GFP - ( n = 4 blocks / animal , 2 - 3 replicates ) and LV . hARSA - injected NHPs ( n = 2 - 4 blocks / animal ; n = 10 and n = 11 total blocks for study group 1 and study group 2 , respectively ) . Graph on the right represent the summary of ARSA activity in each treatment group . One - way ANOVA and Dunnet ' s multiple comparison test . * p < 0 . 05 versus pilot group . ( F ) ARSA activity in the sciatic nerve of LV . GFP - ( n = 2 ) and LV . hARSA - injected NHPs ( n = 3 ) . In B - F data are expressed as floating bars ( line at mean ) . ( G ) ARSA activity in the cerebrospinal fluid ( CSF ) of UT / LV . GFP ( n = 3 ) and LV . hARSA - injected NHP ( n = 5 ) . Data are expressed as mean ± SEM . Student t - test , * p = 0 . 035 .

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Figure 5(A) Schematic representation of ARSA activity in the brain of NHP S1.1 and S2.2 along the rostro-caudal axis (slices 2-9). Data are expressed as fold increase to ARSA activity detected in matched blocks of UT and LV.GFP-injected NHPs (physiological levels). Grading of colors ranges from light yellow (<1.2 fold) to dark green (≥ 2 fold).(B-C) Enzymatic activity measured in single brain slices of NHPs of study group 1 (B) and study group 2 (C) (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere), and in UT and LV.GFP-injected NHPs (white bars; physiological levels). Arrows on x axis indicate the injection sites. n=2-12 tissue blocks/slice. Number of tissue blocks analyzed per animal: 51 (UT/LV.GFP), 98-122 (S1.1 and S2.2), 12-22 (S1.2, S1.3, S2.2, S2.3). Two-way ANOVA followed by Bonferroni's multiple comparison tests, *p<0.05; **p<0.01; ***p<0.001 vs matched slices of UT/LV.GFP.(D) Summary of ARSA activity in the injected and contralateral hemispheres of NHPs in study group 1 and study group 2. Number of tissue blocks analysed: 51 (UT/LV.GFP), 105 (study group 1, injected hemisphere), 52 (study group 1, contralateral hemisphere); 103 (study group 2, injected hemisphere), 72 (study group 2, contralateral hemisphere). One-way ANOVA and Tukey's multiple comparison tests, *** p< 0.001 versus UT/LV.GFP, §p<0.05 versus study group 1.(E) ARSA activity in the spinal cord of individual LV.GFP- (n=4 blocks/animal, 2-3 replicates) and LV.hARSA-injected NHPs (n=2-4 blocks/animal; n=10 and n=11 total blocks for study group 1 and study group 2, respectively). Graph on the right represent the summary of ARSA activity in each treatment group. One-way ANOVA and Dunnet's multiple comparison test. * p<0.05 versus pilot group.(F) ARSA activity in the sciatic nerve of LV.GFP- (n=2) and LV.hARSA-injected NHPs (n=3). In B-F data are expressed as floating bars (line at mean).(G) ARSA activity in the cerebrospinal fluid (CSF) of UT/LV.GFP (n=3) and LV.hARSA-injected NHP (n=5). Data are expressed as mean±SEM. Student t-test, *p=0.035.


Figure 6 ( A ) Vector copy number ( VCN ) indicating the distribution of integrated LV genome along the rostro - caudal axis ( slices 7 - 19 ) in LV . hGALC - injected NHPs ( JT02 and JV02 ) assessed by qPCR . Each dot represent the VCN measured in one block within the slice . Lower threshold ( dotted line ) : VCN < 0 . 001 , corresponding to CT > 37 . ( B ) GALC mRNA expression along the rostro - caudal axis of JT02 ( slices 7 - 13 ) is expressed as fold increase to region - matched blocks of the contralateral hemisphere ( dotted line , y = 1 ) . Data are expressed as floating bars ( min to max , line at mean ; n = 4 - 8 blocks / slice ) . Arrows on x axis in A and B indicate the injection sites . ( C ) Confocal images showing GALC + cells expressing markers of neurons ( NeuN ) and astrocytes ( S100β ) close to the injection site ( slice 10 ) in JT02 . Arrows indicate co - localization of IF signals . ( D - E ) Confocal images showing GALC - expressing astrocytes ( S100β ; D ) and neurons ( NeuN ; E ) in the contralateral hemispheres , anterior ( slice 8 ) and posterior ( slice 12 ) to the injection site . Arrows indicate co - localization of IF signals . Scale bars : C , 70 µm ( upper panel ) and 40µm ( lower panel ) ; D and E , 40µm .

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Figure 6(A) Vector copy number (VCN) indicating the distribution of integrated LV genome along the rostro-caudal axis (slices 7-19) in LV.hGALC-injected NHPs (JT02 and JV02) assessed by qPCR. Each dot represent the VCN measured in one block within the slice. Lower threshold (dotted line): VCN<0.001, corresponding to CT>37.(B) GALC mRNA expression along the rostro-caudal axis of JT02 (slices 7-13) is expressed as fold increase to region-matched blocks of the contralateral hemisphere (dotted line, y=1). Data are expressed as floating bars (min to max, line at mean; n=4-8 blocks/slice). Arrows on x axis in A and B indicate the injection sites.(C) Confocal images showing GALC+ cells expressing markers of neurons (NeuN) and astrocytes (S100β) close to the injection site (slice 10) in JT02. Arrows indicate co-localization of IF signals.(D-E) Confocal images showing GALC-expressing astrocytes (S100β; D) and neurons (NeuN; E) in the contralateral hemispheres, anterior (slice 8) and posterior (slice 12) to the injection site. Arrows indicate co-localization of IF signals. Scale bars: C, 70 µm (upper panel) and 40µm (lower panel); D and E, 40µm.


Figure 7 ( A ) Specific GALC enzymatic activity in single brain slices of WT untreated ( UT ; white bars , physiological levels ) , WT LV . hGALC - treated ( JV02 ) and Krabbe - affected LV . hGALC - treated ( JV02 ) animals ( filled grey bars , injected hemisphere ; striped grey bars , contralateral hemisphere ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison tests ; * p < 0 . 05 , * * p < 0 . 01 vs matched slices of WT UT . ( B - C ) Summary of GALC activity in brain ( B ) samples from JT02 and JV02 , as well as from WT and Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , line at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / animal : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis test and Dunn ' s multiple comparison test ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT . ( B - C ) Summary of GALC activity in spinal cord ( C ; n = 3 blocks / animal ) samples from JT02 and JV02 , as well as from WT and Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , line at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / animal : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis test and Dunn ' s multiple comparison test ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT . ( D ) Chromatographic profile of GALC in selected brain slices and spinal cord ( SC ) tissue of untreated ( UT ) WT and LV . hGALC - treated Krabbe NHP ( JT02 ) . Extracts were run through a Sephadex S - 300 gel filtration column . Fractions ( 0 . 2 ml ) were assayed for enzyme activity using MUGAL substrate in the presence ( ) or absence of 11µM AgNO3 ( ● ) . ( E ) GALC activity in the CSF of JT02 ( pre - and post - treatment ) and JV02 ( post - treatment ) . Dots represent technical replicates . ( F ) Beginning at two months ( m ) of age , animals were evaluated monthly for motor performance using a modified Bayley ' s scale for infant development . For the study animals ( JV02 and JT02 ) , scores are reported as pre - surgery and post - surgery . Historical data for normal and affected animals are presented as means plus one standard deviation . Affected animals score significantly lower than normal animals at any age considered . JT02 ' s motor scores increase over time post LV . GALC injection , being close to the mean observed in normal juveniles at 5 months . Historical data are analyzed by two - way ANOVA and Bonferroni posttests , p < 0 . 05 at 6m , p < 0 . 001 at all other ages . n = 5 - 20 animals / group , except for normal animals at 6 months ( n = 1 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Specific GALC enzymatic activity in single brain slices of WT untreated (UT; white bars, physiological levels), WT LV.hGALC-treated (JV02) and Krabbe-affected LV.hGALC-treated (JV02) animals (filled grey bars, injected hemisphere; striped grey bars, contralateral hemisphere). Two-way ANOVA followed by Bonferroni's multiple comparison tests; *p<0.05, **p<0.01 vs matched slices of WT UT.(B-C) Summary of GALC activity in brain (B) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn's multiple comparison test; *p<0.05, ***p<0.001 vs WT UT.(B-C) Summary of GALC activity in spinal cord (C; n=3 blocks/animal) samples from JT02 and JV02, as well as from WT and Krabbe UT controls. Data in A-C are represented as floating bars (min to max, line at mean; n=2-6 blocks/slice). Number of blocks analysed/animal: 16 (WT UT), 24 (JT02), 22 (JV02). GALC activity in Krabbe UT samples is < 0.001 nmol/h*mg; n=3 blocks. Kruskal-Wallis test and Dunn's multiple comparison test; *p<0.05, ***p<0.001 vs WT UT.(D) Chromatographic profile of GALC in selected brain slices and spinal cord (SC) tissue of untreated (UT) WT and LV.hGALC-treated Krabbe NHP (JT02). Extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 ml) were assayed for enzyme activity using MUGAL substrate in the presence () or absence of 11µM AgNO3 (●).(E) GALC activity in the CSF of JT02 (pre- and post-treatment) and JV02 (post-treatment). Dots represent technical replicates.(F) Beginning at two months (m) of age, animals were evaluated monthly for motor performance using a modified Bayley's scale for infant development. For the study animals (JV02 and JT02), scores are reported as pre-surgery and post-surgery. Historical data for normal and affected animals are presented as means plus one standard deviation. Affected animals score significantly lower than normal animals at any age considered. JT02's motor scores increase over time post LV.GALC injection, being close to the mean observed in normal juveniles at 5 months. Historical data are analyzed by two-way ANOVA and Bonferroni posttests, p<0.05 at 6m, p<0.001 at all other ages. n=5-20 animals/group, except for normal animals at 6 months (n=1).


Figure 1C E139R , E143R , and the combination ER / ER mutant substitutions in UBE2S increase the amount of automodification , monitored by the presence of * Ub ( * denotes fluorescently labeled protein ) and using SDS - PAGE and fluorescent scanning . D ER / ER substitutions increase Ub chain formation in multiple UBE2S constructs ( WT , UBC only , and UBC - UBD fusion ) , monitored by Coomassie - staining of non - reduced SDS - PAGE gels .

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Figure 1C E139R, E143R, and the combination ER/ER mutant substitutions in UBE2S increase the amount of automodification, monitored by the presence of *Ub (* denotes fluorescently labeled protein) and using SDS-PAGE and fluorescent scanning.D ER/ER substitutions increase Ub chain formation in multiple UBE2S constructs (WT, UBC only, and UBC-UBD fusion), monitored by Coomassie-staining of non-reduced SDS-PAGE gels.


Figure 2E , F , G , H Pulse - chase diUb synthesis assays using different E2s , UBE2S ( E ) , UBE2C ( F ) , UBE2D2 ( G ) , or UBE2R2 ( H ) . DiUb formation was monitored by fluorescent scanning ( top ) , quantitated ( bottom ) , and assessed for statistical significance by either an unpaired Welch ' s t - test ( UBE2C and UBE2R2 ) or one - way ANOVA ( UBE2S and UBE2D2 ) ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates for all E2s ) . Error bars : SEM . Data normalized to the maximal WT diUb formation . # denotes a contaminant band .

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Figure 2E, F, G, H Pulse-chase diUb synthesis assays using different E2s, UBE2S (E), UBE2C (F), UBE2D2 (G), or UBE2R2 (H). DiUb formation was monitored by fluorescent scanning (top), quantitated (bottom), and assessed for statistical significance by either an unpaired Welch's t-test (UBE2C and UBE2R2) or one-way ANOVA (UBE2S and UBE2D2) (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates for all E2s). Error bars: SEM. Data normalized to the maximal WT diUb formation. # denotes a contaminant band.


Figure 3C The E139R substitution decreases the stability of UBE2S ~ Ub compared to WT , revealed by fluorescent scanning of a lysine discharge assay ( top ) and its quantification ( bottom ) . Statistical comparison was performed using one - way ANOVA ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , n = 4 independent , technical replicates ) . Error bars : SEM . Data normalized to the initial timepoint of each respective E2 ~ Ub band . D UBE2R2 R149E has a similar E2 ~ Ub stability as WT , monitored by fluorescent scanning ( top ) of the lysine discharge assay and its quantification through an unpaired Welch ' s t - test ( bottom ) ( n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the initial timepoint of each respective E2 ~ Ub band . E Fluorescent scans ( top ) and quantification ( bottom ) of a * LRLRGG peptide transfer assay comparing UBE2S WT and ER / ER reveals no significant difference through an unpaired Welch ' s t - test ( n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the maximal * LRLRGG - Ub formation by WT UBE2S . F UBE2R2 R149E is still defective in the transfer of the * LRLRGG peptide to an acceptor Ub compared to WT , probed by fluorescent scanning ( top ) and its quantitation ( bottom ) . Statistical comparison was performed using an unpaired Welch ' s t - test ( * * p ≤ 0 . 01 , n = 5 independent , technical replicates ) . Error bars : SEM . Data normalized to the maximal * LRLRGG - Ub formation by WT UBE2R2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C The E139R substitution decreases the stability of UBE2S~Ub compared to WT, revealed by fluorescent scanning of a lysine discharge assay (top) and its quantification (bottom). Statistical comparison was performed using one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, n=4 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.D UBE2R2 R149E has a similar E2~Ub stability as WT, monitored by fluorescent scanning (top) of the lysine discharge assay and its quantification through an unpaired Welch's t-test (bottom) (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.E Fluorescent scans (top) and quantification (bottom) of a *LRLRGG peptide transfer assay comparing UBE2S WT and ER/ER reveals no significant difference through an unpaired Welch's t-test (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2S.F UBE2R2 R149E is still defective in the transfer of the *LRLRGG peptide to an acceptor Ub compared to WT, probed by fluorescent scanning (top) and its quantitation (bottom). Statistical comparison was performed using an unpaired Welch's t-test (** p ≤ 0.01, n=5 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2R2.


Figure 4Role of the UBE2RHTH in diUb formation is determined from kinetic parameters ( G - H ) for mutants upon titrating the acceptor Ub in pulse - chase reactions following UBE2R2 ~ * Ub Apparent Km and Vmax were determined using the Michaelis - Menten equation ( n ≥ 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the WT apparent Vmax . Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse - chase reactions following UBE2R2 ~ * Ub monitored by fluorescent scanning of nonreducing SDS - PAGE

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Role of the UBE2RHTH in diUb formation is determined from kinetic parameters (G-H) for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub Apparent Km and Vmax were determined using the Michaelis-Menten equation (n≥3 independent, technical replicates). Error bars: SEM. Data normalized to the WT apparent Vmax.Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub monitored by fluorescent scanning of nonreducing SDS-PAGE gels


Figure 5B Reducing the positive charge of * Ub reduces the autoubiquitination of the E139R - substituted UBE2S variant , monitored by fluorescent scanning of an SDS - PAGE gel . D Plot of 15N - labeled UBE2SUBC WT and ER / ER 1H linewidth changes in the presence of Ub revealing the ER / ER variant has a more dynamic active - site cysteine and HTH , but a more stabilized crossover helix , indicative of the " closed " state . Shown are the best - fit values and standard deviations ( SD ) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension . F C118A substitutions decrease UBE2S autoubiquitination , measured by fluorescent scanning of SDS - PAGE gels , indicating the significance of the " closed " formation of the UBE2S ~ Ub for activity .

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Figure 5B Reducing the positive charge of *Ub reduces the autoubiquitination of the E139R-substituted UBE2S variant, monitored by fluorescent scanning of an SDS-PAGE gel.D Plot of 15N-labeled UBE2SUBC WT and ER/ER 1H linewidth changes in the presence of Ub revealing the ER/ER variant has a more dynamic active-site cysteine and HTH, but a more stabilized crossover helix, indicative of the "closed" state. Shown are the best-fit values and standard deviations (SD) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension.F C118A substitutions decrease UBE2S autoubiquitination, measured by fluorescent scanning of SDS-PAGE gels, indicating the significance of the "closed" formation of the UBE2S~Ub for activity.


Figure 6C Securin ubiquitination by APC / C and either UBE2C WT or K157E , resolved by SDS - PAGE and fluorescent scanning . Quantification of 4 or more ubiquitins added on the substrate ( right ) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch ' s t - test ( * * * * p ≤ 0 . 0001 , n = 5 independent , technical replicates ) . Error bars : SEM . D Similar to C , Ub - securin ubiquitination by APC / C and either UBE2D2 WT or charge - swap variants . Quantification of 4 or more ubiquitins ( right ) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one - way ANOVA ( * * * * p ≤ 0 . 0001 , n = 4 independent , technical replicates ) . Error bars : SEM . F The recombinant UBE2S ER / ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild - type ( WT ) , monitored by immunoblot . G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA ( siFF ) or two UBE2S siRNAs ( siUBE2S ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS-PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch's t-test (**** p ≤ 0.0001, n=5 independent, technical replicates). Error bars: SEM. D Similar to C, Ub-securin ubiquitination by APC/C and either UBE2D2 WT or charge-swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one-way ANOVA (**** p ≤ 0.0001, n=4 independent, technical replicates). Error bars: SEM.F The recombinant UBE2S ER/ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild-type (WT), monitored by immunoblot. G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA (siFF) or two UBE2S siRNAs (siUBE2S).


Figure 7B Representative fluorescent scan of an SDS - PAGE gel ( left ) and its quantification ( right ) where the charged - swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC / Cs harboring APC2 substitutions . Statistical significance was assessed by two - way ANOVA ( * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to the negative control substrate band from reactions lacking an E2 . C APC / C - UBE2S - dependent diUb synthesis was monitored by fluorescent scanning ( left ) , quantitated ( right ) , and assessed for statistical significance by two - way ANOVA ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) , demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2 . Error bars : SEM . Data normalized to WT APC / C - UBE2S diUb reactions . D APC / C Platform - dependent activation of UBE2S - mediated diUb synthesis was monitored by fluorescent scanning ( left ) , quantitated ( right ) , and assessed for statistical significance by two - way ANOVA ( * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to WT APC / C Platform - UBE2S diUb reactions . E Similar to B , the role of the " closed " conformation in UBE2S - dependent Ub chain elongation of the APC / C substrate Ub - CycB * , monitored by the incorporation of C118A - substituted UBE2S variants ( top ) . Quantification of ubiquitinated Ub - CycB * was assessed ( bottom ) and statistical significance by one - way ANOVA between C118A and WT , C118A / E139R , and C118A / E143R ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) . Error bars : SEM . Data normalized to WT UBE2S 4 + Ub additions .

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Figure 7B Representative fluorescent scan of an SDS-PAGE gel (left) and its quantification (right) where the charged-swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC/Cs harboring APC2 substitutions. Statistical significance was assessed by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to the negative control substrate band from reactions lacking an E2.C APC/C-UBE2S-dependent diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates), demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2. Error bars: SEM. Data normalized to WT APC/C-UBE2S diUb reactions. D APC/C Platform-dependent activation of UBE2S-mediated diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT APC/C Platform-UBE2S diUb reactions.E Similar to B, the role of the "closed" conformation in UBE2S-dependent Ub chain elongation of the APC/C substrate Ub-CycB*, monitored by the incorporation of C118A-substituted UBE2S variants (top). Quantification of ubiquitinated Ub-CycB* was assessed (bottom) and statistical significance by one-way ANOVA between C118A and WT, C118A/E139R, and C118A/E143R (** p ≤ 0.01, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT UBE2S 4+ Ub additions.


Figure 1A The mRNA expression of FAM189A2 / ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines . In qRT - PCR analyses , the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16 . Relative expression ratios shown as mean + SD from three biological replicates . B Relative expression of FAM189A2 / ENTREP in the normal ( white ) and cancer ( green ) tissues of breast ( Curtis Breast ) , lung ( Okayama Lung ) , colorectal ( TCGA Colorectal ) and head and neck ( Estilo Head - Neck ) . Data were downloaded from the Oncomine database P - values obtained by Student ' s t - tests and the number of cases are listed on the top and bottom of figures , respectively . P < 0 . 05 was considered as statistically significant . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers . Outlier data are plotted as dots . C Relative expression of FAM189A2 / ENTREP in the primary ( green ) and the metastatic sites ( blue ) including lymph node , bone , liver , lung , soft tissues of prostatic cancer ( Grasso Prostate ) ( left ) and in the normal ( white ) , primary ( green ) and metastatic sites ( three cases of sentinel lymph node ) ( blue ) of breast cancer ( TCGA Breast ) ( right ) . Note that only three cases of the metastatic breast cancer were available in the dataset . Data were downloaded from the Oncomine database P - values obtained by Student ' s t - tests and the number of cases are listed on the top and bottom of figures , respectively . P < 0 . 05 was considered as statistically significant . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers . Outlier data are plotted as dots . D - F Relapse - free survival ( RFS ) of breast cancer patients . Data were downloaded from the Kaplan - Meier

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Figure 1A The mRNA expression of FAM189A2/ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines. In qRT-PCR analyses, the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16. Relative expression ratios shown as mean + SD from three biological replicates.B Relative expression of FAM189A2/ENTREP in the normal (white) and cancer (green) tissues of breast (Curtis Breast), lung (Okayama Lung), colorectal (TCGA Colorectal) and head and neck (Estilo Head-Neck). Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots. C Relative expression of FAM189A2/ENTREP in the primary (green) and the metastatic sites (blue) including lymph node, bone, liver, lung, soft tissues of prostatic cancer (Grasso Prostate) (left) and in the normal (white), primary (green) and metastatic sites (three cases of sentinel lymph node) (blue) of breast cancer (TCGA Breast) (right). Note that only three cases of the metastatic breast cancer were available in the dataset. Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots.D-F Relapse-free survival (RFS) of breast cancer patients. Data were downloaded from the Kaplan-Meier plotter


Figure 2B , C The immunoprecipitation analysis . Co - precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP . Data shown are representative of at least two independent experiments . IP , immunoprecipitation ; IB , immunoblot . E Representative images of the proximity ligation assay ( PLA ) using Cos7 cells transfected with indicated vectors . PLA signals , granular dots of green color , were presented with DAPI images . Arrowheads , the plasma membrane . Scale bar , 10mm . F The box - and - whisker plot presentation of the PLA . A minimum of 10 cells per condition were counted from two independent experiments . P - values obtained by Student ' s t - tests and P < 0 . 05 was considered as statistically significant . * P < 0 . 05 . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers .

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Figure 2B, C The immunoprecipitation analysis. Co-precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP. Data shown are representative of at least two independent experiments. IP, immunoprecipitation; IB, immunoblot.E Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Arrowheads, the plasma membrane. Scale bar, 10mm. F The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.


Figure 3A The nickel - pull down assay using myc / HIS - tagged ENTREP along with HA - tagged ubiquitin . Arrows indicate HA - ubiquitin - incorporating ENTREP . Arrowhead indicates ENTREP without ubiquitination . Data shown are representative of three independent experiments .

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Figure 3A The nickel-pull down assay using myc/HIS-tagged ENTREP along with HA-tagged ubiquitin. Arrows indicate HA-ubiquitin-incorporating ENTREP. Arrowhead indicates ENTREP without ubiquitination. Data shown are representative of three independent experiments.


Figure 4A The immunoprecipitation analysis . The cytoplasmic ENTREP ( wild or mut1 + 2 mutant ) co - precipitated with EPN1myc in the presence of ITCH wild or C830A . The blot bands were semi - quantified using ImageJ software . ENTREP ratio was caluculated as a ratio of ENTREP in the myc - coprecipitaed sample to that of the total lysate . The ratios shown as mean ± SD from three biological replicates . P - values were obtained by Student ' s t - tests and P < 0 . 005 was considered as statistically significant . No significant difference was observed . B Representative images of the proximity ligation assay ( PLA ) using Cos7 cells transfected with indicated vectors . PLA signals , granular dots of green color , were presented with DAPI images . Scale bar , 10mm . C The box - and - whisker plot presentation of the PLA . A minimum of 10 cells per condition were counted from two independent experiments . P - values obtained by Student ' s t - tests and P < 0 . 05 was considered as statistically significant . * P < 0 . 05 . Box - whisker plot represents the interquartile range ( 25th and 75th percentiles ) as a box and the median as a line . The maximn and minimun values within 1 . 5 x interquartile range are shown as whiskers .

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Figure 4A The immunoprecipitation analysis. The cytoplasmic ENTREP (wild or mut1+2 mutant) co-precipitated with EPN1myc in the presence of ITCH wild or C830A. The blot bands were semi-quantified using ImageJ software. ENTREP ratio was caluculated as a ratio of ENTREP in the myc-coprecipitaed sample to that of the total lysate. The ratios shown as mean ± SD from three biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed.B Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Scale bar, 10mm. C The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.


Figure 5B The immunoprecipitation analysis . Cytoplasmic ENTREP co - precipitated with either ITCH C830A , delWW12 or delWW34 but not with delWW14 . Data shown are representative of at least two independent experiments . C The immunoprecipitation analysis . HA - CXCR4SS - DsRed hardly co - precipitated with either ITCHC830A or delWW mutants . HA - CXCR4DD - DsRed co - precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34 . Data shown are representative of at least two independent experiments . F The nickel - pull down assay using indicated vectors along with HISx6 - tagged wild type ubiquitin vector . HA - tagged DsRed was used as a control for HA - CXCR4DD - DsRed . Arrowhead , FLAG - tagged ITCH ( wild or C830A ) ; arrow , FLAG - ENTREP wild cyt . Data shown are representative of at least two independent experiments . G Co - localization of ENTREP and CXCR4 in the endosome . Cos7 cells were transfected with ENTREP - FLAG , CXCR4 - DsRed and Halo - ITCH wild vectors and treated with CXCL12 . ENTREP - FLAG and CXCR4 - DsRed co - localized in the RAB7 - positive endosome . Inserts , the high - magnification images of the marked area indicating the colocalizaion of ENTREP - FLAG , CXCR4 - DsRed and RAB7 . Scale bar 10mm . The image is representative of at least two independent experiments .

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Figure 5B The immunoprecipitation analysis. Cytoplasmic ENTREP co-precipitated with either ITCH C830A, delWW12 or delWW34 but not with delWW14. Data shown are representative of at least two independent experiments. C The immunoprecipitation analysis. HA-CXCR4SS-DsRed hardly co-precipitated with either ITCHC830A or delWW mutants. HA-CXCR4DD-DsRed co-precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34. Data shown are representative of at least two independent experiments.F The nickel-pull down assay using indicated vectors along with HISx6-tagged wild type ubiquitin vector. HA-tagged DsRed was used as a control for HA-CXCR4DD-DsRed. Arrowhead, FLAG-tagged ITCH (wild or C830A); arrow, FLAG-ENTREP wild cyt. Data shown are representative of at least two independent experiments. G Co-localization of ENTREP and CXCR4 in the endosome. Cos7 cells were transfected with ENTREP-FLAG, CXCR4-DsRed and Halo-ITCH wild vectors and treated with CXCL12. ENTREP-FLAG and CXCR4-DsRed co-localized in the RAB7-positive endosome. Inserts, the high-magnification images of the marked area indicating the colocalizaion of ENTREP-FLAG, CXCR4-DsRed and RAB7. Scale bar 10mm. The image is representative of at least two independent experiments.


Figure 6C The expression of endogenous CXCR4 in lentivirally - transduced MCF - 7 ENTREP - KO cells . CXCL12 ( + ) and ( - ) indicate the presence and absence of CXCL12 treatment . In CXCL12 - treated ENTREP - EGFP expressing cells , a non - negligible amount of CXCR4 was observed in the cytoplasm , in which CXCR4 was overlapped with ENTREP - EGFP . CXCR4 expression on the cell surface still remained ( arrows ) . Scale bar 10 mm . The images are representative of at least three independet experiments .

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Figure 6C The expression of endogenous CXCR4 in lentivirally-transduced MCF-7 ENTREP-KO cells. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 treatment. In CXCL12-treated ENTREP-EGFP expressing cells, a non-negligible amount of CXCR4 was observed in the cytoplasm, in which CXCR4 was overlapped with ENTREP-EGFP. CXCR4 expression on the cell surface still remained (arrows). Scale bar 10 mm. The images are representative of at least three independet experiments.


Figure 7A Dot plot presentation of the chemotaxis analyses using the Boyden chamber . CXCL12 ( + ) and ( - ) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers . ENTREP - knockout enhanced the chemotaxis toward CXCL12 , which was blocked by pre - treatment with AMD3100 ( upper panel ) . The doxycyclin ( DOX ) - induced ENTREP expression suppressed the chemotaxis of mouse 4T1 - Luc cells toward CXCL12 ( lower panel ) . Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non - treated cells ( n = 7 , upper panel , and n = 9 , lower panel , from at least two independent experiments , respectively ) . P - values were obtained by Student ' s t - tests and P < 0 . 005 was considered as statistically significant . Insert , the immunoblot analysis of the DOX - induced ENTREP expression in 4T1 - Luc cells . NS , not significant . B Dot plot presentation of mammosphere assay ( left panel ) and the images of mammosphere ( right panel ) . Each mark in dot plot presentation indicates the number of mammosphere from 3 , 000 cells per well in an ultra - low attachment plate from two independent experiments . To estimate the effect of CXCL12 / CXCR4 pathway , 100 ng / ml of CXCL12 and 12 μM of AMD3100 were applied as indicated . For statistical analysis , variance was assessed using two - way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison . NS , not significant . Scale bar in the images of mammosphere 100 mm .

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Figure 7A Dot plot presentation of the chemotaxis analyses using the Boyden chamber. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers. ENTREP-knockout enhanced the chemotaxis toward CXCL12, which was blocked by pre-treatment with AMD3100 (upper panel). The doxycyclin (DOX)-induced ENTREP expression suppressed the chemotaxis of mouse 4T1-Luc cells toward CXCL12 (lower panel). Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non-treated cells (n=7, upper panel, and n=9, lower panel, from at least two independent experiments, respectively). P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. Insert, the immunoblot analysis of the DOX-induced ENTREP expression in 4T1-Luc cells. NS, not significant.B Dot plot presentation of mammosphere assay (left panel) and the images of mammosphere (right panel). Each mark in dot plot presentation indicates the number of mammosphere from 3,000 cells per well in an ultra-low attachment plate from two independent experiments. To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of CXCL12 and 12 μM of AMD3100 were applied as indicated. For statistical analysis, variance was assessed using two-way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison. NS, not significant. Scale bar in the images of mammosphere 100 mm.


Figure 1A Transcriptome re - analysis of ulp2Δ strains compared with WT ( MHY1379 ) from previous RNA - seq experiments Two - dimensional agglomerative hierarchical clustering shows 2 , 903 significantly up - or down - regulated genes ( P < 0 . 05 ) in triplicate RNA samples . Red and blue indicate up and down regulation of genes , respectively , and their intensity represents the relative gene expression changes . B Summery of transcriptome data in ( A ) . White and gray colors indicate up - and down - regulated genes , respectively , in ulp2Δ cells . The pie graph shows the percentages of significantly changed genes , except for 130 ribosomal protein genes ( RPs ) , and are classified by FPKM ( Fragments Per Kilobase of Million reads mapped ) values in the bar graph . C qRT - PCR analysis of the highly transcribed PMA1 , ADH1 and PYK1 genes in ulp2Δ cells . Expression was measured relative to WT cells and data were normalized to SPT15 expression . Error bars indicate the standard deviations ( SDs ) from three independent RNA preparations . FPKMs of each gene in WT are shown in the bottom graph . E ChIP analysis using IgG - Sepharose or anti - Flag - agarose beads in strains expressing TAP - tagged Paf1 , Ubc9 , or Ulp1 or Flag - tagged Ulp2 . An untagged strain ( MHY500 ) was used as a negative control for immunoprecipitation of Ulp2 - Flag The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA . The primer pairs used are indicated in ( D ) . Quantification presented as fold over background ; a value of 1 indicates no signal detected above background signal at a nontranscribed locus , as marked with the horizontal line . Error bars indicate SDs calculated from three independent chromatin preparations . Data information : Asterisks indicate statistically significant differences of Paf1 - TAP and Ubc9 - TAP with Ulp1 - TAP and Ulp2 - Flag with No tag in ( E ) using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) . F Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag - tagged Ulp2 using anti - Rpb3 antibody and anti - Flag agarose beads Mock ' indicates use of Protein G beads without added antibody . For CUP1 induction , cells were harvested at the indicated time points after adding CuSO4 . " Pro " and " ORF " represent the positions of PCR fragments 1 and 2 from the CUP1 gene Black bar indicates a value of 1 , the background signal . Error bars , SD from four independent experiments . Data information : Asterisks indicate significant differences between uninduced and induced cells in ( F ) using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) G A percentage graph of association of Ulp2 - Flag with genes classified by FPKM RPs and ND indicate ribosomal protein genes and genes not detected genes in our earlier RNA - seq experiments , respectively . The numbers above the graph indicate the total number of yeast genes in each expression category . H Average plot of Ulp2 - Flag occupancy at transcribed genes . The values of the y - axis indicate rescaled fold - enrichment , setting the maximum occupancy to 1 and the minimum occupancy to 0 , and the x - axis indicates normalized distance from transcription start sites ( TSS ) and transcription end sites ( TES ) . The dotted lines indicate the TSS and TES . The ChIP - seq data were obtained from duplicate experiments .

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Figure 1A Transcriptome re-analysis of ulp2Δ strains compared with WT (MHY1379) from previous RNA-seq experiments Two-dimensional agglomerative hierarchical clustering shows 2,903 significantly up- or down-regulated genes (P < 0.05) in triplicate RNA samples. Red and blue indicate up and down regulation of genes, respectively, and their intensity represents the relative gene expression changes. B Summery of transcriptome data in (A). White and gray colors indicate up- and down-regulated genes, respectively, in ulp2Δ cells. The pie graph shows the percentages of significantly changed genes, except for 130 ribosomal protein genes (RPs), and are classified by FPKM (Fragments Per Kilobase of Million reads mapped) values in the bar graph. C qRT-PCR analysis of the highly transcribed PMA1, ADH1 and PYK1 genes in ulp2Δ cells. Expression was measured relative to WT cells and data were normalized to SPT15 expression. Error bars indicate the standard deviations (SDs) from three independent RNA preparations. FPKMs of each gene in WT are shown in the bottom graph.E ChIP analysis using IgG-Sepharose or anti-Flag-agarose beads in strains expressing TAP-tagged Paf1, Ubc9, or Ulp1 or Flag-tagged Ulp2. An untagged strain (MHY500) was used as a negative control for immunoprecipitation of Ulp2-Flag The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA. The primer pairs used are indicated in (D). Quantification presented as fold over background; a value of 1 indicates no signal detected above background signal at a nontranscribed locus, as marked with the horizontal line. Error bars indicate SDs calculated from three independent chromatin preparations. Data information: Asterisks indicate statistically significant differences of Paf1-TAP and Ubc9-TAP with Ulp1-TAP and Ulp2-Flag with No tag in (E) using a two-tailed Student's t test (*, P <0.05; **, P <0.01).F Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag-tagged Ulp2 using anti-Rpb3 antibody and anti-Flag agarose beads Mock' indicates use of Protein G beads without added antibody. For CUP1 induction, cells were harvested at the indicated time points after adding CuSO4. "Pro" and "ORF" represent the positions of PCR fragments 1 and 2 from the CUP1 gene Black bar indicates a value of 1, the background signal. Error bars, SD from four independent experiments. Data information: Asterisks indicate significant differences between uninduced and induced cells in (F) using a two-tailed Student's t test (*, P <0.05; **, P <0.01)G A percentage graph of association of Ulp2-Flag with genes classified by FPKM RPs and ND indicate ribosomal protein genes and genes not detected genes in our earlier RNA-seq experiments, respectively. The numbers above the graph indicate the total number of yeast genes in each expression category.H Average plot of Ulp2-Flag occupancy at transcribed genes. The values of the y-axis indicate rescaled fold-enrichment, setting the maximum occupancy to 1 and the minimum occupancy to 0, and the x-axis indicates normalized distance from transcription start sites (TSS) and transcription end sites (TES). The dotted lines indicate the TSS and TES. The ChIP-seq data were obtained from duplicate experiments.


Figure 2A Sensitivity of the indicated mutants to 6 - AU . All strains carried a URA3 plasmid , pRS316 , and were spotted on SD - Ura with or without 6 - AU ( 100 μg / ml ) ; plates were incubated for 2 - 4 days at 30 ° C . ULP2 and ulp2 ( C624A ) represent ulp2Δ : : HIS3 cells containing either pRS314 - ULP2 - FLAG or pRS314 - ulp2 ( C624A ) - FLAG . ChIP analyses using anti - Rpb3 ( B ) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) . ChIP analyses using anti - SUMO ( C ) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) . D qRT - PCR analysis of CUP1 mRNA levels in ulp2Δ cells . Data were normalized to ACT1 mRNA levels . CUP1 gene induction was performed Error bars represent the SD from three RNA samples . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) . ChIP assays using anti - Rpb3 antibodies in ulp2Δ cells Pro " denotes the # 1 PCR product of PMA1 , ADH1 and PYK1 genes , while " ORF " indicates the # 3 products of PMA1 and # 2 of ADH1 and PYK1 ; these are used in all ensuing figures except where specified . Error bars indicate the SD from four ( E ) independent assays . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01ChIP assays using anti - SUMO ( F ) antibodies in ulp2Δ cells ; Pro " denotes the # 1 PCR product of PMA1 , ADH1 and PYK1 genes while " ORF " indicates the # 3 products of PMA1 and # 2 of ADH1 and PYK1 ; these are used in all ensuing figures except where specified . Error bars indicate the SD from three ( F ) independent assays . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two - tailed Student ' s t test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG.ChIP analyses using anti-Rpb3 (B) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).ChIP analyses using anti-SUMO (C) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).D qRT-PCR analysis of CUP1 mRNA levels in ulp2Δ cells. Data were normalized to ACT1 mRNA levels. CUP1 gene induction was performed Error bars represent the SD from three RNA samples. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).ChIP assays using anti-Rpb3 antibodies in ulp2Δ cells Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes , while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from four (E) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01ChIP assays using anti-SUMO (F) antibodies in ulp2Δ cells ;Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from three (F) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


Figure 3A Sensitivity to 6 - AU of the indicated cells expressing chromosomally integrated ULP2 - Myc derivatives . Assays were performed The schematic diagram below shows full - length Ulp2 with a 9Myc epitope tag and the segments defining the N - and C - terminal domains and the catalytic ULP domain ( UD ) . B Immunoblot assay of the Ulp2 - Myc derivatives expressed in the strainsC ChIP analysis of Flag - Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424 - GAL1 plasmids that expressed N - terminally Flag - tagged Ulp2 derivatives - specifically , full - length , NTD ( 1 - 403 ) and CTD ( 667 - 1034 ) - were grown to mid - log phase in SD - Trp medium containing 2 % galactose . Error bars indicate the SD from three independent experiments . Data information : Asterisks indicate statistically significant differences compared with WT , full length of Ulp2 ( * , P < 0 . 05 ) . D Co - IP assay for interaction between Ulp2 - Myc derivatives and Flag - H2B in strains transformed with a pRS314 plasmid expressing Flag - tagged H2B . Immunoprecipitated ( IP ) proteins from anti - Myc agarose beads were analyzed by immunoblotting ( IB ) using anti - Myc or anti - Flag antibodies . The upper and middle panels show anti - Myc blotting and anti - Flag blotting , respectively . The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel . E Chromatin association assays using strains After fractionation into chromatin ( pellet ) and soluble ( supernatant ) components , fractions were analyzed by immunoblotting using the indicated antibodies . F Quantitation of chromatin association of Ulp2 - Myc derivatives in ( E ) . The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD).B Immunoblot assay of the Ulp2-Myc derivatives expressed in the strainsC ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05).D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel.E Chromatin association assays using strains After fractionation into chromatin (pellet) and soluble (supernatant) components, fractions were analyzed by immunoblotting using the indicated antibodies.F Quantitation of chromatin association of Ulp2-Myc derivatives in (E). The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative.


Figure 4A Immunoblot analysis of immunoprecipitated Flag - tagged histone H2B with anti - Flag ( bottom panel ) or anti - SUMO ( upper panel ) antibodies in WT , ulp2Δ , SUMO - all KR or ulp2Δ SUMO - all KR strains carrying empty vector or pRS426 - FLAG - HTB1 . One , two or three asterisks represent mono , di and polysumoylated histones , respectively . Note that the apparent diSUMO - H2B species ( * * ) in ulp2Δ SUMO - all KR cells migrates more slowly than H2B linked to a SUMO dimer , suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines ( lane 8 ; also see weaker band in lane 6 ) . B Yeast strains of the indicated genotypes and carrying a YCplac33 - ULP2 cover plasmid were streaked on SD + 5 - FOA to evict YCplac33 - ULP2 ; cells were grown at 30 ° C for 3 days . C Mutant htb1 - K123R ulp2Δ and htb1 - K123R ULP2 strains with a pRS316 - ULP2 cover plasmid were grown on the indicated plates at 30 ° C for 2 - 3 days . Three double mutant isolates were tested in parallel . D Mutant ulp2Δ bre1Δ strains carrying a pRS426 - ULP2 ( URA3 ) cover plasmid and the indicated plasmids were grown on SD - Ura and SD + 5 - FOA at 30 ° C for 2 - 3 days . E Yeast strains of the indicated genotypes and carrying a YCplac33 - ULP2 cover plasmid were streaked on SD + 5 - FOA to evict YCplac33 - ULP2 ; cells were grown at 30 ° C for 3 days .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B with anti-Flag (bottom panel) or anti-SUMO (upper panel) antibodies in WT, ulp2Δ, SUMO-all KR or ulp2Δ SUMO-all KR strains carrying empty vector or pRS426-FLAG-HTB1. One, two or three asterisks represent mono, di and polysumoylated histones, respectively. Note that the apparent diSUMO-H2B species (**) in ulp2Δ SUMO-all KR cells migrates more slowly than H2B linked to a SUMO dimer, suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines (lane 8; also see weaker band in lane 6).B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.C Mutant htb1-K123R ulp2Δ and htb1-K123R ULP2 strains with a pRS316-ULP2 cover plasmid were grown on the indicated plates at 30°C for 2-3 days. Three double mutant isolates were tested in parallel.D Mutant ulp2Δ bre1Δ strains carrying a pRS426-ULP2 (URA3) cover plasmid and the indicated plasmids were grown on SD-Ura and SD+5-FOA at 30°C for 2-3 days.E Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.


Figure 5A Immunoblot analysis of immunoprecipitated Flag - tagged histone H2B using anti - Flag or anti - SUMO antibodies in the indicated mutants , The upper and lower panels show Flag - H2B - SUMO and Flag - H2B - ub , respectively . One , two or three asterisks indicate mono - , di - , and poly - sumoylated histones ( upper panel ) , respectively , and an arrowhead and bullet point represent mono - ubiquitylated H2B , and a non - specific band , respectively . B Immunoblot assay of sumoylated proteins in extracts prepared from the strains Anti - Flag blotting for Flag - H2B was used to verify the similar loading . C Chromatin double immunoprecipitation ( ChDIP ) of Flag - H2B - ub in the indicated strains expressing Flag - H2B . The first ChIP was performed with anti - Flag agarose , and the eluted samples were immunoprecipitated with an antibody against ubiquitin . PCR signals from the indicated genes were normalized to an internal control and the input DNA . The error bars indicate the SD from three independent chromatin samples . Data information : Asterisks indicate statistically significant differences compared with WT in * , P < 0 . 05 ; * * , P < 0 . 01D ChDIP of Flag - H2B - SUMO in the indicated strains expressing both Flag - H2B and HA - SUMO . Chromatin samples were sequentially immunoprecipitated with anti - HA agarose followed by anti - Flag agarose . PCR signals were normalized to the input DNA . The error bars indicate the SD from three independent experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 . 01E ChDIP of Flag - H2B - SUMO in the indicated mutant strains during CUP1 gene induction The CUP1 induction was performed CUP1 OFF and ON indicate uninduced and induced ( 5 min ) conditions , respectively . The error bars represent the SD from three independent ChDIP assays . Data information : Asterisks indicate significant differences between uninduced and induced cells in ( E ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ) . F Immunoblot assay of Flag - tagged Ulp2 in the indicated strains . Anti - PGK blotting shows similar loading . ChIP assays in the indicated strains expressing Flag - tagged Ulp2 The error bars indicate the SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 . 01ChIP assays in the indicated strains expressing Flag - tagged Ulp2 The error bars indicate the SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 . 01ChIP assays in the indicated strains expressing Flag - tagged Ulp2 The error bars indicate the SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively.B Immunoblot assay of sumoylated proteins in extracts prepared from the strains Anti-Flag blotting for Flag-H2B was used to verify the similar loading.C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples. Data information: Asterisks indicate statistically significant differences compared with WT in *, P <0.05; **, P <0.01D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01E ChDIP of Flag-H2B-SUMO in the indicated mutant strains during CUP1 gene induction The CUP1 induction was performed CUP1 OFF and ON indicate uninduced and induced (5 min) conditions, respectively. The error bars represent the SD from three independent ChDIP assays. Data information: Asterisks indicate significant differences between uninduced and induced cells in (E) (*, P <0.05; **, P <0.01).F Immunoblot assay of Flag-tagged Ulp2 in the indicated strains. Anti-PGK blotting shows similar loading.ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01).