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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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

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 .

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-CONTROLLED_VAR O O 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

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 .

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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 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 .

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 .

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 .

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 .

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 .

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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-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

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 .

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 ( - ) .

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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 ( - ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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.


Chromosome parameters obtained for laggard - producing chromosomes are shown in A - D on the vertical axis , time in min on the horizontal axis relative to the anaphase II onset . Light blue lines indicate 12 laggard - producing chromosomes with correct ( equational ) chromatid segregation ; darker line highlights one chromosome with chromatid non - disjunction . 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 black dashed lines . Changes in the distance to the equator plane ( shown by a black arrowed line on the scheme ) for 13 laggard - producing chromosomes . All laggard - producing chromosomes are positioned above the equator at the start of analysis period . Black dotted lines indicate the average thickness of the metaphase plate . Changes in the distance to spindle axis ( shown by a black arrowed line on the scheme ) for 13 laggard - producing chromosomes . Black dotted line indicates the average radius of the metaphase plate . Changes in the angle to the spindle axis ( highlighted in black on the scheme ) for 13 laggard - producing chromosomes . Dashed line on the chart indicates the mean angle for normally segregating chromosomes . Changes in the inter - centromere distances ( shown by black arrowed line on the scheme ) for 13 laggard - producing chromosomes . Dashed line indicates the mean inter - centromere distance for normally segregating chromosomes .

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Chromosome parameters obtained for laggard-producing chromosomes are shown in A-D on the vertical axis, time in min on the horizontal axis relative to the anaphase II onset. Light blue lines indicate 12 laggard-producing chromosomes with correct (equational) chromatid segregation; darker line highlights one chromosome with chromatid non-disjunction. 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 black dashed lines. Changes in the distance to the equator plane (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. All laggard-producing chromosomes are positioned above the equator at the start of analysis period. Black dotted lines indicate the average thickness of the metaphase plate. Changes in the distance to spindle axis (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. Black dotted line indicates the average radius of the metaphase plate. Changes in the angle to the spindle axis (highlighted in black on the scheme) for 13 laggard-producing chromosomes. Dashed line on the chart indicates the mean angle for normally segregating chromosomes. Changes in the inter-centromere distances (shown by black arrowed line on the scheme) for 13 laggard-producing chromosomes. Dashed line indicates the mean inter-centromere distance for normally segregating chromosomes.


( a ) RT - PCR analysis of LC3 , IL - 8 , CHOP and GADD34 in U2OS cells subjected to glutamine deprivation ( − Q ) or exposed to TPG ( 1 μM ) . (

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(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). (


( b ) Immunoblot analysis of ER stress - and autophagy - related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment . Samples are run in triplicate .

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(b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate.


( c ) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence ( + Q ) or absence ( − Q ) of glutamine .

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(c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine.


( d ) IL - 8 enzyme - linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence ( + Q ) or absence ( − Q ) of glutamine . Error bars in all figures represent s . d . of three biological replicates .

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(d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.


( a ) Autophagosomes were visualized in U2OS cells stably expressing a GFP - LC3 reporter construct . GFP - labelled puncta were examined after 24 h in the presence ( + Q ) or absence ( − Q ) of glutamine , with or without co - addition of 100 nM CCI - 779 ( CCI ) and 400 nM BafA1 . Scale bar , 10 μm . ( b ) Graphical summary of experiments performed as described in a . Percentage of cells with > 10 puncta per cell from three independent experiments is depicted . Bars represent mean ± s . d . from three independent experiments ( > 50 cells per experiment ) . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . * P0 . 05 .

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(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05.


( c ) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1 . Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine - deficient medium . Autophagic activity was monitored by detection of p62 and LC3 - II proteins .

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(c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins.


d ) U2OS mCherry - GFP - LC3 cells were cultured in the presence ( + Q ) or absence ( − Q ) of glutamine for 18 h . Red vesicles denote autolysosomes , whereas yellow vesicles represent autophagosomes . Bars indicate numbers of yellow vesicles ( autophagosomes ) or red vesicles ( autolysosomes ) per cell ± s . d . ( e ) Images of U2OS mCherry - GFP - LC3 cells cultured for 18 h in the presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

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d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


( f ) Phagophore formation in mCherry - ULK1 . Scale bar , 10 μm . ( g ) mCherry - ATG5 - expressing U2OS cells after 24 h in the presence ( + Q ) or absence ( − Q ) of glutamine . Scale bar , 10 μm .

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(f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.


( a ) Colocalization of lysosomes ( Lysotracker Green ) with phagophores ( mCherry - ULK1 and mCherry - glutamine puncta ) in glutamine - deprived U2OS cells . Scale bar , 25 μm .

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(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm.


( b ) Colocalization of LAMP1 - positive lysosomes with GFP - LC3 - labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells . Scale bar , 15 μm .

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(b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm.


( c ) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment . Nuclei were stained with DAPI . Scale bar , 15 μm .

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(c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm.


( d ) Immunofluorescence images of cells stained with LAMP1 ( lysosomes ) and the Golgi marker , RCAS1 , following glutamine deprivation . Nuclei were stained with DAPI . Scale bar , 15 μm .

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(d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm.


( e ) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids . Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62 . (

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(e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. (


( f ) Immunofluorescence staining for LAMP1 ( green ) and mTOR ( red ) in U2OS cells subjected to glutamine deprivation ( 24 h ) , in the presence of 100 nM WYE - 125132 ( WYE - 132 ) . − AA denotes cells deprived of all amino acids . Scale bar , 15 μm . Error bars in all figures represent s . d . of three biological replicates . Ess . , essential .

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(f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.


( a ) Immunoblot of autophagy - related proteins in U2OS cells expressing a control TALEN ( U2OS - TAL2 - C1 ) or two independent subcloned TALEN cell lines targeting ATG7 ( U2OS - TAL2 - A7 , U2OS - TAL2 - A8 ) . p62 long and short refer to either long or short exposure times during film development .

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(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development.


( b ) U2OS cells expressing an ATG7 - TALEN ( TAL2 - ATG7 ) or control TALEN ( TAL2 - CTL ) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine - replete medium . Cells were then stained with SRB and absorbance was read at 540 nM . Graph shows average results from five clonal sub - lines for each condition . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . P0 . 05 . ( c ) Representative images of SRB - stained clones in

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(b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in b


d ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of U2OS - TAL2 - CTL - C1 ( control ) and U2OS - TAL2 - ATG7 - A8 ( ATG7 knockout ) following 24 h glutamine deprivation .

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d) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.


( e ) IL - 8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE - 125132 . Scale bar , 15 μm .

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(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.


( b ) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE - 125132 .

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(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.


( c ) IL - 8 enzyme - linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 .

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(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.


( d ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) Immunoblot analysis of autophagy , ER stress - and mTOR - related proteins , and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 .

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(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.


( b ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK ( JNK # 1 and JNK # 2 ) and subjected to 24 h glutamine deprivation .

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(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.


( c ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of conditioned media from the experiment described in b .

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(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.


( d ) Immunoblot analysis of JNK protein expression in JNK siRNA - treated cells .

(d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells.


e ) RT - PCR analysis of IL - 8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 ( JNKi ) .

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e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).


( f ) IL - 8 ELISA of conditioned medium from the experiment described in e .

(f) IL-8 ELISA of conditioned medium from the experiment described in e.


( g ) IL - 8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 ( IRE - A and IRE - B ) and subjected to glutamine deprivation for 24 h .

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(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.


( h ) RT - PCR analysis of IL - 8 mRNA expression from experiment described in g . ( i ) RT - PCR analysis of CHOP mRNA expression from experiment described in g . ( j ) RT - PCR analysis of IRE1 mRNA expression from experiment described in g .

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(h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g.


( k ) Immunoblot analysis of experiment described in g showing phosphorylation of JNK . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


( a ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) on U2OS cells starved of glutamine or treated with BPTES ( 10 μM ) for 24 h .

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(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.


( b ) RT - PCR analysis of IL - 8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG .

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(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.


( c ) Metabolite analysis of U2OS cells starved of glutamine , treated with BPTES or glutamine - starved co - treated with DMαKG .

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(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.


( d ) IL - 8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co - treated with DMαKG .

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(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.


( e ) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


Representative density plots of Ca2 + flux from conjugated OptoCAR ‑ T cells over time at 37ºC . Conjugated cells were stimulated with dimerizer addition for 0 ( F ) , 5 ( G ) , 10 ( H ) , 15 ( I ) or 30 ( J ) minutes prior to disrupting signaling by illuminating cells .

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Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.


OptoCAR ‑ T cells were conjugated with ligand - presenting cells and activated as in the upper regime shown in ( A ) . The median of ppERK intensity distribution was plotted with time , after subtraction of background intensity . Bars show mean ± SEM ( n = 3 ) . An equivalent experiment was performed as in ( B ) but following the middle regime shown in ( A ) , with sample illumination after 5 minutes for the remainder of the dataset . Indicated half - life was calculated from the time taken for the median ppERK to decrease to 50 % of maximum . Bars show mean ± SEM ( n = 4 ) . An equivalent experiment was performed as in ( B ) but following the lower regime shown in ( A ) , with sample illumination after 5 minutes for 3 minutes . Bars show mean ± SEM ( n = 3 ) .

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OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).


Representative fluorescent Western blot showing the dynamics of FOS phosphorylation ( p - FOSS32 ) on light - modulated control . Conjugated OptoCAR ‑ T cells were stimulated for a defined period after dimerizer addition ( denoted above blots ) before either being left in the dark ( dark line ) or illuminated ( blue line ) . Full blot image with total protein normalization control shown in Figure EV3A . Representative fluorescent Western blot showing the dynamics of FOS expression ( FOSTotal ) on light - modulated control , from the same dataset as in ( A ) . Full blot image with total protein normalization control shown in Figure EV3B .

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Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A. Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.


RT - qPCR analysis of relative mRNA levels for four representative genes following OptoCAR - mediated T cell activation . OptoCAR ‑ T cell conjugates were activated in the dark ( signaling competent ) state for a period of 3 hours before light - mediated disruption of signaling ( open circles ) or maintained for a further 60 minutes in the dark ( filled circles ) . The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3 - 4 hours . Individual datasets are presented as gray lines , with bars showing mean ± SEM ( n = 3 ) . Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1 .

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RT-qPCR analysis of relative mRNA levels for four representative genes following OptoCAR-mediated T cell activation. OptoCAR‑T cell conjugates were activated in the dark (signaling competent) state for a period of 3 hours before light-mediated disruption of signaling (open circles) or maintained for a further 60 minutes in the dark (filled circles). The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3-4 hours. Individual datasets are presented as gray lines, with bars showing mean ± SEM (n=3). Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1.


Single pulses of varying duration were applied to OptoCAR ‑ T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling , with illumination started once the signal pulse had ended . All values have been corrected for background GFP fluorescence from non - activated OptoCAR ‑ T cells and shown relative to the output at 24 hours . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 ) Equivalent datasets as in ( B ) for the geometric mean of CD69 upregulation . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 )

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Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)


The cRQ values from RT - qPCR of indicated cells . Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells . Conjugated OptoCARCD19 ‑ T cells were also assayed either before or after 24 hours stimulation in the dark state . Individual values from three biological replicates are shown , with mean depicted by bar plot .

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The cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot.


Determination of maximum growth rate ( Vmax ) using a label - free cell count - based proliferation assay . Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi - log scale displaying total numbers of cells over time . Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints .

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Determination of maximum growth rate (Vmax) using a label-free cell count-based proliferation assay. Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi-log scale displaying total numbers of cells over time. Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints.