( B ) Representative confocal showing neurons ( NeuN ) overexpressing ARSA ( green ) close to posterior injection site of NHP S2 . 2 . Arrows indicate co - localization of IF signals . Note granular perinuclear ARSA , likely representing localized in Golgi / lysosomal vesicles .

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(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 showing presence of ARSA - positive ( green ) co - stained with oligodendrocyte markers CNPase APC ( arrows ) . ( D ) Undetectable ARSA immunoreactivity in tissues of untreated . In pictures nuclei are counterstained with ToPro ( blue ) . Scale bars : A , 80 µm ; B , D 60µm , C ,

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(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


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

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Vector 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).


( A ) ARSA overexpression detected by in cortical subcortical regions of injected hemispheres of NHP S2 . 3 .

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(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 subcortical regions of injected hemisphere in LV . GFP - injected NHP P1 . Scale bar , 100 µm . Insets show ARSA - expressing at higher magnification . Note perinuclear accumulation of ARSA .

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(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 ) 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 Bonferroni post - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs physiological .

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(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.


( A ) Schematic representation of ARSA activity in brain of NHP S1 . 1 S2 . 2 along rostro - caudal axis ( slices 2 - 9 ) . Data are expressed as fold increase to ARSA activity detected in matched blocks of UT LV . GFP - injected NHPs ( physiological ) . Grading of colors ranges from yellow ( < 1 . 2 fold ) to green ( ≥ 2 fold ) .

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(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 ) study group 2 ( C ) ( filled grey bars , injected hemisphere ; striped grey bars , hemisphere ) , in UT LV . GFP - injected NHPs ( bars ; physiological ) . Arrows on x axis indicate injection sites . n = 2 - 12 tissue blocks / slice . Number of tissue blocks analyzed per : 51 ( UT / LV . GFP ) , 98 - 122 ( S1 . 1 S2 . 2 ) , 12 - 22 ( S1 . 2 , S1 . 3 , S2 . 2 , S2 . 3 ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs matched slices of UT / LV . GFP .

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(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 injected hemispheres of NHPs in study group 1 study group 2 . Number of tissue blocks analysed : 51 ( UT / LV . GFP ) , 105 ( study group 1 , injected hemisphere ) , 52 ( study group 1 , hemisphere ) ; 103 ( study group 2 , injected hemisphere ) , 72 ( study group 2 , hemisphere ) . One - way ANOVA Tukey ' s multiple comparison , * * * p < 0 . 001 versus UT / LV . GFP , § p < 0 . 05 versus study group 1 .

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(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 spinal cord of LV . GFP - ( n = 4 blocks / , 2 - 3 replicates ) LV . hARSA - injected NHPs ( n = 2 - 4 blocks / ; n = 10 n = 11 total blocks for study group 1 study group 2 , respectively ) . Graph on right represent summary of ARSA activity in each treatment group . One - way ANOVA Dunnet ' s multiple comparison . * p < 0 . 05 versus pilot group .

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(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 sciatic nerve of LV . GFP - ( n = 2 ) LV . hARSA - injected NHPs ( n = 3 ) . In B - F data are expressed as floating bars ( at mean ) .

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(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 cerebrospinal fluid ( CSF ) of UT / LV . GFP ( n = 3 ) LV . hARSA - injected NHP ( n = 5 ) . Data are expressed as mean ± . Student t - , * p = 0 . 035 .

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(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.


( A ) Vector copy number ( VCN ) indicating distribution of integrated LV genome along rostro - caudal axis ( slices 7 - 19 ) in LV . hGALC - injected NHPs ( JT02 JV02 ) assessed by qPCR . Each dot represent VCN measured in one block within slice . Lower threshold ( dotted ) : VCN < 0 . 001 , corresponding to CT > 37 .

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(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 rostro - caudal axis of JT02 ( slices 7 - 13 ) is expressed as fold increase to region - matched blocks of hemisphere ( dotted , y = 1 ) . Data are expressed as floating bars ( min to max , at mean ; n = 4 - 8 blocks / slice ) . Arrows on x axis in A B indicate injection sites .

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(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 showing GALC + expressing markers of neurons ( NeuN ) astrocytes ( S100β ) close to injection site ( slice 10 ) in JT02 . Arrows indicate co - localization of IF signals .

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(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 showing GALC - expressing astrocytes ( S100β ; D ) neurons ( NeuN ; E ) in hemispheres , anterior ( slice 8 ) posterior ( slice 12 ) to injection site . Arrows indicate co - localization of IF signals . Scale bars : C , 70 µm ( upper panel ) 40µm ( lower panel ) ; D E , 40µm .

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(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.


( A ) Specific GALC enzymatic activity in single brain slices of WT untreated ( UT ; bars , physiological ) , WT LV . hGALC - treated ( JV02 ) Krabbe - affected LV . hGALC - treated ( JV02 ) ( filled grey bars , injected hemisphere ; striped grey bars , hemisphere ) . Two - way ANOVA followed by Bonferroni ' s multiple comparison ; * p < 0 . 05 , * * p < 0 . 01 vs matched slices of WT UT .

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(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 JV02 , as well as from WT Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis Dunn ' s multiple comparison ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT .

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(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 / ) samples from JT02 JV02 , as well as from WT Krabbe UT controls . Data in A - C are represented as floating bars ( min to max , at mean ; n = 2 - 6 blocks / slice ) . Number of blocks analysed / : 16 ( WT UT ) , 24 ( JT02 ) , 22 ( JV02 ) . GALC activity in Krabbe UT samples is < 0 . 001 nmol / h * mg ; n = 3 blocks . Kruskal - Wallis Dunn ' s multiple comparison ; * p < 0 . 05 , * * * p < 0 . 001 vs WT UT .

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(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 spinal cord ( SC ) tissue of untreated ( UT ) WT 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 presence ( ) or absence of 11µM AgNO3 ( ● ) .

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(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 CSF of JT02 ( pre - post - treatment ) JV02 ( post - treatment ) . Dots represent technical replicates .

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(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 , were evaluated monthly for motor performance using a modified Bayley ' s scale for infant development . For study ( JV02 JT02 ) , scores are reported as pre - surgery post - surgery . Historical data for normal affected are presented as means plus one standard deviation . Affected score significantly lower than normal at any considered . JT02 ' s motor scores increase over time post LV . GALC injection , being close to mean observed in normal juveniles at 5 months . Historical data are analyzed by two - way ANOVA Bonferroni posttests , p < 0 . 05 at 6m , p < 0 . 001 at other ages . n = 5 - 20 / group , except for normal at 6 months ( n = 1 ) .

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(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).


E . Ribosomal pull down shows lack of occupancy of ribosomes on veal2 . fli1a actb were used as positive controls .

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E. Ribosomal pull down shows lack of occupancy of ribosomes on veal2. fli1a and actb were used as positive controls.


F - I . e - GFP fusion confirms lack of peptide formation from veal2 . ( F - G ) mitfa - eGFP fused transcript . ( H - I ) veal2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected . Scale bar - 100μm .

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F-I. e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (F-G) mitfa-eGFP fused transcript. (H-I) veal2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μm.


F - T . Representative of 2 dpf zebrafish which displayed rescue of vascular integrity defects in progeny of an outcross of veal2gib005Δ8 / + zebrafish upon complementing with wild type ( WT ) veal2 . ( F - J ) Control zebrafish . ( K - O ) veal2gib005Δ8 / + injected with vehicle control . ( P - T ) veal2gib005Δ8 / + complemented with veal2 . ( F , K , P ) bright field . ( G , L , Q ) mRFP . ( H , M , R ) stained with O - dianisidine stain . ( I , N , S ) eGFP . ( J , O , T ) merged eGFP mRFP filters . Arrowheads show presence of hemorrhage due to vascular integrity defects . ( F - H , K - M , P - R ) magnification - 5X , scale bar - 100μm ( I - J , N - O , S - T ) magnification - 20X , Scale bar - 50μm .

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F-T. Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8/+ zebrafish upon complementing with the wild type (WT) veal2 RNA. (F-J) Control zebrafish embryos. (K-O) veal2gib005Δ8/+ embryos injected with vehicle control. (P-T) veal2gib005Δ8/+ embryos complemented with veal2 RNA. (F,K,P) bright field. (G,L,Q) mRFP. (H,M,R) Animals stained with O-dianisidine stain. (I,N,S) eGFP. (J,O,T) merged eGFP and mRFP filters. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (F-H,K-M,P-R) magnification-5X, scale bar-100μm (I-J,N-O,S-T) magnification-20X, Scale bar-50μm.


C . immunoprecipitation of veal2 was performed by pull - down of Prkcbb Epbhb3 , followed by qRT - PCR . IgG pull - down was performed as control . Gel image shows amplification of veal2 in different immunoprecipitation samples , along with 10 % input control , as marked by arrowheads .

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C. RNA immunoprecipitation of veal2 was performed by pull-down of Prkcbb and Epbhb3, followed by qRT-PCR. IgG pull-down was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads.


D . qRT - PCR of veal2 transcript across Prkcbb , Ephb3 IgG immunoprecipitations . actb was used as normalization control . Data from three different biological replicates represented as mean fold change values ± standard deviation .

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D. qRT-PCR based quantification of veal2 transcript across Prkcbb, Ephb3 and IgG immunoprecipitations. actb was used as normalization control. Data from three different biological replicates represented as mean fold change values ± standard deviation.


F - K . Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8 / + zebrafish indicated by rescue of vascular integrity defects in veal2gib005Δ8 / + . Arrowheads show presence of hemorrhage due to vascular integrity defects . Experiment was repeated in biological replicates total no of scored are mentioned on figure . ( F - I ) Magnification - 5X , Scale bar - 100μm , ( J - K ) Magnification - 20X , Scale bars - 50μm .

O O O O B-SMALL_MOLECULE O O B-DISEASE O O B-GENEPROD O O B-ORGANISM O O O O B-TISSUE O O O B-GENEPROD O O O O O O O B-DISEASE 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

F-K. Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure. (F-I) Magnification-5X, Scale bar-100μm, (J-K) Magnification-20X, Scale bars-50μm.


F . Abundance of Prkcbb in total ( T ) , ( C ) ( M ) fractions of from 2 dpf gib004Tg ( fli1a : EGFP ; gata1a : DsRed ) veal2gib005Δ8 / + zebrafish . Arrowhead indicates Prkcbb enrichment in fraction of veal2gib005Δ8 / + zebrafish .

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F. Abundance of Prkcbb protein in total cell (T), cytoplasmic (C) and membrane (M) fractions of cells from 2 dpf gib004Tg(fli1a:EGFP;gata1a:DsRed) and veal2gib005Δ8/+ zebrafish embryos. Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2gib005Δ8/+ zebrafish embryos.


E - H e - GFP fusion confirms lack of peptide formation from veal2 . ( E - F ) mitfa - eGFP fused transcript . ( G - H ) VEAL2 - eGFP fused transcript . Arrowheads indicating e - GFP expression in mitfa - eGFP injected . Scale bar -

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E-H e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (E-F) mitfa-eGFP fused transcript. (G-H) VEAL2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μ


I - N . Complementation of VEAL2 in veal2gib005Δ8 / + significantly rescued hemorrhage phenotype . Arrowheads show presence of hemorrhage due to vascular integrity defects . ( I - L ) Magnification - 5X , Scale bar - 100μm ( M - N ) Magnification - 20X , Scale bars - 50μm .

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

I-N. Complementation of VEAL2 in veal2gib005Δ8/+ embryos significantly rescued hemorrhage phenotype. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (I-L) Magnification-5X, Scale bar-100μm (M-N) Magnification-20X, Scale bars-50μm.


R - T . Co IF for PRKCB smFISH of VEAL2 highlight their colocalization . ( R ) PRKCB in GFP channel . ( S ) VEAL2 in CAL - fluor red ( 610nM ) . ( T ) Merged image for PRKCB VEAL2 . Magnification - 100X , scale bar - 20μm .

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

R-T. Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization. (R) PRKCB in the GFP channel. (S) VEAL2 in CAL-fluor red (610nM). (T) Merged image for PRKCB and VEAL2. Magnification-100X, scale bar-20μm.


K - AB . VEAL2 regulates junctional dynamics by interacting with PRKCB . Overexpression of VEAL2 retains PRKCB mostly in cytoplasm keeps strong junctional assembly formation of CDH5 CTNNB1 on . Knockdown of VEAL2 led to migration of PRKCB on henceforth degradation of junctional assembly of CDH5 CTNNB1 . ( K - N , W - X ) PRKCB . ( O - R , Y - Z ) CDH5 . ( S - V , AA - AB ) CTNNB1 . ( K - V ) Magnification - 60X , Scale bar - 15μm . Arrowheads indicate representation of signals of in HUVECs . ( W - AB ) Dot plot representing of signal localization in / total fraction . was done using imageJ . Data from of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as values ; middle bar represents mean error bar represents ± standard deviation .

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

K-AB. VEAL2 regulates junctional dynamics by interacting with PRKCB. Overexpression of VEAL2 retains PRKCB mostly in cytoplasm and keeps strong junctional assembly formation of CDH5 and CTNNB1 on the membrane. Knockdown of VEAL2 led to migration of PRKCB on membrane and henceforth degradation of junctional assembly of CDH5 and CTNNB1. (K-N,W-X) PRKCB. (O-R,Y-Z) CDH5. (S-V,AA-AB) CTNNB1. (K-V) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (W-AB) Dot plot representing quantification of protein signal localization in membrane/total fraction. The quantification was done using imageJ. Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.


E - S . Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 CTNNB1 increased localization of PRKCB . Complementation of VEAL2 veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 CTNNB1 also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia . ( E - H , Q ) CDH5 , ( I - L , R ) CTNNB1 , ( M - P , S ) PRKCB . ( E - P ) Magnification - 60X , Scale bar - 15μm . Arrowheads indicate representation of signals of in HUVECs . ( Q - S ) Data from of different fields of 3 technical replicates of 1 biological replicate is presented as representation . Data is shown as values ; middle bar represents mean error bar represents ± standard deviation .

O O O O O B-DISEASE O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-DISEASE O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-DISEASE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD 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 B-CELL_LINE O O O O O O O O O O O O O O O O O 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-S. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E-H,Q) CDH5 protein, (I-L,R) CTNNB1 protein, (M-P,S) PRKCB protein. (E-P) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q-S) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.


C Wild - type tel1 ∆ strains expressing a fully functional Top1 - HA tagged were arrested in G1 with α - factor ( αf ) released into YEPD supplemented with CPT ( 50 μM ) . Western blot with anti - HA antibodies ( top ) Coomassie ( bottom ) of extracts prepared at indicated time points

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

C Wild-type and tel1∆ strains expressing a fully functional Top1-HA tagged protein were arrested in G1 with α-factor (αf) and released into YEPD supplemented with CPT (50 μM). Western blot with anti-HA antibodies (top) and Coomassie staining (bottom) of protein extracts prepared at the indicated time points


A - C Exponentially growing cultures ( exp ) were arrested in G1 with α - factor ( αf ) released into YEPD with or without CPT ( 50 μM ) . samples were harvested at indicated time points to evaluat Rad53 phosphorylation by western blot with anti - Rad53 antibodies ( C )

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

A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C)


E , F G1 - arrested cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) ( E ) or in YEPD supplemented with CPT ( 10 μM ) with ( + noc ) or without ( - noc ) nocodazole ( F ) . Western blot with anti - Rad53 antibodies

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

E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies


A - C Exponentially growing cultures were arrested in G1 with α - factor ( αf ) released in YEPD supplemented with CPT ( 50 μM ) . ( A ) Western blot with anti - Rad53 antibodies

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

A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies


( B - E ) G1 - arrested cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at indicated time points were subjected to western blot with anti - Rad53 antibodie

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(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodie


( B - E ) G1 - arrested cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at indicated time points were subjected to western blot with anti - Rad53 antibodies ( B , D

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(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D


F , G G1 - arrested cultures ( αf ) were released into YEPD supplemented with CPT ( 50 μM ) . Western blot with anti - Rad53 antibodies

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F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies


HeLa cyclin F Knock - Out ( CCNF K / O ) were treated with Kinase ChemoGenomic Set ( KSGS ) in 384 well format . After 72 Hours ( h ) viability was measured using rezasurin . Flowchart representation ( left ) . Robust Z - score difference is plotted for data acquired at 1 μM ( right ) .

B-CELL_LINE B-GENEPROD I-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 B-EXP_ASSAY 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

HeLa and cyclin F Knock-Out (CCNF K/O) cells were treated with Kinase ChemoGenomic Set (KSGS) in 384 well format. After 72 Hours (h) viability was measured using rezasurin. Flowchart representation (left). Robust Z-score difference is plotted for data acquired at 1 μM concentration (right).


survival measured using rezasurin compared to controls treated with DMSO ( expressed as relative proliferation % ) . were treated with indicated inhibitors at 0 . 1 1 μM .

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Cell survival measured using rezasurin and compared to controls treated with DMSO (expressed as relative proliferation %). Cells were treated with the indicated inhibitors at 0.1 and 1 μM concentration.


survival measured using rezasurin compared to controls ( 0 ) treated with DMSO ( expressed as relative proliferation % ) . were treated with UCN - 01 at indicated concentrations .

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Cell survival measured using rezasurin and compared to controls (0) treated with DMSO (expressed as relative proliferation %). Cells were treated with UCN-01 at the indicated concentrations.


Differences in viability of HeLa CCNF K / O after treatment with specific Chk1i ( LY2603618 ) at indicated concentrations plotted on a log10 scale to measure differences in IC50 .

O O B-EXP_ASSAY O B-CELL_LINE B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O

Differences in viability of HeLa and CCNF K/O cells after treatment with specific Chk1i (LY2603618) at the indicated concentrations plotted on a log10 scale to measure differences in IC50.


Number of was measured in HeLa CCNF K / O left untreated or treated with Chk1i ( LY2603618 ) for 24h , 48h , or 72h as indicated . ( n . s . = non - significant ) .

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Number of cells was measured in HeLa and CCNF K/O cells left untreated or treated with Chk1i (LY2603618) for 24h, 48h, or 72h as indicated. (n.s. = non-significant).


viability measurements using Propidium Iodide for HeLa CCNF K / O after treatment with Chk1i ( LY2603618 ) .

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Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K/O cells after treatment with Chk1i (LY2603618).


U - 2 - OS were seeded transfected with non - targeting siRNA siNC ( Negative Control ) or siCyc F . 24 hours after transfection , were treated with UCN - 01 , or Chk1i ( LY2603618 ) at indicated concentrations for 3 days before viability was measured .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O

U-2-OS cells were seeded and transfected with non-targeting siRNA siNC (Negative Control) or siCyc F. 24 hours after transfection, cells were treated with UCN-01, or Chk1i (LY2603618) at indicated concentrations for 3 days before viability was measured.


viability measurements using Propidium Iodide in U - 2 - OS transfected with non - targeting siNC siCyc F treated with Chk1i ( LY2603618 ) as indicated .

B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O

Cell viability measurements using Propidium Iodide staining in U-2-OS cells transfected with non-targeting siNC and siCyc F and treated with Chk1i (LY2603618) as indicated.


treated with 1μM Chk1i ( LY2603618 ) for indicated hours ( h ) were harvested lysed using SDS . Indicated were resolved by SDS page detected by Western blot ( WB ) . TFIIH was used as a loading control .

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

Cells treated with 1μM Chk1i (LY2603618) for the indicated hours (h) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by Western blot (WB). TFIIH was used as a loading control.


( % ) of γ - H2AX positive in G1 after Chk1i ( LY2603618 ) treatment for indicated hours ( h ) measured using Activated Sorting ( FACS ) . G1 were considered having DAPI 2n EdU negative . ( % ) of γ - H2AX positive in replicating ( EdU + ) after Chk1i ( LY2603618 ) treatment at indicated time points . ( % ) of γ - H2AX positive in G2 / M after Chk1i ( LY2603618 ) treatment . G2 / M were considered having DAPI 4n EdU negativity .

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

Percentage (%) of γ-H2AX positive cells in G1 cells after Chk1i (LY2603618) treatment for the indicated hours (h) measured using Fluorescence Activated Cell Sorting (FACS). G1 cells were considered having DAPI 2n staining and EdU negative. Percentage (%) of γ-H2AX positive cells in replicating (EdU+) cells after Chk1i (LY2603618) treatment at the indicated time points. Percentage (%) of γ-H2AX positive cells in G2/M cells after Chk1i (LY2603618) treatment. G2/M cells were considered having DAPI 4n staining and EdU negativity.


of γ - H2AX measured as Relative Units ( RFU ) in early S phase treated with Chk1i . Mean of γ - H2AX was evaluated from EdU + with 2N DAPI content . of γ - H2AX measured as Relative Units ( RFU ) in replicating treated with Chk1i . Mean of γ - H2AX was evaluated from EdU + . Overall , cycle profile of HeLa CCNF K / O after Chk1i treatment . with 2n DAPI but EdU - were gated as G1 ; with > 2n but < 4n DAPI but EdU - were gated as EdU - S ; with > 2n but < 4n DAPI but EdU + were gated as EdU + S phase ; with 4n DAPI pH3 S10 negative were gated as G2 phase with 4n DAPI pH3 S10 positive as M . Data from at least 3 independent replicates plotted as Mean ± SD .

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 B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-CELL_LINE B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in early S phase cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+ cells with 2N DAPI content. Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in replicating cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+. Overall, cell cycle profile of HeLa and CCNF K/O cells after Chk1i treatment. Cells with 2n DAPI staining but EdU- were gated as G1 cells; cells with >2n but <4n DAPI staining but EdU- were gated as EdU- S cells; cells with >2n but <4n DAPI staining but EdU+ were gated as EdU+ S phase; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells. Data from at least 3 independent replicates and plotted as Mean ± SD.


Representative plots of γ - H2AX vs RPA2 signal of HeLa CCNF K / O after treatment with 1μM Chk1i ( LY2603618 ) at indicated hours ( h ) . Blue labeled dots represent γ - H2AX positive . Relates to materials section for details on representation .

O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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

Representative plots of γ-H2AX vs RPA2 signal of HeLa and CCNF K/O cells after treatment with 1μM Chk1i (LY2603618) at the indicated hours (h). Blue labeled dots represent γ-H2AX positive cells. Relates to materials section for details on representation.


Representative plots of γ - H2AX vs DAPI signal of CCNF K / O transfected with empty GFP GFP - CCNF untreated ( top panels ) after treatment with 1μM Chk1i ( LY2603618 ) for 20h ( bottom panels ) . Normalised γ - H2AX in CCNF K / O transfected with either GFP empty vector or GFP - Cyc F treated with 1μM Chk1i for 20 hours . GFP positive were gated . Data from 3 independent experiments were plotted with Mean % ± SD .

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

Representative plots of γ-H2AX intensity vs DAPI signal of CCNF K/O cells transfected with empty GFP and GFP-CCNF untreated (top panels) and after treatment with 1μM Chk1i (LY2603618) for 20h (bottom panels). Normalised γ-H2AX intensity in CCNF K/O cells transfected with either GFP empty vector or GFP-Cyc F and treated with 1μM Chk1i for 20 hours. GFP positive cells were gated. Data from 3 independent experiments were plotted with Mean % ± SD.


Representative of neutral alkaline comet from HeLa CCNF K / O treated with Chk1i . were treated with 1μM Chk1i for 24h before being harvested for neutral alkaline comet assays as indicated . of comets in alkaline gels . At least 100 , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using Mann - Whitney ( two - ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % / 100 . of comets in neutral gels . At least 100 , across two slides , were analysed in each condition in two biological replicates . Data are shown as medians , with 25 / 75 % percentile range ( box ) 10 - 90 % percentile range ( whiskers ) . Fold changes of median values are shown in bold . p - values were calculated using Mann - Whitney ( two - ) . Olive tail moment = ( Tail . mean - Head . mean ) * Tail % / 100 .

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

Representative images of neutral and alkaline comet assay from HeLa and CCNF K/O cells treated with Chk1i. Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated. Quantification of comets in alkaline gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100. Quantification of comets in neutral gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.


( % ) of γ - H2AX positive in HeLa CCNF K / O after transfection of indicated siRNAs treated with Chk1i for 20 hours . of γ - H2Ax positive were plotted as Mean % ± SD . ( % ) of hyper - RPA positive in HeLa CCNF K / O after transfection of indicated siRNAs treated with Chk1i for 20 hours . of hyper RPA positive were plotted as Mean % ± SD .

O O O O O O B-GENEPROD O O B-CELL_LINE B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE B-GENEPROD 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

Percentage (%) of γ-H2AX positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of γ-H2Ax positive cells were plotted as Mean % ± SD. Percentage (%) of hyper-RPA positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of hyper RPA positive cells were plotted as Mean % ± SD.


( % ) of γ - H2AX positive in S phase by DAPI content after Chk1i ( LY2603618 )

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Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618)


Relative γ - H2AX in transfected with either Empty Vector ( EV ) or HA - E2F1 treated with Chk1i for 20 hours . γ - H2AX in HA positive was normalized to empty vector transfected compared to not expressing HA .

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Relative γ-H2AX intensity in cells transfected with either Empty Vector (EV) or HA-E2F1 treated with Chk1i for 20 hours. γ-H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA.


HeLa CCNF K / O transfected with indicated siRNA treated with 1μM Chk1i ( LY2603618 ) for 20 hours were harvested lysed using SDS . Indicated were resolved by SDS page detected by WB . TFIIH served as a loading control .

B-CELL_LINE B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O

HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.


HeLa CCNF K / O were harvested lysed using SDS . Indicated were resolved by SDS page detected by WB . Ponceau S was used as a loading control .

B-CELL_LINE B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control.


U - 2 - OS transfected with non - targeting siRNA ( siNC ) or with an siRNA targeting Cyc F were lysed using SDS . Whole lysates were analyzed by immunoblotting 48h after transfection . H3 was used as a loading control .

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O

U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control.


showing gene set variance ( GSVA ) score for gene sets related to E2F targets ( PID _ E2F _ PATHWAY , ISHIDA _ E2F _ TARGETS HALLMARK _ E2F _ TARGETS ) .

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

Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS).


HEK293T transfected with empty vector ( EV ) , FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) FLAG - cyclin F M309A were treated with MLN4924 for 4 hours harvested for immunoprecipitation using FLAG antibodies . Immunoprecipitates were immunoblotted as indicated .

B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY O O O

HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.


HEK293T were cotransfected with MYC - tagged ubiquitin HA - E2F1 in presence of FLAG - cyclin F WT , FLAG - cyclin F ( ∆ F ) FLAG - cyclin F M309A were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions immunoblotted .

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

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HeLa CCNF K / O treated with 10 μM Chk1i ( LY2603618 ) for indicated time points ( h = hours ) were harvested lysed using SDS . Indicated were resolved by SDS page detected by WB . H2B served as a loading control .

B-CELL_LINE B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O

HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control.


HEK293 transfected with empty vector ( EV ) or HA - E2F1 were treated with Chk1i for 20 hours harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated .

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

HEK293 cells transfected with empty vector (EV) or HA-E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.


of immunoprecipitation results of Figure 5G from three independent experiments . Data from 3 independent experiments were plotted with Mean % ± SD . * < 0 . 05 p - value calculated by two - paired t - .

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Quantification of immunoprecipitation results of Figure 5G from three independent experiments. Data from 3 independent experiments were plotted with Mean % ± SD. *< 0.05 p-value and calculated by two-tailed paired t-test.


HEK293T were cotransfected with MYC - tagged ubiquitin HA - E2F1 were treated with Chk1i for 20 hours siRNA of cyclin F were indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions immunoblotted .

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HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HEK293T transfected with HA - E2F1 Wild Type ( WT ) HA - E2F1 lacking a CY motif ( ∆ RxL ) harvested for immunoprecipitation using HA antibodies . Immunoprecipitates were immunoblotted as indicated .

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

HEK293T cells transfected with HA-E2F1 Wild Type (WT) and HA-E2F1 lacking a CY motif (∆RxL) harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.


HEK293T were cotransfected with MYC - tagged ubiquitin HA - E2F1 or HA - E2F1 ∆ RxL lacking CY motif as indicated ( + ) . HA - E2F1 was immunoprecipitated with an anti - HA resin under denaturing conditions immunoblotted .

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

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 or HA-E2F1 ∆RxL lacking the CY motif as indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.


HeLa stably expressing HA - E2F1 WT HA - E2F1 ∆ RxL under control of a retroviral promoter were treated with Cycloheximide ( CHX ) at 50 μg / ml for indicated minutes ( min ) of E2F1 half -

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HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-life


HeLa stably expressing an Empty Vector ( EV ) , HA - E2F1 WT HA - E2F1 ∆ RxL under control of a retroviral promoter were treated with Chk1i for indicated hours ( h ) . Indicated were resolved by SDS page detected by WB . H3 was used as a loading control .

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

HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated protein were resolved by SDS page and detected by WB. H3 was used as a loading control.


Relative survival ( % ) of HeLa stably expressing an Empty Vector ( EV ) , HA - E2F1 WT HA - E2F1 ∆ RxL under control of a retroviral promoter measured using rezasurin compared to EV treated with Chk1i at indicated concentrations .

O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-EXP_ASSAY O

Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.


A . Representative plots of splenocytes from CD45 . 1 mice adoptively transferred with CD45 . 2 OT - II WT or CCR5 - / - lymph node suspensions , five after infection with rVACV - OVA . gating strategy used to identify memory CD4 + T subtypes is shown ( n = 5 ) .

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

A. Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT-II WT or CCR5-/- lymph node cell suspensions, five weeks after infection with rVACV-OVA virus. The gating strategy used to identify the memory CD4+ T cell subtypes is shown (n = 5).


B . Absolute number of OT - II recovered in spleens of mice as in A ( n = 5 ) .

O O O B-EXP_ASSAY O O O O O O B-TISSUE O B-ORGANISM O O O O O O O O O

B. Absolute number of OT-II cells recovered in spleens of mice as in A (n = 5).


C , of CD4 + ( C ) in OT - II WT CCR5 - / - populations ( n = 5 ) .

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

C, Percentage of CD4+ TEM (C) in the OT-II WT and CCR5-/- populations (n = 5).


D . of TCM ( D ) in OT - II WT CCR5 - / - populations ( n = 5 ) .

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

D. Percentage of TCM (D) in the OT-II WT and CCR5-/- populations (n = 5).


E . IFNγ - producing OT - II WT CCR5 - / - memory isolated from mice as in A restimulated ex vivo with OVA323 - 339 ( 1 μM ) ( n = 4 ) .

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

E. IFNγ-producing OT-II WT and CCR5-/- memory cells isolated from mice as in A and restimulated ex vivo with OVA323-339 (1 μM) (n = 4).


G - I . Representative plots ( G ) of frequency ( H ) absolute number ( I ) of Tfh ( CD4 + CD44 + PD - 1 + CXCR5 + ) in spleen after immunization ( day 7 ) with NIP - OVA ( n = 7 ) .

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

G-I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7).


J , K . ELISA of high - ( J ) low - affinity ( K ) isotype - specific anti - NIP antibodies in sera from OVA / OVA - OVA / KLH - immunized mice ( day 15 post - challenge ; n = 5 mice / group ) . Data representative of one experiment of two .

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

J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two.


A , Representative histograms of mean ( MFI ; A ) in OT - II WT CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± ( n ≥ 3 ) .

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

A, Representative histograms and quantification of mean fluorescence intensity (MFI; A) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).


B . of positive for indicated memory markers ( B ) in OT - II WT CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± ( n ≥ 3 ) .

B. the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).


C - F . IL - 2 - ( C , D ) IL - 15 - expanded lymphoblasts ( E , F ) were restimulated with indicated concentrations of OVA323 - 339 ; proliferation ( thymidine incorporation into ; C , E ) IL - 2 production ( by ELISA ; D , F ) were measured after 72 h . Data are presented as mean ± ( n = 5 ) .

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

C-F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323-339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5).


A - C . of TCR nanoclustering by EM in OT - II WT CCR5 - / - naïve ( A ; n = 6 / genotype ; WT : 3427 , CCR5 - / - : 3528 particles ) , IL - 2 - ( B ; WT , n = 8 , 15419 particles ; CCR5 - / - , n = 6 , 5410 particles ) or IL - 15 - expanded lymphoblasts ( C ; WT , n = 8 , 27518 particles ; CCR5 - / - , n = 7 , 22696 particles ) . A representative small field image at top of each panel shows gold particle distribution in surface replicas of anti - CD3ε - labeled ; at bottom , ( mean ± ) of gold particles in clusters of indicated in WT ( gray bars ) CCR5 - / - ( red ) . Insets show distribution of clusters of one , two , three , four , or more than four particles , statistical .

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

A-C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5-/- naïve cells (A; n = 6 cells/genotype; WT: 3427, CCR5-/-: 3528 particles), and IL-2- (B; WT, n = 8 cells, 15419 particles; CCR5-/-, n = 6 cells, 5410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27518 particles; CCR5-/-, n = 7 cells, 22696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5-/- cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis.


D , E . Posterior distribution in naïve ( D ) IL - 2 - expanded lymphoblasts ( E ) of clustering parameter b for WT ( gray ) CCR5 - / - ( red ) ; randomly generated distributions of receptors are shown in blue . mean value of b parameter is indicated for each condition . probability of a chance distribution similar to that determined in is nearly 0 % by ROPE .

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

D, E. Posterior distribution in naïve (D) and IL-2-expanded lymphoblasts (E) of the clustering parameter b for WT (gray) and CCR5-/- cells (red); randomly generated distributions of receptors are shown in blue. The mean value of the b parameter is indicated for each condition. The probability of a chance distribution similar to that determined in cells is nearly 0% by the ROPE.


F . Comparison of TCR oligomer using BN - PAGE anti - CD3ζ immunoblotting in day 10 , IL - 2 - expanded WT CCR5 - / - OT - II lymphoblasts lysed in buffer containing digitonin or Brij - 96 . marker is ferritin ( f1 , 440 f2 , 880 kDa forms ) . of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry ( right ; n = 5 ) .

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

F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5-/- OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5).


G . Top , representative small field EM showing gold particle distribution in surface replicas of CD4 + T isolated from OVA / OVA - immunized WT CCR5 - / - mice . Bottom , ( mean ± ) of gold particles in clusters of indicated ( WT , gray bars ; n = 5 , 14680 particles ; CCR5 - / - , red ; n = 7 , 15374 particles ) . Insets show distribution between clusters of one , two , three , four or more than four particles , statistical .

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

G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


A . OT - II WT were activated with OVA323 - 339 , alone or with TAK - 779 . After three days , antigen TAK - 779 were removed lymphoblasts expanded in IL - 2 - containing medium . TCR nanoclustering was analyzed in anti - CD3ε - labeled surface replicas of day 10 lymphoblasts . Top , representative small field EM showing gold particle distribution in surface replicas of WT CD4 + T alone or with TAK - 779 . Bottom , of gold particles in clusters of indicated . Inset , distribution of gold particles between clusters of one , two , three , four or more than four particles in vehicle - ( gray bars ; n = 5 , 11266 particles ) TAK - 779 - treated ( black ; n = 6 , 5138 particles ) . B . OT - II WT were activated with OVA323 - 339 , TAK - 779 was added at days 3 , 5 , 7 after antigen removal . as above , untreated ( gray bars ; n = 5 , 6400 particles ) TAK - 779 - treated ( black ; n = 6 , 7153 particles ) . Inset show distribution between clusters of one , two , three , four or more than four particles , statistical .

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

A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


C . OT - II WT naïve were activated with antigen in presence or not of CXCR4 inhibitor AMD3100 . Left , representative EM showing gold particle distribution in surface replicas . Right , of gold particles in clusters as above , vehicle - ( gray bars ; n = 6 , 12339 particles ) AMD3100 - treated ( black ; n = 7 , 17059 particles ) . Inset show distribution between clusters of one , two , three , four or more than four particles , statistical .

O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 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 O O O O O O O O O O O O O O O O

C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


A . Total Chol in WT CCR5 - / - OT - II lymphoblasts ( day 10 , IL - 2 - expanded ) as determined by a fluorometric ( n = 6 ) .

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

A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6).


B - D . SM ( B ) , Cer ( C ) dhCer ( D ) in WT CCR5 - / - OT - II 10 - day lymphoblasts , as determined by UPLC - TOF MS . Values , after normalization with C17 standards number in each sample , are mean of two independent experiments ( n = 6 ) .

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

B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6).


E . RT - qPCR determination of CerS mRNA in naïve IL - 2 - expanded WT CCR5 ‑ / - OT - II 10 - day lymphoblasts ( n = 3 - 5 ) .

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

E. RT-qPCR determination of CerS mRNA levels in naïve and IL-2-expanded WT and CCR5‑/- OT-II 10-day lymphoblasts (n = 3-5).


F . Representative immunoblot showing CerS2 in naïve WT CCR5 - / - OT - II 10 - day lymphoblasts , densitometric of blots as above ( n = 10 ) .

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

F. Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5-/- OT-II 10-day lymphoblasts, and densitometric quantification of blots as above (n = 10).


G . ChIP of CerS2 promoter using an anti - H3K9Ac antibody . Scheme of CerS2 promoter showing CpG islands primers used for amplification . Relative ChIP of CerS2 promoter in WT CCR5 - / - OT - II 10 - day lymphoblasts ( n = 3 ) .

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

G. ChIP analysis of the CerS2 promoter using an anti-H3K9Ac antibody. Scheme of the CerS2 promoter showing CpG islands and primers used for amplification. Relative ChIP of the CerS2 promoter in WT and CCR5-/- OT-II 10-day lymphoblasts (n = 3).


H . Relative CerS2 mRNA level in CD4 T treated with PTx ( n = 3 ) .

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

H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3).


L . Representative immunofluorescence showing pSer142 - GATA - 1 ( green ) of OT - II WT CCR5 - / - lymphoblasts . green channel ( top ) merge with DAPI ( blue ; bottom ) are shown . Scale bar , 10 μm . M . of of plotted as integrated density in DAPI - stained ; n ≥ 50 / condition ) .

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

L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm. M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition).


N . Top , basic scheme of CerS2 promoter , indicating putative GATA - 1 binding site ( blue ) location of primers used for amplification in ChIP assays ( black arrows ) . Bottom , relative anti - GATA - 1 ChIP in OT - II WT CCR5 - / - lymphoblasts ( n = 5 ) .

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

N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).


A . Scheme of strategy used to form TCR proteoliposomes , of LUV generated at indicated lipid molar . Polydispersity index values are shown as black squares for each condition ( n = 3 ) . SBP , streptavidin - binding - peptide - tagged TCR .

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

A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.


B . Representative immunoblots comparing TCR nanocluster sizes via BN - PAGE anti - CD3ζ immunoblotting in TCR proteoliposomes lysed in presence of Brij - 96 or digitonin . marker is ferritin ( f1 , 440 f2 , 880 kDa forms ) .

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

B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).


C . of nanocluster monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B ( n ≥ 4 ) .

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

C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B (n ≥ 4).


D . Cer in OT ‑ II 10 - day lymphoblasts , untreated or treated with SMase ( n = 4 ) .

O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O

D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).


E . Representative small field showing gold particle distribution , ( mean ± ) of gold particles in clusters of indicated in surface replicas from untreated ( gray bars ; n = 5 , 8126 particles ) SMase - treated ( 1h ) OT - II lymphoblasts ( black cyan ; n = 6 , 8457 particles ) after CD3ε labeling , as determined by EM . inset shows distribution between clusters of one , two , three , four or more than four particles .

O O O O O O B-SMALL_MOLECULE 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 O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD 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

E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.


F . GFP expression in shCtrl - ( black ) shCerS2 ( orange ) - transduced 2B4 after puromycin selection , as determined by FACS .

O O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O

F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.


G . Relative CerS2 mRNA in TAK - 779 - treated 2B4 as in F . Values were normalized to those obtained in untransduced TAK - 779 - treated 2B4 ( n = 3 ) .

O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O O

G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).


H . Representative immunoblot with anti - CerS2 antibody to determine CerS2 in shCtrl ShCerS2 - transduced 2B4 as in G . Filters were rehybridized with β - actin as loading control .

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

H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G. Filters were rehybridized with β-actin as loading control.


I . TCR nanoclustering of shCtrl - shCerS2 transduced 2B4 in presence of TAK - 779 as determined by EM . Representative small field ( mean ± ) of gold particles in clusters of indicated sizes in surface replicas shCtrl ( gray black bars ; n = 6 , 12337 particles ) shCerS2 2B4 lymphoblasts ( orange ; n = 7 , 13456 particles ) . Inset , distribution between clusters of indicated statistical .

O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY 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 O O O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779 as determined by EM. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray black bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (orange; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size and statistical analysis.


J . of CD69 + shCtrl ( black ) shCerS2 ( orange ) 2B4 restimulated with plate - bound anti - CD3ε antibody in presence of TAK - 779 ( n = 3 ) .

O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

J. Percentage of CD69+ shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779 (n = 3).


A . of TCR nanoclustering in lymphoblasts from healthy WT ccr5Δ32 homozygous donors by EM . Top , representative small field image showing gold particle distribution in surface replicas of anti - CD3ε - labeled ; bottom , ( mean ± ) of gold particles in clusters of indicated in WT ( gray bars ; n = 5 , 17689 particles ) Δ32 / Δ32 ( red ; n = 4 , 16938 particles ) . Insets show distribution between clusters of one , two , three , four or more than four particles , statistical .

O O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O 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. Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM. Top, representative small field image showing gold particle distribution in cell surface replicas of anti-CD3ε-labeled cells; bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size in the WT (gray bars; n = 5 cells, 17689 particles) and Δ32/Δ32 cells (light red; n = 4 cells, 16938 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


B . Normalized SM , Cer dhCer in lymphoblasts obtained as in A . A representative experiment is shown ( n = 3 donors / genotype ; n = 2 independent experiments )

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

B. Normalized SM, Cer and dhCer levels in lymphoblasts obtained as in A. A representative experiment is shown (n = 3 donors/genotype; n = 2 independent experiments)


C . Relative CerS2 mRNA in day 8 WT ccr5Δ32 lymphoblasts . Each data point is average of a technical triplicate from 3 donors in two independent experiments ( n = 6 )

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

C. Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts. Each data point is the average of a technical triplicate from 3 donors in two independent experiments (n = 6)


OTULIN chromatograms showing homozygous single base substitution ( c . 841G > A , p . Gly281Arg , arrowhead ) . Data are representative to two independent experiments .

B-GENEPROD 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

OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.


Affinity measurements by polarisation ( FP ) with recombinant catalytically inactive OTULINC129A or OTULINC129A / G281R FlAsH - labelled M1 - linked diUb . Data represent mean ± SD of one experiment performed in technical triplicate . Data are representative of three independent experiments . a . u . , arbitrary units . K D , dissociation constant .

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

Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. KD, dissociation constant.


M1 - linked tetraUb hydrolysis by recombinant OTULINWT OTULING281R using indicated OTULIN concentrations visualised on silver - stained SDS - PAGE gels . Data are representative of three independent experiments .

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

M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.


First derivative of tryptophan upon thermal unfolding of recombinant OTULINcatWT OTULINcatG281R ( 1 . 0 mg / mL ) measured by nano differential scanning fluorimetry ( nanoDSF ) . Apparent melting temperatures ( Tm ) are indicated ( dashed lines ) . Data are representative to two independent experiments .

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

First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.


Immunoblot of whole lysates from untreated healthy control fibroblasts . Data are representative of three independent experiments .

B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O O O O O

Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts. Data are representative of three independent experiments.


Immunoblot of whole lysates from healthy control fibroblasts either left untreated or treated with proteasome inhibitor MG132 ( 10 μM ) for 24 h . Data are representative of two independent experiments .

B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.


Immunoblot of whole lysates from untreated healthy control or fibroblasts . Data are representative of three independent experiments .

B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O

Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts. Data are representative of three independent experiments.


Immunoblot ( left ) densitometry ( right ) of IκBα in healthy control fibroblasts treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments .

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

Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


Immunoblot of IκBα phosphorylation degradation as well as p65 / RelA phosphorylation in healthy control or OTULING281R fibroblasts fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Immunoblot of phosphorylation of MAP kinases p38 JNK in healthy control or OTULING281R fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments .

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

Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments. Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.


Densitometry of p65 / RelA phosphorylation as presented in ( A ) from three independent experiments .

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

Densitometry analysis of p65/RelA phosphorylation as presented in (A) from three independent experiments.


ELISA of IL - 8 secretion in response to TNF stimulation ( 10 ng / mL ) in healthy control or OTULING281R fibroblasts . Bars represent mean ± ( n = 4 ) .

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

ELISA analysis of IL-8 secretion in response to TNF stimulation (10 ng/mL) in healthy control or OTULING281R primary fibroblasts. Bars represent mean ± SEM (n=4).


Immunoblot of native TNF - RSC purified by immunoprecipitation from healthy control or OTULING281R fibroblasts . Data are representative of three independent experiments .

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

Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.


Immunoblot of whole lysate from untreated human THP - 1 monocytes with stable expression of a non - targeting control shRNA or shRNA targeting OTULIN . Data are representative of two independent experiments .

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

Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments.


Immunoblot ( left ) densitometry ( right ) of IκBα in shControl shOTULIN THP - 1 treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments .

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

Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


ELISA of spontaneous TNF secretion over 96 h in shControl shOTULIN THP - 1 . Data represents mean ± ( n = 4 ) were analysed using two - way ANOVA of statistical significance with Sidak ' s correction for multiple comparisons .

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

ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.


Immunoblot of IκBα phosphorylation degradation as well as p65 / RelA phosphorylation in shControl shOTULIN THP - 1 in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments .

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

Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.


Densitometry of p65 / RelA phosphorylation as presented in ( D ) from three independent experiments .

Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.


Viability of healthy control or OTULING281R fibroblasts after 24 h 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , as indicated was analysed using MTT reduction assays .

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

Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), as indicated was analysed using MTT reduction assays.