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 .

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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 .

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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 .

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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 .

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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

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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 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

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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

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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 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

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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 .

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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 ) .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 .

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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 .

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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.


Colocalization of novel nSB proteins with HSATIII . THRAP3 ( F ) , were stained with anti - THRAP3 , HSATIII was visualized by RNA - FISH using a dig - labeled HSATIII ASO , and the nuclei were stained with DAPI . Scale bar : 10 μm .

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Colocalization of novel nSB proteins with HSATIII. THRAP3 (F), were stained with anti-THRAP3, HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.


Colocalization of novel nSB proteins with HSATIII . FLAG - tagged PPHLN1 ( G ) were stained with anti - FLAG antibodies HSATIII was visualized by RNA - FISH using a dig - labeled HSATIII ASO , and the nuclei were stained with DAPI . Scale bar : 10 μm .

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Colocalization of novel nSB proteins with HSATIII. FLAG-tagged PPHLN1 (G) were stained with anti-FLAG antibodies HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.


Specific interaction of CLK1 with HSATIII during thermal stress recovery . HSATIII - ChIRP at various time points during and after thermal stress exposure . Endogenous CLK1 , SRSF7 , and SRSF9 were detected using anti - CLK1 , anti - SRSF7 , and anti - SRSF9 antibodies , respectively . HSATIII was detected by ChIRP - RNA purification , followed by RT - PCR . Input : 0 . 1 % for CLK1 , 1 % for SRSF7 and SRSF9 , and 100 % for HSATIII .

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Specific interaction of CLK1 with HSATIII during thermal stress recovery. HSATIII-ChIRP at various time points during and after thermal stress exposure. Endogenous CLK1, SRSF7, and SRSF9 were detected using anti-CLK1, anti-SRSF7, and anti-SRSF9 antibodies, respectively. HSATIII was detected by ChIRP-RNA purification, followed by RT-PCR. Input: 0.1% for CLK1, 1% for SRSF7 and SRSF9, and 100% for HSATIII.


Specific interaction of HSATIII with CLK1 proteins during stress recovery . HSATIII - ChIRP / western blotting was performed using HeLa cells expressing FLAG - CLK1 WT or the KR mutant . FLAG - tagged CLK1 and SRSF9 were detected by western blotting using an anti - FLAG and an anti - SRSF9 antibody , respectively . HSATIII was detected by ChIRP followed by RT - PCR . Input : 1 % for western blotting , 100 % for RT - PCR .

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Specific interaction of HSATIII with CLK1 proteins during stress recovery. HSATIII-ChIRP/western blotting was performed using HeLa cells expressing FLAG-CLK1 WT or the KR mutant. FLAG-tagged CLK1 and SRSF9 were detected by western blotting using an anti-FLAG and an anti-SRSF9 antibody, respectively. HSATIII was detected by ChIRP followed by RT-PCR. Input: 1% for western blotting, 100% for RT-PCR.


Re - translocation of FLAG - CLK1 to nSBs after thermal stress removal . HeLa cells were transfected with a FLAG - CLK1 expression vector , cultured for 16 h , and then exposed to thermal stress ( 42 ° C for 2 h ) with or without recovery ( 37 ° C for 1 h ) . FLAG - CLK1 was visualized with an anti - FLAG antibody . Nuclear stress bodies and nuclei were visualized by HSATIII - FISH and DAPI staining , respectively . Scale bar : 10 μm .

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Re-translocation of FLAG-CLK1 to nSBs after thermal stress removal. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and then exposed to thermal stress (42°C for 2 h) with or without recovery (37°C for 1 h). FLAG-CLK1 was visualized with an anti-FLAG antibody. Nuclear stress bodies and nuclei were visualized by HSATIII-FISH and DAPI staining, respectively. Scale bar: 10 μm.


Box plot of colocalization correlation coefficients between HSATIII and FLAG - CLK1 WT . The first and third quartiles are the ends of the box , the median is indicated by the vertical line in the box , and the minimum and maximum are the ends of the whiskers .

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Box plot of colocalization correlation coefficients between HSATIII and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated by the vertical line in the box, and the minimum and maximum are the ends of the whiskers.


Recovery phase - specific interaction of HSATIII with the CLK1 N - terminal region . HSATIII - ChIRP / western blotting was performed on HeLa cells expressing FLAG - CLK1 ∆ C or FLAG - CLK1 ∆ N Asterisk : an unidentified partial fragment of FLAG - CLK1 ∆ N .

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Recovery phase-specific interaction of HSATIII with the CLK1 N-terminal region. HSATIII-ChIRP/western blotting was performed on HeLa cells expressing FLAG-CLK1∆C or FLAG-CLK1∆N Asterisk: an unidentified partial fragment of FLAG-CLK1∆N.


Dependency of SRSF9 on the interaction of CLK1 ( G ) with HSATIII . HSATIII - ChIRP was performed using control and SRSF9 - depleted cells expressing FLAG - CLK1 proteins ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) . Input : 1 % . Graphs represent coprecipitation ratios of FLAG - CLK1 proteins to HSATIII in the control and SRSF9 - depleted cells .

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Dependency of SRSF9 on the interaction of CLK1 (G) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells.


H Dependency of SRSF9 on the interaction of CLK1 ∆ C ( H ) with HSATIII . HSATIII - ChIRP was performed using control and SRSF9 - depleted cells expressing FLAG - CLK1 proteins ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) . Input : 1 % . Graphs represent coprecipitation ratios of FLAG - CLK1 proteins to HSATIII in the control and SRSF9 - depleted cells .

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H Dependency of SRSF9 on the interaction of CLK1∆C (H) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells.


A Localization of SRSF9 with HSATIII during thermal stress and recovery .

A Localization of SRSF9 with HSATIII during thermal stress and recovery.


B Recovery phase - specific colocalization of CLK1 and SRSF9 . HeLa cells were transfected with a FLAG - CLK1 expression vector , cultured for 16 h , and exposed to thermal stress ( 42 ° C for 2 h ) followed by recovery ( 37 ° C for 1 h ) . FLAG - CLK1 and SRSF9 were co - stained using anti - FLAG and anti - SRSF9 antibodies , respectively , and the nuclei were stained with DAPI .

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B Recovery phase-specific colocalization of CLK1 and SRSF9. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and exposed to thermal stress (42°C for 2 h) followed by recovery (37°C for 1 h). FLAG-CLK1 and SRSF9 were co-stained using anti-FLAG and anti-SRSF9 antibodies, respectively, and the nuclei were stained with DAPI.


C Box plot of colocalization correlation coefficients between SRSF9 and FLAG - CLK1 WT . The first and third quartiles are the ends of the box , the median is indicated with a vertical line in the box , and the minimum and maximum are the ends of the whiskers .

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C Box plot of colocalization correlation coefficients between SRSF9 and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum are the ends of the whiskers.


F The effect of HSATIII knockdown on time course - dependent changes in SRSF9 phosphorylation . The phosphorylation states were estimated by Phos - tag SDS - PAGE , followed by western blotting using an anti - SRSF9 antibody . SRSF9 detected by western blotting of a normal SDS - PAGE gel is shown in the bottom panel . The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress - induced hypo - and de - phosphorylated forms , respectively .

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F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively.


F The effect of HSATIII knockdown on time course - dependent changes in SRSF9 phosphorylation . The phosphorylation states were estimated by Phos - tag SDS - PAGE , followed by western blotting using an anti - SRSF9 antibody . SRSF9 detected by western blotting of a normal SDS - PAGE gel is shown in the bottom panel . The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress - induced hypo - and de - phosphorylated forms , respectively . G Quantification of the data shown in ( F ) . Graphs show the time - course changes in four representative SRSF9 phosphorylated forms in ( F ) . Relative levels of band intensity were calculated as the ratio of each band to α - tubulin and were normalized to those of control at 37 ° C ( bands ( a ) and ( b ) ) and 42 ° C ( bands ( c ) and ( d ) ) . Since band ( d ) partially overlapped with the other band ( asterisk in ( F ) ) at 37 ° C and recovery 4h , band ( d ) at those time points was not measured .

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F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively. G Quantification of the data shown in (F). Graphs show the time-course changes in four representative SRSF9 phosphorylated forms in (F). Relative levels of band intensity were calculated as the ratio of each band to α-tubulin and were normalized to those of control at 37°C (bands (a) and (b)) and 42°C (bands (c) and (d)). Since band (d) partially overlapped with the other band (asterisk in (F)) at 37°C and recovery 4h, band (d) at those time points was not measured.


H The re - phosphorylation status of SRSF9 within nSBs . Phosphorylated ( arrows ) and hypo - and de - phosphorylated ( arrowheads ) SRSF9 in the nSB fractions ( lanes 4 , 6 , and 8 ) were analyzed by western blotting on Phos - tag gels . KH - CB19 ( 10 μM ) or DMSO as a control was administrated during the recovery period . The lower panel shows a shorter exposure image of the phosphorylated forms . The asterisks indicate bands different from band ( a ) or ( d ) . I Quantification of the data shown in ( H ) . Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS - PAGE , and were normalized to those at 42 ° C .

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H The re-phosphorylation status of SRSF9 within nSBs. Phosphorylated (arrows) and hypo- and de-phosphorylated (arrowheads) SRSF9 in the nSB fractions (lanes 4, 6, and 8) were analyzed by western blotting on Phos-tag gels. KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The lower panel shows a shorter exposure image of the phosphorylated forms. The asterisks indicate bands different from band (a) or (d). I Quantification of the data shown in (H). Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS-PAGE, and were normalized to those at 42°C.


A , B Validation of the effect of SRSF9 on retention / splicing of HSATIII target introns by qRT - PCR . The relative expression levels of intron - retaining ( IR ) forms ( A ) and spliced forms ( B ) of newly transcribed RNAs during the recovery period are shown . Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the control .

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A, B Validation of the effect of SRSF9 on retention/splicing of HSATIII target introns by qRT-PCR. The relative expression levels of intron-retaining (IR) forms (A) and spliced forms (B) of newly transcribed RNAs during the recovery period are shown. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the control.


C , D Time course analysis of the splicing patterns of CLK1 and TAF1D intron - retaining RNAs in control and HSATIII knockdown cells by semi - quantitative RT - PCR . Arrows indicate the positions of primers for RT - PCR . GAPDH mRNA was used as an internal control . The experiment was performed in triplicate . Quantification of the data shown in ( C ) ,

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C, D Time course analysis of the splicing patterns of CLK1 and TAF1D intron-retaining RNAs in control and HSATIII knockdown cells by semi-quantitative RT-PCR. Arrows indicate the positions of primers for RT-PCR. GAPDH mRNA was used as an internal control. The experiment was performed in triplicate. Quantification of the data shown in (C),


F Validation of the requirement of CLK1 catalytic activity for promotion of intron retention by qRT - PCR . Data are shown as the mean ± SD ( n = 3 ) .

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F Validation of the requirement of CLK1 catalytic activity for promotion of intron retention by qRT-PCR. Data are shown as the mean±SD (n=3).


G The diagrams show the domain structures of wild - type ( WT ) and mutant ( mut ) SRSF9 , in which all SR / SP dipeptides were replaced by AR / AP . FLAG - SRSF9 - WT and FLAG - SRSF9 - mut were extracted from transfected HeLa cells under normal ( 37 ° C ) and thermal stress ( 42 ° C for 2 h ) conditions , separated in a Phos - tag gel , and detected by western blot using an anti - FLAG antibody . The arrowhead indicates the thermal stress - induced de - phosphorylated form of FLAG - SRSF9 - WT .

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G The diagrams show the domain structures of wild-type (WT) and mutant (mut) SRSF9, in which all SR/SP dipeptides were replaced by AR/AP. FLAG-SRSF9-WT and FLAG-SRSF9-mut were extracted from transfected HeLa cells under normal (37°C) and thermal stress (42°C for 2 h) conditions, separated in a Phos-tag gel, and detected by western blot using an anti-FLAG antibody. The arrowhead indicates the thermal stress-induced de-phosphorylated form of FLAG-SRSF9-WT.


The role of phosphorylation of SRSF9 on splicing of the TAF1D ( H ) splicing reporter plasmid was transfected into HeLa cells along with a siRNA ( control or SRSF9 - specific ) and a siRNA - resistant SRSF9 - WT or SRSF9 - mut expression plasmid ( or pEGFP - C1 vector as the empty vehicle ( - ) ) 48 h before the assay . Semi - quantitative RT - PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress - exposed cells ( 42 ° C for 2 h and 37 ° C for 1 h ) . The positions of the PCR primers are indicated by arrows . Graphs represent the relative changes in the spliced / unspliced ratios , normalized to the control sample with control siRNA and empty plasmid ( - ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The role of phosphorylation of SRSF9 on splicing of the TAF1D (H) splicing reporter plasmid was transfected into HeLa cells along with a siRNA (control or SRSF9-specific) and a siRNA-resistant SRSF9-WT or SRSF9-mut expression plasmid (or pEGFP-C1 vector as the empty vehicle (-)) 48 h before the assay. Semi-quantitative RT-PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress-exposed cells (42°C for 2 h and 37°C for 1 h). The positions of the PCR primers are indicated by arrows. Graphs represent the relative changes in the spliced/unspliced ratios, normalized to the control sample with control siRNA and empty plasmid (-).


I The role of phosphorylation of SRSF9 on splicing of the DNAJB9 ( I ) reporters . Each splicing reporter plasmid was transfected into HeLa cells along with a siRNA ( control or SRSF9 - specific ) and a siRNA - resistant SRSF9 - WT or SRSF9 - mut expression plasmid ( or pEGFP - C1 vector as the empty vehicle ( - ) ) 48 h before the assay . Semi - quantitative RT - PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress - exposed cells ( 42 ° C for 2 h and 37 ° C for 1 h ) . The positions of the PCR primers are indicated by arrows . Graphs represent the relative changes in the spliced / unspliced ratios , normalized to the control sample with control siRNA and empty plasmid ( - ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The role of phosphorylation of SRSF9 on splicing of the DNAJB9 (I) reporters. Each splicing reporter plasmid was transfected into HeLa cells along with a siRNA (control or SRSF9-specific) and a siRNA-resistant SRSF9-WT or SRSF9-mut expression plasmid (or pEGFP-C1 vector as the empty vehicle (-)) 48 h before the assay. Semi-quantitative RT-PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress-exposed cells (42°C for 2 h and 37°C for 1 h). The positions of the PCR primers are indicated by arrows. Graphs represent the relative changes in the spliced/unspliced ratios, normalized to the control sample with control siRNA and empty plasmid (-).


Time - lapse imaging of an oocyte expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF - dependent MII arrest . The 3D positions of centromeres are shown as black dots in a side view ( along the spindle equator , middle row ) and top view ( perpendicular to the equator plane , bottom row ) . Tracks of individual centromeres are color - coded according to the speed , as indicated by the colour bar . Time is shown after anaphase I onset ( min : sec ) . Scale bars , 5 μm .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-lapse imaging of an oocyte expressing CENP-C-EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF-dependent MII arrest. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown after anaphase I onset (min:sec). Scale bars, 5 μm.


Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . On the schemes above the charts in ( A ) and ( C ) , chromosomes are red with green centromeres . Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1 . The black arrow on the scheme above the chart indicates chromosome - centre distance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1. The black arrow on the scheme above the chart indicates chromosome-centre distance.


Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . Changes in the speed of all chromosomes observed for the oocyte from Fig 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. Changes in the speed of all chromosomes observed for the oocyte from Fig 1.


Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . On the schemes above the charts in ( A ) and ( C ) , chromosomes are red with green centromeres . ( C ) Changes in inter - centromere distance ( indicated on a scheme by the black arrow ) , observed for the oocyte from Fig 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. (C) Changes in inter-centromere distance (indicated on a scheme by the black arrow), observed for the oocyte from Fig 1.


Kinetochore - MT attachments at the prometaphase II stage were visualized using an anti - tubulin antibody ( white ) , an anti - centromeric ACA antibody ( green ) and DAPI to label chromatin ( red ) . The image represents maximum intensity projection through all z - planes containing MTs . Representative chromosomes displaying syntelic , amphitelic or merotelic / lateral attachments are enclosed in white frames and their enlarged single z - plane images are shown to the right . PB : polar body . Scale bar , 10μm . Chromosomes with syntelic ( red ) , amphitelic ( green ) and merotelic / lateral ( grey ) attachments were plotted according to their inter - centromere distance ( on the horizontal axis ) and distance to spindle equator ( vertical axis ) . Data shown for 156 chromosomes taken from 8 prometaphase II oocytes .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm. Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.


Time - lapse imaging of a representative MII oocyte with a normal segregation pattern ( i . e . all 20 chromosomes show synchronous equational chromatid separation at anaphase onset , leaving no chromatids behind at the midzone ) , expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II . Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles . The 3D positions of centromeres are shown as black dots in a side view ( along the spindle equator , middle row ) and top view ( perpendicular to the equator plane , bottom row ) . Tracks of individual centromeres are color - coded according to their speed , as indicated by the colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-lapse imaging of a representative MII oocyte with a normal segregation pattern (i.e. all 20 chromosomes show synchronous equational chromatid separation at anaphase onset, leaving no chromatids behind at the midzone), expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to their speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.


Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A - D on the vertical axis , and time in min on the horizontal axis relative to the anaphase II onset . Thin grey lines in A - D represent individual chromosomes ; thick black line in C - D shows the mean value for each timepoint . On the schemes near the charts chromosomes are red with green centromeres ; spindle equator in A and spindle axis in B - C are indicated by dashed lines . Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern , shown in Fig 3 . The analysed distance is indicated by a black arrowed line on the scheme above the chart . Changes in distance to the spindle axis ( shown on the scheme by a black arrowed line ) for all chromosomes in oocyte from Fig 3 . Changes in chromosome orientation for all chromosomes in oocyte from Fig 3 . The orientation is characterized by the angle ( highlighted black ) between the spindle axis and the line connecting the sister centromeres ( black solid line on the scheme ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by dashed lines. Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern, shown in Fig 3. The analysed distance is indicated by a black arrowed line on the scheme above the chart. Changes in distance to the spindle axis (shown on the scheme by a black arrowed line) for all chromosomes in oocyte from Fig 3. Changes in chromosome orientation for all chromosomes in oocyte from Fig 3. The orientation is characterized by the angle (highlighted black) between the spindle axis and the line connecting the sister centromeres (black solid line on the scheme).


Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A - D on the vertical axis , and time in min on the horizontal axis relative to the anaphase II onset . Thin grey lines in A - D represent individual chromosomes ; thick black line in C - D shows the mean value for each timepoint . ( D ) Changes in inter - centromere distance , shown on the scheme by black arrowed line , for all chromosomes in oocyte from Fig 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. (D) Changes in inter-centromere distance, shown on the scheme by black arrowed line, for all chromosomes in oocyte from Fig 3.


Kinetochore - MT attachments in oocytes released from the CSF - dependent metaphase arrest were visualized using an anti - tubulin antibody ( white ) , an anti - centromeric ACA antibody ( green ) and DAPI to label chromatin ( red ) . The image represents maxiumum intensity projection through all z - planes containing MTs . Single z - planes of representative chromosomes displaying amphitelic or merotelic / lateral attachments are shown to the right . Scale bar , 10μm . Chromosomes with amphitelic ( green ) and merotelic / lateral ( grey ) attachments were plotted according to their inter - centromere distance ( on the horizontal axis ) and distance to spindle equator ( vertical axis ) . The inter - centromere distance in fixed oocytes is 1 . 2 ± 0 . 2 μm for amphitelic and 1 . 0 ± 0 . 2 μm for merotelic attachments ( Mean ± SD ) . Data shown for 95 chromosomes taken from 5 oocytes released from the CSF - dependent metaphase II arrest .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetochore-MT attachments in oocytes released from the CSF-dependent metaphase arrest were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maxiumum intensity projection through all z-planes containing MTs. Single z-planes of representative chromosomes displaying amphitelic or merotelic/lateral attachments are shown to the right. Scale bar, 10μm. Chromosomes with amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). The inter-centromere distance in fixed oocytes is 1.2±0.2 μm for amphitelic and 1.0±0.2 μm for merotelic attachments (Mean±SD). Data shown for 95 chromosomes taken from 5 oocytes released from the CSF-dependent metaphase II arrest.


A time - lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II . A lagging chromatid is indicated by a blue arrowhead . Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole . The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots , and centromeres of laggard - producing chromosome as red dots in a side view along the spindle equator . One of the centromeres is left behind at anaphase ( blue arrowhead ) , but will segregate equationally . Tracks of individual centromeres are color - coded according to the speed , as indicated by the colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 time-lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. A lagging chromatid is indicated by a blue arrowhead. Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole. The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots, and centromeres of laggard-producing chromosome as red dots in a side view along the spindle equator. One of the centromeres is left behind at anaphase (blue arrowhead), but will segregate equationally. Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.