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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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

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


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

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


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

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


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

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


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

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


Relative expression of Rab39a in siRNA - treated cells after 48 hours of knockdown . Rab39a expression in I - Ab or Rab39a siRNA - treated cells was normalized to expression in cells treated with control β2m siRNA . Error bars show SD between three independent experiments . * p < 0 . 05 using two - way Student ' s t - test .

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Relative expression of Rab39a in siRNA-treated cells after 48 hours of knockdown. Rab39a expression in I-Ab or Rab39a siRNA-treated cells was normalized to expression in cells treated with control β2m siRNA. Error bars show SD between three independent experiments. * p < 0.05 using two-way Student's t-test.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

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XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - latex The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

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XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-latex The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments .

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XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 Cells were then fed with Ova - Fe and exposed to reporter T cells as in ( B ) . Error bars show SD of duplicate wells . * p < 0 . 05 using two - way ANOVA . Representative plot of 3 independent experiments .

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DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.


MHC - II presentation of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe . The cells were then exposed to MF2 . 2D9 - Luc Reporter CD4 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control β2m using two - way ANOVA . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m . Representative plot of 3 independent

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MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experiments


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with Ova - Fe and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m using two - way ANOVA . Representative plot of 3 independent

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DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experiments


MHC - I Presentation of SIINFEKL - H2 - Kb complexes on siRNA treated DC3 . 2 NS - Ova ( C ) or DC3 . 2 UbS8L ( D ) cells . 48 hours after siRNA transfection , the indicated amounts of dox was added to the cells to induce Ova expression . After 2 hours at 37oC , RF33 . 70 - Luc CD8 T cells were added along with Brefeldin A ( Golgiplug ) to a final concentration of 1 : 1000 . The cells were incubated overnight for reporter T cell luciferase expression . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control I - Ab using two - way ANOVA . None of the dox concentrations are p < 0 . 05 for Rab39a siRNA vs I - Ab . Representative plot of 3 independent

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MHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experiments


Flow cytometric analysis of surface H2 - Kb and I - Ab levels of siRNA treated DC3 . 2R cells . Shown are results after 48 hours of knockdown . Representative plot of 3 independent

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Flow cytometric analysis of surface H2-Kb and I-Ab levels of siRNA treated DC3.2R cells. Shown are results after 48 hours of knockdown. Representative plot of 3 independent experiments


Phagocytosis assay after 48 hours of siRNA knockdown . DC3 . 2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads . The mixture was then placed in media at 37oC for 1 hour . Cells were harvested and incubated on ice with 1 mg / ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry . As a control , some cells were incubated with 10 µM Cytochalasin D during the feeding period . Shown are percentages of cells that have taken up beads . Representative plot of two independent experiments .

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Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments.


Rab39a was induced in DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) via overnight incubation with 1µg / ml dox . KO and rescued cells were then harvested and assayed as in ( A ) for phagocytosis . Representative plot of two independent experiments .

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Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments.


DC3 . 2 - Rab39a cells were induced to express Rab39a overnight . Cells on coverslips were washed , fixed and stained according to the listed protocol and antibodies . Green = Rab39a ( HA tag ) , Magenta = Lamp2 . Arrows highlight punctate structures with colocalization . Scale bar = 10 µm . Representative image of two independent experiments .

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

DC3.2-Rab39a cells were induced to express Rab39a overnight. Cells on coverslips were washed, fixed and stained according to the listed protocol and antibodies. Green = Rab39a (HA tag), Magenta = Lamp2. Arrows highlight punctate structures with colocalization. Scale bar = 10 µm. Representative image of two independent experiments.


DC3 . 2 Rab39a KO cells were transduced with dox inducible mcherry - tagged Rab39a . Cells were plated in 35 mm glass bottom dishes and induced with 1 µg / ml dox . The next day , 1 µm latex beads conjugated with Alexa 488 - NHS ( 1 bead / cell ) were added . After 2 hours at 37oC , cells were imaged under a Leica TCS SP5 confocal microscope . Green = Alexa 488 labeled beads . Magenta = mcherry - Rab39a . Arrows highlight internalized beads surrounded by Rab39a . Scale bar = 10 µm . Representative image of two independent experiments .

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

DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments.


5x105 of DC3 . 2 - Rab39a cells were induced to express Rab39a overnight with 1 µg / ml dox in a 6 well plate . Cells were fed at 1 bead / cell of biotinylated - 6µm magnetic beads for the indicated lengths of time . Magnetic - bead - phagosomes were isolated using the listed protocol . Phagosomes were washed and stained for Rab39a ( HA - tag ) . Representative data for 3 independent experiments .

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

5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments.


1 . 25 x 105 of DC3 . 2 - Rab39a cells were incubated in a 12 well plate overnight ± dox ( 1µg / ml ) to induce Rab39a . Cells were fed at 4 beads / cell of biotinylated - ova - 6µm - magnetic - beads for 3 hours with or without 1 : 1000 BFA ( golgiplug , BD ) . Phagosomes were isolated using the listed protocol . Isolated phagosomes were washed , permeabilized and stained . Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes . Representative plot of two independent experiments .

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

1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments


XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts o Ova ( B ) conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent

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

XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments


DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . * p < 0 . 05 between no dox and + dox using two - way ANOVA . Representative plot of 3 independent

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

DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experiments


Same as in ( A ) but cells were fed with 8 µg of A9M peptide bead and exposed to 12 . 64 - Luc Reporter T cells . Experiments were combined by normalizing RLU values to the average of the negative control I - Ab . Error bars show SD of 3 independent experiments . * p < 0 . 05 for Rab39a or β2m siRNA vs negative control I - Ab using

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

Same as in (A) but cells were fed with 8 µg of A9M peptide bead and exposed to 12.64-Luc Reporter T cells. Experiments were combined by normalizing RLU values to the average of the negative control I-Ab. Error bars show SD of 3 independent experiments. * p < 0.05 for Rab39a or β2m siRNA vs negative control I-Ab using ANOVA


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms shown in ( B ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2 - Ld ( dox / no dox ) was calculated . Three independent experiments were combined for analysis and error bars show the SD between experiments . Open H2 - Ld , but not Closed H2 - Ld was statistically significant ( p < 0 . 05 ) between KO and rescued cells using two - tailed ratio - paired Student ' s t - test .

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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test.


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . H2 - Ld cell surface levels of KO and induced cells as in ( A ) .

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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. H2-Ld cell surface levels of KO and induced cells as in (A).


Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains .

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

Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains.


Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains . Flow cytometry plots of ( D ) .

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

Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains. Flow cytometry plots of (D).


Analysis of phagosomes shown in ( D ) and ( E ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated .

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

Analysis of phagosomes shown in (D) and (E). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated.


DC3 . 2 Rab39a - H2 - Ld cells were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours in the presence or absence of BFA . Isolated phagosomes were stained for H2 - Ld . Representative of two independent experiments .

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

DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.


DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours with the indicated inhibitors . Magnetic - bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms of two independent experiments shown .

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

DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours with the indicated inhibitors. Magnetic-bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open and Closed (30-5-7s) forms of H2-Ld. Representative histograms of two independent experiments shown.


Cells as in ( A ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours . Phagosomes were isolated and stained for Ova and Open H2 - Ld . Representative of 3 independent experiments .

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

Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours. Phagosomes were isolated and stained for Ova and Open H2-Ld. Representative of 3 independent experiments.


Quantification of Ova remaining in bead - phagosomes ( C ) and Nox2 staining ( D ) . Representative Histogram shown . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein ( dox / no dox ) was calculated . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments .

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

Quantification of Ova remaining in bead-phagosomes (C) and Nox2 staining (D). Representative Histogram shown. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Cells as in ( A ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were incubated on ice with 4 beads / cell of biotinylated - Ova - beads for bead attachment . Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red . After 4 hours of incubation , unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change ( dox / no dox ) was calculated . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments .

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

Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were incubated on ice with 4 beads/cell of biotinylated-Ova-beads for bead attachment. Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red. After 4 hours of incubation, unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Same as in C with Sec22b staining . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments .

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

Same as in C with Sec22b staining. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Cells as in ( A ) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH . After 4 hours of incubation , whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads . For analysis , the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox / no dox was calculated . Three independent experiments were combined . p < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Also shown are histograms of whole cells compared to background fluorescence of pH beads .

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

Cells as in (A) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH. After 4 hours of incubation, whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads. For analysis, the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox/no dox was calculated. Three independent experiments were combined. p<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments. Also shown are histograms of whole cells compared to background fluorescence of pH beads.


DC3 . 2R cells were treated with the indicated siRNA . 48 hours after transfection , cells were fed with Ova - Fe ( 2 µg Ova ) with or without 100 µM leupeptin . RF33 . 70 - Luc reporter T cells were added . After overnight incubation , T cell luciferase was measured . Representative plot of two independent experiments . Error bars show SD between 3 replicate wells . p values were calculated using 2 - way ANOVA .

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

DC3.2R cells were treated with the indicated siRNA. 48 hours after transfection, cells were fed with Ova-Fe (2 µg Ova) with or without 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.


5x104 DC3 . 2 - Rab39a - H2 - Ld cells were fed with Ova - Fe ( 1µg Ova ) with or without 1 µg dox and / or 100 µM leupeptin . RF33 . 70 - Luc reporter T cells were added . After overnight incubation , T cell luciferase was measured . Representative plot of two independent experiments . Error bars show SD between 3 replicate wells . p values were calculated using 2 - way ANOVA .

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

5x104 DC3.2-Rab39a-H2-Ld cells were fed with Ova-Fe (1µg Ova) with or without 1 µg dox and/or 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.


Rab39a expression in splenocyte subsets was measured using an FDG assay . Shown are relative FDG fluorescence of Rab39a KO cells / WT background . Summary of two independent experiments .

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

Rab39a expression in splenocyte subsets was measured using an FDG assay. Shown are relative FDG fluorescence of Rab39a KO cells/WT background. Summary of two independent experiments.


Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16 - Flt3L tumor . 10 days post tumor implantation , DCIR2 + ( Cd8α - ) or XCR1 + ( Cd8α + ) dendritic cells were isolated from spleens via magnetic positive selection . 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova - Fe and 1 : 1 of Reporter CD8 or CD4 T - cells overnight before measuring luciferase activity . Error bars indicate SD of 2 replicate wells . Shown are representative graphs of 3 independent experiments . * p < 0 . 05 using two - way ANOVA .

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

Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16-Flt3L tumor. 10 days post tumor implantation, DCIR2+ (Cd8α-) or XCR1+ (Cd8α+) dendritic cells were isolated from spleens via magnetic positive selection. 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova-Fe and 1:1 of Reporter CD8 or CD4 T-cells overnight before measuring luciferase activity. Error bars indicate SD of 2 replicate wells. Shown are representative graphs of 3 independent experiments. * p < 0.05 using two-way ANOVA.


CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm biotin - ova - magnetic beads for 4 hours . Phagosomes were extracted , permeabilized and stained for the indicated proteins . For ROS measurements , phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target ( dox / no dox ) was calculated . Shown are three independent experiments combined . Error bars show SD between experiments . p values were calculated using two - tailed ratio - paired Student ' s t - test .

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

CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm biotin-ova-magnetic beads for 4 hours. Phagosomes were extracted, permeabilized and stained for the indicated proteins. For ROS measurements, phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target (dox/no dox) was calculated. Shown are three independent experiments combined. Error bars show SD between experiments. p values were calculated using two-tailed ratio-paired Student's t-test.


CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm magnetic beads conjugated to pHrodo . After 4 hours , cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH . For analysis , the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change ( dox / no dox ) was calculated . Shown are three independent experiments with error bars indicating the SD . p values were calculated using two - tailed ratio - paired Student ' s t - test .

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

CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to pHrodo. After 4 hours, cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH. For analysis, the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change (dox/no dox) was calculated. Shown are three independent experiments with error bars indicating the SD. p values were calculated using two-tailed ratio-paired Student's t-test.


CD11b + CD11c + dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection . Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye . After 4 hours , cells were harvested and resuspended in 1 mg / ml Trypan blue to quench fluorescence of uneaten beads . Cells were run for flow cytometry to measure phagocytosis . The ratio of KO cells with beads / WT cells with beads was then obtained . Graphs show three independent experiments and p values were calculated using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments .

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

CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye. After 4 hours, cells were harvested and resuspended in 1 mg/ml Trypan blue to quench fluorescence of uneaten beads. Cells were run for flow cytometry to measure phagocytosis. The ratio of KO cells with beads / WT cells with beads was then obtained. Graphs show three independent experiments and p values were calculated using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.


Rab39a KO and WT mice were injected intravenously with 1 x 105 A20 - Ova cells . The next day , CFSE labeled OTI - splenocytes were injected i . v . . 3 days post OT - I transfer , spleens were harvested and assayed for OT - I proliferation . Three independent experiments ( WT vs KO ) were performed . To combine the data , the OT - I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment . Error bars show SD between individual mice and p value was derived from two - tailed student ' s t - test . n = 14 ( WT ) or 9 ( KO ) .

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

Rab39a KO and WT mice were injected intravenously with 1 x 105 A20-Ova cells. The next day, CFSE labeled OTI-splenocytes were injected i.v.. 3 days post OT-I transfer, spleens were harvested and assayed for OT-I proliferation. Three independent experiments (WT vs KO) were performed. To combine the data, the OT-I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment. Error bars show SD between individual mice and p value was derived from two-tailed student's t-test. n = 14 (WT) or 9 (KO).


B HSATIII ASO - mediated depletion of nSBs . Thermal stress - exposed HeLa cells ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) were visualized by HSATIII - FISH and immunofluorescence using an anti - SAFB antibody or anti - HNRNPM antibody . The nuclei were stained with DAPI . Scale bar : 10 μm .

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B HSATIII ASO-mediated depletion of nSBs. Thermal stress-exposed HeLa cells (42°C for 2 h and recovery for 1 h at 37°C) were visualized by HSATIII-FISH and immunofluorescence using an anti-SAFB antibody or anti-HNRNPM antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.


C qRT - PCR - validation of HSATIII knockdown . The graph shows the qRT - PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions : 37 ° C , 42 ° C for 2 h , and thermal stress followed by recovery at 37 ° C for 1 h ( Recovery ) . Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions ( 42 ° C for 2 h ) . Data are shown as the mean ± SD ( n = 3 ) . HSATIII RNAs ratios ( % ) are indicated .

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C qRT-PCR-validation of HSATIII knockdown. The graph shows the qRT-PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions: 37°C, 42°C for 2 h, and thermal stress followed by recovery at 37°C for 1 h (Recovery). Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions (42°C for 2 h). Data are shown as the mean±SD (n=3). HSATIII RNAs ratios (%) are indicated.


D MA - plot ( log2 fold - change over the average expression level ) of all detected introns in HSATIII KD and control cells ( n = 3 ) . The introns with significant changes in their expression levels ( adjusted p - value < 0 . 01 ) are shown as green dots . Among these introns , up - regulated ( log2 fold - change > 1 ) and down - regulated ( log2 fold - change < - 1 ) introns are shown with magenta and light blue dots , respectively . Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol - i ( intron ) # . Total numbers of introns and genes utilized were 144769 and 32623 , respectively .

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D MA-plot (log2 fold-change over the average expression level) of all detected introns in HSATIII KD and control cells (n=3). The introns with significant changes in their expression levels (adjusted p-value < 0.01) are shown as green dots. Among these introns, up-regulated (log2 fold-change > 1) and down-regulated (log2 fold-change < -1) introns are shown with magenta and light blue dots, respectively. Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol-i (intron) #. Total numbers of introns and genes utilized were 144769 and 32623, respectively.


Examples of RNA - seq read maps of HSATIII target RNAs . Affected introns and exons are indicated by blue ( down - regulated upon HSATIII knockdown )

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Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown)


G Examples of RNA - seq read maps of HSATIII target RNAs . Affected introns and exons are indicated by blue ( down - regulated upon HSATIII knockdown ) and magenta ( up - regulated ) boxes .

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G Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown) and magenta (up-regulated) boxes.


Validation of HSATIII target introns by qRT - PCR . The graphs show the relative amounts of the intron - retaining ( IR ) ( upper ) and spliced ( lower ) forms in control and HSATIII knockdown cells under three conditions : 37 ° C ( normal ) , 42 ° C for 2 h ( thermal stress ) , and thermal stress followed by recovery at 37 ° C for 1 h . Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions ( 37 ° C ) . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( Sidak ' s multiple comparison test ) .

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

Validation of HSATIII target introns by qRT-PCR. The graphs show the relative amounts of the intron-retaining (IR) (upper) and spliced (lower) forms in control and HSATIII knockdown cells under three conditions: 37°C (normal), 42°C for 2 h (thermal stress), and thermal stress followed by recovery at 37°C for 1 h. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions (37°C). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).


B Nuclear localization of the intron - retaining RNAs . The relative amounts of intron - retaining RNAs in the nuclear and cytoplasmic fractions were quantified by qRT - PCR and are represented as the ratio ( % of the total ) . GAPDH mRNA and U1 snRNA were used as cytoplasmic and nuclear controls , respectively . Data are shown as the mean ± SD ( n = 3 ) .

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B Nuclear localization of the intron-retaining RNAs. The relative amounts of intron-retaining RNAs in the nuclear and cytoplasmic fractions were quantified by qRT-PCR and are represented as the ratio (% of the total). GAPDH mRNA and U1 snRNA were used as cytoplasmic and nuclear controls, respectively. Data are shown as the mean±SD (n=3).


D , E The levels of HSATIII target introns in newly synthesized RNAs within 1 h after thermal stress removal , as determined by qRT - PCR . The graphs show the changes in the expression levels of the intron - retaining ( IR ) ( D ) and spliced ( E ) forms . Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the level in the control cells . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( multiple t - test modified by Holm - Sidak ' s method ) .

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D, E The levels of HSATIII target introns in newly synthesized RNAs within 1 h after thermal stress removal, as determined by qRT-PCR. The graphs show the changes in the expression levels of the intron-retaining (IR) (D) and spliced (E) forms. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the level in the control cells. Data are shown as the mean±SD (n=3); *p<0.05 (multiple t-test modified by Holm-Sidak's method).


A Splicing isoforms of the CLK1 pre - mRNA . The retained introns are indicated by red lines . An asterisk indicates the position of the premature termination codon in the nonsense - mediated mRNA decay - targeted isoform . B Time course analysis of the splicing pattern of CLK1 pre - mRNAs in control and HSATIII knockdown ( HSATIII KD ) cells by semi - quantitative RT - PCR . Arrows indicate the positions of PCR primers . The GAPDH mRNA was used as an internal control . C Quantification of the data shown in ( B ) . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( Sidak ' s multiple comparison test ) .

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A Splicing isoforms of the CLK1 pre-mRNA. The retained introns are indicated by red lines. An asterisk indicates the position of the premature termination codon in the nonsense-mediated mRNA decay-targeted isoform. B Time course analysis of the splicing pattern of CLK1 pre-mRNAs in control and HSATIII knockdown (HSATIII KD) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control. C Quantification of the data shown in (B). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).


D Thermal stress - induced expression of HSATIII in CHO ( His9 ) cells . The cells were visualized by HSATIII - FISH ( green ) , and the nuclei were stained with DAPI ( blue ) . Scale bar : 10 μm .

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D Thermal stress-induced expression of HSATIII in CHO (His9) cells. The cells were visualized by HSATIII-FISH (green), and the nuclei were stained with DAPI (blue). Scale bar: 10 μm.


E Ectopic HSATIII - induced nSB assembly in CHO ( His9 ) cells . The nSBs ( yellow arrowheads ) were visualized by HSATIII - FISH and immunofluorescence using an anti - SRSF1 antibody . The nuclei were stained with DAPI . Scale bar : 10 μm .

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E Ectopic HSATIII-induced nSB assembly in CHO (His9) cells. The nSBs (yellow arrowheads) were visualized by HSATIII-FISH and immunofluorescence using an anti-SRSF1 antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.


F Time course analysis of the splicing pattern of Chinese hamster Clk1 pre - mRNAs in control ( CHO ) and CHO ( His9 ) cells by semi - quantitative RT - PCR . Arrows indicate the positions of PCR primers . The GAPDH mRNA was used as an internal control .

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

F Time course analysis of the splicing pattern of Chinese hamster Clk1 pre-mRNAs in control (CHO) and CHO (His9) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control.


Time course analysis of the splicing pattern of Chinese hamster Clk1 pre - mRNAs in control ( CHO ) and CHO ( His9 ) cells by semi - quantitative RT - PCR . Arrows indicate the positions of PCR primers . The GAPDH mRNA was used as an internal control . G Quantification of the data shown in ( F ) . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( Sidak ' s multiple comparison test ) .

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

Time course analysis of the splicing pattern of Chinese hamster Clk1 pre-mRNAs in control (CHO) and CHO (His9) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control. G Quantification of the data shown in (F). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).


H The effect of HSATIII knockdown on pooling of intron - retaining RNAs in control ( CHO ) and CHO ( His9 ) cells ( 42 ° C for 2 h and recovery for 2 h at 37 ° C ) . Arrows indicate the positions of PCR primers .

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H The effect of HSATIII knockdown on pooling of intron-retaining RNAs in control (CHO) and CHO (His9) cells (42°C for 2 h and recovery for 2 h at 37°C). Arrows indicate the positions of PCR primers.


B RT - PCR validation of the specific precipitation of HSATIII by ChIRP . The NEAT1 ncRNA and GAPDH mRNA were used as negative controls . Input : 100 % .

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B RT-PCR validation of the specific precipitation of HSATIII by ChIRP. The NEAT1 ncRNA and GAPDH mRNA were used as negative controls. Input: 100%.


C Silver staining of the coprecipitated proteins . The predicted mobilities of five known nSB proteins are indicated . Input : 0 . 1 % .

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C Silver staining of the coprecipitated proteins. The predicted mobilities of five known nSB proteins are indicated. Input: 0.1%.


D Western blot analyses of proteins identified by the HSATIII - ChIRP experiment . Known nSB proteins ( SAFB , SRSF1 , SRSF7 , SRSF9 , and HNRNPM ) were used as positive controls , and ELAVL1 , PSPC1 and α - tubulin were used as negative controls . SRSF3 , SRSF5 , TRA2B and THRAP3 are newly identified nSB components .

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

D Western blot analyses of proteins identified by the HSATIII-ChIRP experiment. Known nSB proteins (SAFB, SRSF1, SRSF7, SRSF9, and HNRNPM) were used as positive controls, and ELAVL1, PSPC1 and α-tubulin were used as negative controls. SRSF3, SRSF5, TRA2B and THRAP3 are newly identified nSB components.


Colocalization of novel nSB proteins with HSATIII . Endogenous TRA2B ( E ) were stained with anti - TRA2B HSATIII was visualized by RNA - FISH using a dig - labeled HSATIII ASO , and the nuclei were stained with DAPI . Scale bar : 10 μm .

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

Colocalization of novel nSB proteins with HSATIII. Endogenous TRA2B (E) were stained with anti-TRA2B HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.