A . PINK1 antagonizes OTULIN activity in cells . HEK293T cells expressing HOIP and HOIL - 1L , were transfected with PINK1 , WT OTULIN or the inactive OTULIN mutant W96A , as indicated . The cells were lysed 24 h later under denaturing conditions , and lysates were subjected to affinity purification using the Strep - tagged UBAN domain of NEMO to enrich proteins modified with M1 - linked ubiquitin . Proteins affinity - purified by Strep - Tactin beads were immunoblotted against ubiquitin .

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

A. PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L, were transfected with PINK1, WT OTULIN or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.


B . Catalytically active PINK1 increases p - S65 - ubiquitin at mitochondria . HEK293T cells were transfected with wildtype PINK1 or kinase - dead ( K / D ) PINK1 . 48 h after transfection , the cells were treated with TNF ( 25 ng / ml , 15 min ) and lysed . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p - S65 - ubiquitin , PINK1 , and VDAC ( loading control ) .

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B. Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. 48 h after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).


C . The TNF - induced increase in p - S65 - ubiquitin is abolished in PINK1 - deficient cells . HEK293T cells were transfected with control or PINK1 - specific siRNA and treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) 48 h after transfection . The whole cell lysates were analyzed by immunoblotting using the indicated antibodies .

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

C. The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.


D . TNF - induced M1 ubiquitination is reduced in PINK1 - deficient cells . WT and PINK1 - KO MEFs were treated with TNF ( 25 ng / ml , 15 min ) and then harvested . Purified mitochondrial fractions were subjected to affinity purification using the Strep - tagged UBAN domain , as described in A , followed by immunoblotting using M1 - ubiquitin - specifc antibodies , Strep - Tactin conjugated to horse radish peroxidase ( to control the UBAN pulldown efficiency ) , and TIM23 ( input control ) . Quantification of the M1 - ubiquitin - positive signal intensities is shown in the lower panel . Data represent the mean values with standard deviations of 3 independent experiments ; n . s . not significant , * * * p < 0 . 001 . A two - way ANOVA test was used to analyze statistical significance .

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

D. TNF-induced M1 ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in A, followed by immunoblotting using M1-ubiquitin-specifc antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of 3 independent experiments; n.s. not significant, ***p < 0.001. A two-way ANOVA test was used to analyze statistical significance.


A . STS - induced mitochondrial Bax recruitment is reduced by TNF . HeLa cells were treated with STS ( 1 μM , 1 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) and then harvested . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1 - ubiquitin . The input was immunoblotted for TIM23 ( upper panel ) . Bax - specific signal intensities were quantified and normalized to TIM23 - specific signals ( lower panel ) .

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

A. STS-induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 μM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1-ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax-specific signal intensities were quantified and normalized to TIM23-specific signals (lower panel).


B . STS - induced cytochrome C release is decreased by TNF . HeLa cells were treated as described in A . The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies . GAPDH was used as a reference . Quantification of 5 biological replicates is shown in the lower panel . Signal intensities were quantified and normalized to that of GAPDH . * p < 0 . 05 . Kolmogorow - Smirnov normality test , paired t - test , two - tailed , n = 5 .

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

B. STS-induced cytochrome C release is decreased by TNF. HeLa cells were treated as described in A. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of 5 biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. *p < 0.05. Kolmogorow-Smirnov normality test, paired t-test, two-tailed, n=5.


C , D . TNF treatment prevents damage of mitochondrial cristae under pro - apoptotic conditions . C . SH - SY5Y cells were treated as described in A , fixed , embedded and the mitochondrial ultrastructure was imaged by electron microscopy . Scale bar : 400 nm . D . Cristae abundance ( average cristae number per mitochondrial area ) and cristae length ( average cristae length per mitochondrium ) were analyzed by Imaris 9 . 8 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 . Cristae abundance : Shapiro - Wilk normality test , One - way ANOVA followed by Tukey ' s multiple comparison test , n = 27 . Cristae length : Kruskal - Wallis test followed by Dunn ' s multiple comparison test , n = 24 - 27 . Bars represent mean ±

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

C, D. TNF treatment prevents damage of mitochondrial cristae under pro-apoptotic conditions. C. SH-SY5Y cells were treated as described in A, fixed, embedded and the mitochondrial ultrastructure was imaged by electron microscopy. Scale bar: 400 nm. D. Cristae abundance (average cristae number per mitochondrial area) and cristae length (average cristae length per mitochondrium) were analyzed by Imaris 9.8. *p < 0.05, **p < 0.01, ***p < 0.01. Cristae abundance: Shapiro-Wilk normality test, One-way ANOVA followed by Tukey's multiple comparison test, n = 27. Cristae length: Kruskal-Wallis test followed by Dunn's multiple comparison test, n=24-27. Bars represent mean ± SEM


E . The fast anti - apoptotic effect of TNF is not affected by the NF - κΒ inhibitor IκΒα . SH - SY5Y cells were transiently transfected with the NF - κΒ super - repressor IᴋBα - 2S or luciferase as a control . 24 h later , the cells were treated with STS ( 5 µM , 2 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) . Cells were fixed and stained by antibodies against active caspase - 3 . Signal intensities were quantified by immunocytochemistry and fluorescence microscopy . Expression of IᴋBα - 2S was tested by immunoblotting using antibodies against the HA tag . Data represent the standard deviation of three independent experiments ; at least 300 cells were counted per experiment . * p < 0 . 05 , * * p < 0 . 01 . Student ' s t - test , two - tailed , n = 3 .

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

E. The fast anti-apoptotic effect of TNF is not affected by the NF-κΒ inhibitor IκΒα. SH-SY5Y cells were transiently transfected with the NF-κΒ super-repressor IᴋBα-2S or luciferase as a control. 24 h later, the cells were treated with STS (5 µM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase-3. Signal intensities were quantified by immunocytochemistry and fluorescence microscopy. Expression of IᴋBα-2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. *p < 0.05, **p < 0.01. Student's t-test, two-tailed, n=3.


F . The protective effect of TNF is dependent on HOIP expression . HeLa cells were transiently transfected with control or HOIP siRNA . 48 h after transfection , cells were treated with STS ( 1 μM , 1 h ) with or without a 15 min TNF pretreatment ( 25 ng / ml ) and then harvested . The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies . HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP . GAPDH and actin were immunoblotted as input controls .

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

F. The protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. 48 h after transfection, cells were treated with STS (1 μM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls.


G . The protective effect of TNF is dependent on PINK1 expression . HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F . The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies . GAPDH was immunoblotted as input control .

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G. The protective effect of TNF is dependent on PINK1 expression. HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F. The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies. GAPDH was immunoblotted as input control.


A . NF - κB signaling components are recruited to mitochondria upon TNF treatment . A . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) , harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated ; p - p65 : phospho - S536 - p65 ; p - TBK1 : phospho - S172 - TBK1 .

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

A. NF-κB signaling components are recruited to mitochondria upon TNF treatment. A. HEK293T cells were treated with TNF (25 ng/ml, 15 min), harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated; p-p65: phospho-S536-p65; p-TBK1: phospho-S172-TBK1.


B . Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment . Representative immunofluorescence images of SH - SY5Y cells using the proximity ligation assay ( PLA ) between phospho - S536 - p65 ( rabbit ) and TOM20 ( mouse ) couple antibodies . One set of cells were treated with TNF ( 25 ng / ml , 15 min ) before fixation . Nuclei were stained with DAPI , mitochondria were stained with MitoTrackerTM Red CMXRos ( red ) , and the PLA amplification reaction was visualized by green foci . As a positive control , fixed cells were incubated with primary TOM20 ( mouse ) and TOM70 ( rabbit ) antibodies and subjected to the PLA assay . As negative controls , fixed cells were incubated with either TOM20 or p - p65 antibodies prior to the PLA assay .

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

B. Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH-SY5Y cells using the proximity ligation assay (PLA) between phospho-S536-p65 (rabbit) and TOM20 (mouse) couple antibodies. One set of cells were treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTrackerTM Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p-p65 antibodies prior to the PLA assay.


C . Quantification of the phospho - S536 - p65 / TOM20 PLA foci per cells . Data represent the number of PLA foci per cell ( mean ± SD ) ; at least 84 cells in total were analyzed per condition . * * * * p < 0 . 0001 . A two - tailed non - parametric Mann - Whitney test was used to analyze statistical significance .

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C. Quantification of the phospho-S536-p65/TOM20 PLA foci per cells. Data represent the number of PLA foci per cell (mean ±SD); at least 84 cells in total were analyzed per condition. ****p < 0.0001. A two-tailed non-parametric Mann-Whitney test was used to analyze statistical significance.


A . Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression . SH - SY5Y cells were transfected with control or PINK1 - specific siRNAs . For rescue experiments , cells were co - transfected with wildtype PINK1 , PINK1Δ77 , or kinase - dead PINK1 - K / D . Two days after transfection the cells were treated with TNF ( 25 ng / ml , 25 min ) , fixed and stained with p65 antibodies . The fraction of cells showing nuclear translocation of p65 was determined for each condition . Data represent the mean ± SEM of 3 independent experiments . The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition .

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

A. Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH-SY5Y cells were transfected with control or PINK1-specific siRNAs. For rescue experiments, cells were co-transfected with wildtype PINK1, PINK1Δ77, or kinase-dead PINK1-K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of 3 independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition.


B . PINK1 phosphorylates ubiquitinated NEMO . HEK293T cells were co - transfected with V5 - tagged wildtype PINK1 or kinase - dead PINK1 - K / D and HA - tagged NEMO . One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA . Precipitated proteins were then detected by immunoblotting using p - S65 - ubiquitin antibodies . The input was immunoblotted for NEMO , PINK1 , p - S65 - ubiquitin , and GAPDH .

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

B. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.


C . Linear ubiquitination of NEMO is reduced in PINK1 - deficient cells . HEK293T cells were co - transfected with HA - NEMO and control or PINK1 - specific siRNAs . 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA . Precipitated proteins were then detected by immunoblotting using M1 - ubiquitin antibodies . The input was immunoblotted for NEMO and actin . PINK1 silencing efficiency was determined by real - time RT - PCR . Bars represent mean ± SD with three technical replicates .

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

C. Linear ubiquitination of NEMO is reduced in PINK1-deficient cells. HEK293T cells were co-transfected with HA-NEMO and control or PINK1-specific siRNAs. 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1-ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real-time RT-PCR. Bars represent mean± SD with three technical replicates.


A . TNF induces the movement of peripheral mitochondria towards the nucleus . Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF ( 25 ng / ml ) . The motility of mitochondria was analyzed using Imaris 9 . 8 spot function . The graphical representation of the top 25 % peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment . Exemplified dataset shows the median distance with the upper and lower quartile ( shaded area ) of n = 105 - 187 mitochondrial spots per time point .

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

A. TNF induces the movement of peripheral mitochondria towards the nucleus. Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF (25 ng/ml). The motility of mitochondria was analyzed using Imaris 9.8 spot function. The graphical representation of the top 25% peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment. Exemplified dataset shows the median distance with the upper and lower quartile (shaded area) of n=105-187 mitochondrial spots per time point.


B . Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1 / 2 expression . HeLa cells were transfected with control or Miro1 and Miro2 siRNAs . 48 h later , cells were treated with TNF ( 25 ng / ml , 15 min ) , fixed and stained using antibodies against p65 and tubulin and DAPI . Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4 . 2 . 1 . Quantification is based on 3 biological replicates . Kruskal - Wallis test followed by Dunn ' s multiple comparison test , n = 79 - 157 , * p < 0 . 05 .

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

B. Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1/2 expression. HeLa cells were transfected with control or Miro1 and Miro2 siRNAs. 48 h later, cells were treated with TNF (25 ng/ml, 15 min), fixed and stained using antibodies against p65 and tubulin and DAPI. Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4.2.1. Quantification is based on 3 biological replicates. Kruskal-Wallis test followed by Dunn's multiple comparison test, n=79-157, *p < 0.05.


E , F , G . TNF increases mitochondria - nucleus contact sites . Representative immunofluorescence images ( E , F ) and quantification ( G ) of mitochondria - nucleus contact sites in HeLa cells transfected with the SPLICSNU - MT and treated with 25 ng / ml TNF or PBS for 15 min . TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation . Nuclei were stained with Hoechst33342 ( ThermoFisher , 1 μg / ml ) . Unpaired two - tailed t - test , n = 43 ( control ) or 51 ( TNF ) ,

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E, F, G. TNF increases mitochondria-nucleus contact sites. Representative immunofluorescence images (E, F) and quantification (G) of mitochondria-nucleus contact sites in HeLa cells transfected with the SPLICSNU-MT and treated with 25 ng/ml TNF or PBS for 15 min. TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation. Nuclei were stained with Hoechst33342 (ThermoFisher, 1 μg/ml). Unpaired two-tailed t-test, n=43 (control) or 51 (TNF),


H , I , J . TNF increases mitochondria - nucleus contact sites in a HOIP - dependent manner . Representative immunofluorescence images ( H , I ) and quantification ( J ) of mitochondria - nucleus contact sites in control or HOIP - silenced ( HOIP siRNA ) HeLa cells transfected with the SPLICSNU - MT probe treated with 25 ng / ml TNF or PBS for 15 minutes . Mitochondria and nuclei were stained as described in E and F .

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H, I, J. TNF increases mitochondria-nucleus contact sites in a HOIP-dependent manner. Representative immunofluorescence images (H, I) and quantification (J) of mitochondria-nucleus contact sites in control or HOIP-silenced (HOIP siRNA) HeLa cells transfected with the SPLICSNU-MT probe treated with 25 ng/ml TNF or PBS for 15 minutes. Mitochondria and nuclei were stained as described in E and F.


( C ) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 ( ACT2 ) as a loading control .

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(C) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control.


( D ) qPCR quantification of shGAG and flGAG - POL normalized to ACT2 and to GLYCERALDEHYDE - 3 - PHOSPHATE DEHYDROGENASE C SUBUNIT ( GAPC ) levels . qPCR was performed on n = 3 biological replicates ; bars : standard error . ( * * ) = p - value < 0 . 01 ( two - sided t - test between indicated values ) .

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(D) qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n=3 biological replicates; bars: standard error. (**) = p-value < 0.01 (two-sided t-test between indicated values).


( E ) Western analysis of GAG and RDR6 with Coomassie ( coom . ) staining as a loading control . Arrow indicates cognate RDR6 protein band .

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(E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band.


( F - G ) sRNA - seq profiles from EVD de - repressed in the ddm1 ( F ) or ddm1 dcl1 ( G ) backgrounds . Different siRNA size categories are stacked . Nomenclature as in ( B ) .

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(F-G) sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B).


( B ) Expression levels of shGFP ( spliced ) and GFP - EVDint / ter - GUS ( unspliced ) transcripts , relative to ACT2 and AT4G26410 ( RHIP1 ) , in the WT or rdr6 background . qPCR was performed on three biological replicates and error bars represent the standard error on . ( * ) = p - value < 0 . 05 ( two - sided t - test against corresponding controls ) .

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(B) Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. (*) = p-value < 0.05 (two-sided t-test against corresponding controls).


( C ) sRNA - seq profile mapped on the genomic 35S : GFP - EVDint / ter - GUS locus . ( RPM ) Reads per million . Positions indicated in nucleotides ( nt ) from the start of the 35S sequence . Dashed vertical lines : shGFP and GFP - GUS 3 ' ends .

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(C) sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends.


( D ) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2 . qPCR was performed on three biological replicates and error bars represent the standard error . ( ns . ) = non - significant , ( * ) = p - value < 0 . 05 , ( * * ) = p - value < 0 . 01 , ( two - sided t - test between indicated samples / targets ) .

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(D) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2. qPCR was performed on three biological replicates and error bars represent the standard error. (ns.) = non-significant, (*) = p-value < 0.05, (**) = p-value < 0.01, (two-sided t-test between indicated samples/targets).


( E ) High and low molecular - weight RNA analysis of EVD GAG ( GAG - ex1 ) and EVD intron ( GAG - in ) in two independent T1 bulks from each indicated line . The filled arrows on the right hand - side or with an asterisk on the blots correspond to the transcripts depicted in ( A - C ) . ACT2 : loading control for mRNAs ; tasiR255 , miR173 and U6 : loading controls for sRNAs . Hybridizations for GAG - ex2 and POL probes are found in Supp . Fig . 5A .

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(E) High and low molecular-weight RNA analysis of EVD GAG (GAG-ex1) and EVD intron (GAG-in) in two independent T1 bulks from each indicated line. The filled arrows on the right hand-side or with an asterisk on the blots correspond to the transcripts depicted in (A-C). ACT2: loading control for mRNAs; tasiR255, miR173 and U6: loading controls for sRNAs. Hybridizations for GAG-ex2 and POL probes are found in Supp. Fig.5A.


( B ) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in ( A ) .

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(B) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in (A).


( A ) Comparison of RNA isoforms and sRNA patterns generated by 35S : GFP - GUS and 35S : GFP - EVDint / ter - GUS . High and low molecular - weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background . mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig . 2A . EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs , respectively .

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(A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively.


( B ) . Western blot analysis of the translation products from GFP and GFP - GUS transcripts . Coomassie ( coom . ) staining as a loading control . Black arrow : GFP - GUS fusion protein ; white arrow : GFP protein .

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(B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein.


( C ) Nucleo - cytosolic distribution of 35S : EVD and 35S : GFP - EVDint / ter - GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR . RNA extracted from Total , nuclear ( Nucl ) and cytoplasmic ( Cyto ) fractions was reverse transcribed with random hexamers and oligo ( dT ) . snoRNA U5 is shown as a nuclear - only RNA control . ( D ) Same as in ( C ) but using exclusively oligo ( dT ) to reverse transcribe poly ( A ) + RNAs .

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(C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control. (D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.


( B ) RIBO - seq coverage profiles from 35S : EVD in WT or rdr6 . RPM : Reads per million . ( C ) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P - sites to calculate codon coverage . The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence .

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(B) RIBO-seq coverage profiles from 35S:EVD in WT or rdr6. RPM: Reads per million. (C) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P-sites to calculate codon coverage. The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence.


C . Autoradiograph of hTrmt13 - RNA complexes in the MDA - MB - 231 cells immunoprecipitated with an antibody against hTrmt13 ( right lane ) and IgG as a negative control ( left lane ) . * RNA residing in the regions between marked lines was recovered for sequencing .

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C. Autoradiograph of hTrmt13-RNA complexes in the MDA-MB-231 cells immunoprecipitated with an antibody against hTrmt13 (right lane) and IgG as a negative control (left lane). * RNA residing in the regions between marked lines was recovered for sequencing.


H . Mass chromatograms of the nucleosides , Um ( Q1 / Q3 = 259 . 1 / 113 . 1 ) , Cm ( Q1 / Q3 = 258 . 1 / 112 . 1 ) , and G ( Q1 / Q3 = 284 . 1 / 152 . 2 ) of HctRNAGly ( GCC ) , HctRNAGly ( CCC ) , HctRNAGly ( UCC ) , and HctRNAPros isolated from WT and hTrmt13 KO cells . Um , 2 ′ - O - methyluridine , Cm , 2 ′ - O - methylcytidine . Q1 / Q3 : the mass of the precursor ion and the mass of the product ion . These endogenous tRNAs were purified separately from total cellular RNAs using sequence - specific probes .

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H. Mass chromatograms of the nucleosides, Um (Q1/Q3 = 259.1/113.1), Cm (Q1/Q3 = 258.1/112.1), and G (Q1/Q3 = 284.1/152.2) of HctRNAGly(GCC), HctRNAGly(CCC), HctRNAGly(UCC), and HctRNAPros isolated from WT and hTrmt13 KO cells. Um, 2′-O-methyluridine, Cm, 2′-O-methylcytidine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. These endogenous tRNAs were purified separately from total cellular RNAs using sequence-specific probes.


I . Quantification of the Um / G and Cm / G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells . Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for three independent experiments .

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I. Quantification of the Um/G and Cm/G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.


A . De novo protein synthesis measured by puromycin incorporation in shSCR , hTrmt13 knockdown cells ( sh - 1 and sh - 2 ) , and sh - 1 with stable expression of wild type hTrmt13 ( sh - 1 + hTrmt13 ) or enzymatically - inactive hTrmt13 ( sh - 1 + E463A ) . Puromycin - labeled proteins were detected by immunoblotting . Representative images ( left ) and statistical analysis ( right ) are shown . Knockdown and overexpression efficiency of hTrmt13 in MDA - MB - 231 verified by Western blots . Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments .

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

A. De novo protein synthesis measured by puromycin incorporation in shSCR, hTrmt13 knockdown cells (sh-1 and sh-2), and sh-1 with stable expression of wild type hTrmt13 (sh-1+hTrmt13) or enzymatically-inactive hTrmt13 (sh-1+E463A). Puromycin-labeled proteins were detected by immunoblotting. Representative images (left) and statistical analysis (right) are shown. Knockdown and overexpression efficiency of hTrmt13 in MDA-MB-231 verified by Western blots. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.


C . The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot . The samples were also deacylated ( DA ) by incubating with alkaline buffer ( pH 9 . 0 ) to obtain fully uncharged tRNA control .

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C. The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot. The samples were also deacylated (DA) by incubating with alkaline buffer (pH 9.0) to obtain fully uncharged tRNA control.


D . RT - qPCR using a stem - loop primer to quantify the tRFs levels in shSCR , sh - 1 , and sh - 2 . Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments .

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D. RT-qPCR using a stem-loop primer to quantify the tRFs levels in shSCR, sh-1, and sh-2. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.


G . Quantification of de novo protein synthesis in cells treated with different concentration of tRFs H . Quantification of de novo protein synthesis in cells treated with different concentration of tRNAGly ( CCC ) Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments .

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G. Quantification of de novo protein synthesis in cells treated with different concentration of tRFs H. Quantification of de novo protein synthesis in cells treated with different concentration of tRNAGly(CCC) Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.


A , B . Representative images showing endogenous hTrmt13 ( A ) and HA - tagged hTrmt13 ( HA - hTrmt13 , B ) subcellular localization by confocal microscopy analysis . DAPI staining was included to visualize the cell nucleus ( Blue ) .

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A, B. Representative images showing endogenous hTrmt13 (A) and HA-tagged hTrmt13 (HA-hTrmt13, B) subcellular localization by confocal microscopy analysis. DAPI staining was included to visualize the cell nucleus (Blue).


C . Representative confocal microscopy images showing the subcellular localization of hTrmt13 in MDA - MB - 231 cells cultured with 10 % FBS ( left ) or no FBS ( right ) . DAPI staining was included to visualize the cell nucleus ( Blue ) .

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C. Representative confocal microscopy images showing the subcellular localization of hTrmt13 in MDA-MB-231 cells cultured with 10% FBS (left) or no FBS (right). DAPI staining was included to visualize the cell nucleus (Blue).


E . Confocal microscopy images of MDA - MB - 231 cells were co - immunolabeled with antibodies against hTrmt13 and histone H3 ( left ) . The images were captured by GE DeltaVision OMX SR . Co - localization of H3 and hTrmt13 was analyzed by ImageJ ( right , n = 20 cells , center line , the median of the data ) . DAPI staining was included to visualize the cell nucleus ( Blue ) .

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E. Confocal microscopy images of MDA-MB-231 cells were co-immunolabeled with antibodies against hTrmt13 and histone H3 (left). The images were captured by GE DeltaVision OMX SR. Co-localization of H3 and hTrmt13 was analyzed by ImageJ (right, n = 20 cells, center line, the median of the data). DAPI staining was included to visualize the cell nucleus (Blue).


I . The binding profiles of hTrmt13 , H3K4me3 , H3K27Ac , and other transcription factors ( MYC , E2F1 , TP53 , FOSL1 , EZH2 , FOXM1 ) from ChIP - seq and ATAC - seq on representative target genes TGFB1 , MEF2A , and PKN2 . hTrmt13 track were average across 2 biological replicates .

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I. The binding profiles of hTrmt13, H3K4me3, H3K27Ac, and other transcription factors (MYC, E2F1, TP53, FOSL1, EZH2, FOXM1) from ChIP-seq and ATAC-seq on representative target genes TGFB1, MEF2A, and PKN2. hTrmt13 track were average across 2 biological replicates.


N . EMSA of hTrmt13 for synthesized DNA ( 0 . 25 μM ) carrying consensus motif or mutated motif .

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N. EMSA of hTrmt13 for synthesized DNA (0.25 μM) carrying consensus motif or mutated motif.


B . Transwell migration assays in MDA - MB - 231 cells . Control with siRNA scramble ( NC ) , treated with siRNA against hTrmt13 ( si - 1 , si - 2 ) and stably expressing wild - type hTrmt13 ( si - 1 + WT ) or enzymatic inactive hTrmt13 mutant ( si - 1 + E463A ) . Representative images ( left panel ) and statistical analysis ( middle panel ) are shown . Data information : for each group , five different fields were chosen and counted using a microscope with 20 - fold magnification . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for four independent experiments .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transwell migration assays in MDA-MB-231 cells. Control with siRNA scramble (NC), treated with siRNA against hTrmt13 (si-1, si-2) and stably expressing wild-type hTrmt13 (si-1+WT) or enzymatic inactive hTrmt13 mutant (si-1+E463A). Representative images (left panel) and statistical analysis (middle panel) are shown. Data information: for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for four independent experiments.


C . Transwell migration assays in HCC38 cells infected with scrambled siRNA ( NC ) or siRNA against hTrmt13 ( si - 1 , si - 2 ) . Data information : for each group , five different fields were chosen and counted using a microscope with 20 - fold magnification . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for three independent experiments .

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

C. Transwell migration assays in HCC38 cells infected with scrambled siRNA (NC) or siRNA against hTrmt13 (si-1, si-2). Data information: for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.


E . Volcano plot of differential expressed genes analysis results for RNA - seq data comparing hTrmt13 knockdown MDA - MB - 231 cells ( N = 2 ) to control cells ( N = 2 ) , the dots for significant regulated genes ( FDR corrected p - value < 0 . 05 ) were colored red , the dots and labels of interesting genes with oncogenic implications have been colored in blue or red .

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

E. Volcano plot of differential expressed genes analysis results for RNA-seq data comparing hTrmt13 knockdown MDA-MB-231 cells (N=2) to control cells (N=2), the dots for significant regulated genes (FDR corrected p-value < 0.05) were colored red, the dots and labels of interesting genes with oncogenic implications have been colored in blue or red.


B . The mRNA level of the indicated genes was measured by RT - qPCR . Data information : statistical analysis was performed using t - tests , and the error bars indicate mean ± SD for three independent experiments .

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

B. The mRNA level of the indicated genes was measured by RT-qPCR. Data information: statistical analysis was performed using t-tests, and the error bars indicate mean ± SD for three independent experiments.


B . Interaction of FLAG - hTrmt13 with endogenous FOSL1 , E2F1 , and USF1 . Whole - cell lysates from MDA - MB - 231 cells stably expressing FLAG - hTrmt13 were prepared and immunoprecipitation was performed with anti - FLAG followed by immunoblotting with antibodies against indicated proteins .

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

B. Interaction of FLAG-hTrmt13 with endogenous FOSL1, E2F1, and USF1. Whole-cell lysates from MDA-MB-231 cells stably expressing FLAG-hTrmt13 were prepared and immunoprecipitation was performed with anti-FLAG followed by immunoblotting with antibodies against indicated proteins.


F . ChIP - qPCR analysis of the selected promoters was performed using antibodies against indicated proteins in control and hTrmt13 knockdown MDA - MB - 231 cells . Results are represented as fold change over control IgG . Statistical analysis was performed using t - tests . Error bars indicate mean ± SD for three independent experiments .

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

F. ChIP-qPCR analysis of the selected promoters was performed using antibodies against indicated proteins in control and hTrmt13 knockdown MDA-MB-231 cells. Results are represented as fold change over control IgG. Statistical analysis was performed using t-tests. Error bars indicate mean ± SD for three independent experiments.


C . The binding shift assays of hTrmt13 , USF1 , and their mixture for synthesized DNA from the TGFB1 promoter . In the reaction system , 10 ng / μL DNA was used , and 1 μM hTrmt13 or 1 μM USF1 or a mixture ( 0 . 5 μM hTrmt13 + 0 . 5 μM USF1 ) was applied . D . The binding shift assays of hTrmt13 for HctRNAGly ( GCC ) with the presence of various concentrations of USF1 . In the reaction system , 0 . 3 μM HctRNAGly ( GCC ) and 0 . 5 μM hTrmt13 were used , and a varied amount of USF1 ( 0 , 0 . 5 , 1 , 2 , 3 , 4 μM ) were used . E . The binding shift assays of hTrmt13 ( 0 . 5 μM ) , or USF1 ( 0 . 5 μM ) or the hTrmt13 - USF1 complex ( 0 . 5 μM ) for both tRNA ( 0 . 1 μM ) and DNA ( 15 ng / μL ) in the same reaction .

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

C. The binding shift assays of hTrmt13, USF1, and their mixture for synthesized DNA from the TGFB1 promoter. In the reaction system, 10 ng/μL DNA was used, and 1 μM hTrmt13 or 1 μM USF1 or a mixture (0.5 μM hTrmt13 + 0.5 μM USF1) was applied. D. The binding shift assays of hTrmt13 for HctRNAGly(GCC) with the presence of various concentrations of USF1. In the reaction system, 0.3 μM HctRNAGly(GCC) and 0.5 μM hTrmt13 were used, and a varied amount of USF1 (0, 0.5, 1, 2, 3, 4 μM) were used. E. The binding shift assays of hTrmt13 (0.5 μM), or USF1 (0.5 μM) or the hTrmt13-USF1 complex (0.5 μM) for both tRNA (0.1 μM) and DNA (15 ng/μL) in the same reaction.


A . Volcano plot of differential expressed genes analysis results for RNA - seq data on TCGA comparing hTrmt13 expression low group patients ( first quartile ) versus high group patients ( fourth quartile ) . Significant differentially expressed genes ( FDR corrected p - value < 0 . 05 ) were colored by red , interesting genes with oncogenic implications are labelled in blue . Pseudo values 1e - 15 were added to FDR correlated p - value for better visualization .

O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Volcano plot of differential expressed genes analysis results for RNA-seq data on TCGA comparing hTrmt13 expression low group patients (first quartile) versus high group patients (fourth quartile). Significant differentially expressed genes (FDR corrected p-value < 0.05) were colored by red, interesting genes with oncogenic implications are labelled in blue. Pseudo values 1e-15 were added to FDR correlated p-value for better visualization.


E . Immunohistochemical staining of tissue arrays containing 24 breast carcinoma samples paired with adjacent normal mammary tissues for hTrmt13 expression . Representative images are shown ( left ) , and the mean staining intensity was calculate using Image - Pro Plus software ( right ) . Statistical analysis was performed using t - tests .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE I-DISEASE O O O O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

E. Immunohistochemical staining of tissue arrays containing 24 breast carcinoma samples paired with adjacent normal mammary tissues for hTrmt13 expression. Representative images are shown (left), and the mean staining intensity was calculate using Image-Pro Plus software (right). Statistical analysis was performed using t-tests.


F . Kaplan - Meier survival analysis of Kaplan - Meier plotter ( http : / / kmplot . com / analysis / ) for the relationship between the relapse - free survival ( RFS ) or overall survival time ( OS ) of breast cancer patients ( BRCA , n = 349 ( low ) / 205 ( high ) ) , liver cancer patients ( LIHC , n = 270 ( low ) / 100 ( high ) ) , patients with kidney renal clear cell carcinoma ( KRIC , n = 156 ( low ) / 374 ( high ) ) , and patients with kidney renal papillary cell carcinoma ( KIRP , n = 208 ( low ) / 79 ( high ) ) and their expression of hTrmt13 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-DISEASE I-DISEASE B-ORGANISM O B-DISEASE O O O O O O O O O O O O O O B-DISEASE I-DISEASE B-ORGANISM O B-DISEASE O O O O O O O O O O O O O O B-ORGANISM O B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O O B-ORGANISM O B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O

F. Kaplan-Meier survival analysis of Kaplan-Meier plotter (http://kmplot.com/analysis/) for the relationship between the relapse-free survival (RFS) or overall survival time (OS) of breast cancer patients (BRCA, n=349 (low)/205 (high)), liver cancer patients (LIHC, n=270 (low)/100 (high)), patients with kidney renal clear cell carcinoma (KRIC, n=156 (low)/374 (high)), and patients with kidney renal papillary cell carcinoma (KIRP, n=208 (low)/79 (high)) and their expression of hTrmt13.


( a ) HEK293 cells stably expressing GFP - DFCP1 were transfected with or without shRNA against Atg14L . The cell lysates were subjected to immunoblotting with the indicated antibodies .

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

(a) HEK293 cells stably expressing GFP-DFCP1 were transfected with or without shRNA against Atg14L. The cell lysates were subjected to immunoblotting with the indicated antibodies.


( b ) GFP signals were observed in these cells before or after starvation . ( c ) The number of GFP - positive puncta per cell was counted ; mean ± SD values are presented . N , nutrient rich ; S , starvation .

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

(b) GFP signals were observed in these cells before or after starvation. (c) The number of GFP-positive puncta per cell was counted; mean ± SD values are presented. N, nutrient rich; S, starvation.


( d ) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods . Equivalent amounts of total , cytosolic , and ER fractions were subjected to immunoblotting with each organelle marker antibody . The percentage of recovery in each fraction is presented as mean ± SD values .

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

(d) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods. Equivalent amounts of total, cytosolic, and ER fractions were subjected to immunoblotting with each organelle marker antibody. The percentage of recovery in each fraction is presented as mean ± SD values.


( e ) HEK293 cells stably expressing GFP - DFCP1 were transiently transformed with Flag - tagged Atg14L and subjected to starvation . The cells were fixed and stained with the indicated antibodies . Insets show higher magnification of the boxed areas .

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

(e) HEK293 cells stably expressing GFP-DFCP1 were transiently transformed with Flag-tagged Atg14L and subjected to starvation. The cells were fixed and stained with the indicated antibodies. Insets show higher magnification of the boxed areas.


( f ) HEK293 cells were transfected with GFP - Atg14L with or without mCherry - linked dominant - negative ULK1 , and these cells were cultured in complete or starvation medium for 60 min . Bars , 10 µm .

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

(f) HEK293 cells were transfected with GFP-Atg14L with or without mCherry-linked dominant-negative ULK1, and these cells were cultured in complete or starvation medium for 60 min. Bars, 10 µm.


( a ) HEK293 cells were simultaneously transfected with four plasmids harboring One - Strep - Flag ( OSF ) - tagged Atg14L , Beclin - 1 , hVps34 , and hVps15 , and Atg14L was pulled down using Strep - Tactin Sepharose beads . Empty plasmid was used as control instead of Atg14L . The precipitates were subjected to SDS - PAGE and Coomassie brilliant blue staining .

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

(a) HEK293 cells were simultaneously transfected with four plasmids harboring One-Strep-Flag (OSF)-tagged Atg14L, Beclin-1, hVps34, and hVps15, and Atg14L was pulled down using Strep-Tactin Sepharose beads. Empty plasmid was used as control instead of Atg14L. The precipitates were subjected to SDS-PAGE and Coomassie brilliant blue staining.


( c ) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti - GFP antibody . Each precipitant was subjected to immunoblotting with the indicated antibodies . IP , immunoprecipitation .

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

(c) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti-GFP antibody. Each precipitant was subjected to immunoblotting with the indicated antibodies. IP, immunoprecipitation.


( e ) NRK cells were transiently transfected with adenovirus expressing GFP - Atg14L or GFP - Atg14L4C4A and fixed under nutrient - rich conditions . The cells were stained with anticalnexin antibody as an ER membrane marker . Higher magnification of the boxed areas is shown below . Bars , 10 µm .

O O O B-CELL_LINE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O

(e) NRK cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14L4C4A and fixed under nutrient-rich conditions. The cells were stained with anticalnexin antibody as an ER membrane marker. Higher magnification of the boxed areas is shown below. Bars, 10 µm.


( a ) A549 cells were transiently transfected with adenovirus expressing GFP - Atg14L or GFP - Atg14LCAAX and subjected to starvation . The cells were fixed and stained with anti - Beclin - 1 antibody . Higher magnification of the boxed areas is shown below .

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

(a) A549 cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14LCAAX and subjected to starvation. The cells were fixed and stained with anti-Beclin-1 antibody. Higher magnification of the boxed areas is shown below.


( b ) HEK293 cells were cotransfected with MEF - hVps34 and GFP or GFP - Atg14LCAAX and stained with anti - Flag antibody . Bars , 10 µm .

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

(b) HEK293 cells were cotransfected with MEF-hVps34 and GFP or GFP-Atg14LCAAX and stained with anti-Flag antibody. Bars, 10 µm.


Autophagy induction by overexpressed Atg14L . A549 cells were transiently transfected with adenovirus harboring GFP - Atg14L or GFP - Atg14L4C4A . ( a and b ) The cells before starvation ( a ) or after starvation ( b ) were fixed and stained with anti - LC3 antibody . ( c ) The number of LC3 - positive puncta per cell was counted , and mean ± SD values are presented . Bars , 10 µm .

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

Autophagy induction by overexpressed Atg14L. A549 cells were transiently transfected with adenovirus harboring GFP-Atg14L or GFP-Atg14L4C4A. (a and b) The cells before starvation (a) or after starvation (b) were fixed and stained with anti-LC3 antibody. (c) The number of LC3-positive puncta per cell was counted, and mean ± SD values are presented. Bars, 10 µm.


The effect of Atg14L localization on its function . ( a ) Atg14L knockout mouse ES cells were transfected with GFP , GFP - Atg14L , GFP - Atg14L4C4A , or GFP - ER - Atg14L4C4A of the pCAG vector and subjected to starvation . The cells were fixed and immunostained with anti - Atg16L or anti - LC3 antibodies . ( b and c ) The number of Atg16L - positive puncta ( b ) and LC3 - positive puncta ( c ) per cell was counted in at least 10 GFP - positive cells , and mean ± SD values are presented . Bars , 10 µm .

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

The effect of Atg14L localization on its function. (a) Atg14L knockout mouse ES cells were transfected with GFP, GFP-Atg14L, GFP-Atg14L4C4A, or GFP-ER-Atg14L4C4A of the pCAG vector and subjected to starvation. The cells were fixed and immunostained with anti-Atg16L or anti-LC3 antibodies. (b and c) The number of Atg16L-positive puncta (b) and LC3-positive puncta (c) per cell was counted in at least 10 GFP-positive cells, and mean ± SD values are presented. Bars, 10 µm.


A ) Oocytes were fixed for chromosome spreads 8 hours after GVBD , and stained for PP2A - c ( red ) , Sgo2 ( green ) , CREST ( blue ) and DNA ( Hoechst , shown in grey ) . Spreads were classified into metaphase I and anaphase I , depending on whether chromosome segregation had taken place , or not . Arrowhead indicates fused sister kinetochores , arrows indicate separated sister kinetochores .

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

A) Oocytes were fixed for chromosome spreads 8 hours after GVBD, and stained for PP2A-c (red), Sgo2 (green), CREST (blue) and DNA (Hoechst, shown in grey). Spreads were classified into metaphase I and anaphase I, depending on whether chromosome segregation had taken place, or not. Arrowhead indicates fused sister kinetochores, arrows indicate separated sister kinetochores.


B ) Example of a whole mount oocyte staining , in early anaphase I after cold - treatment to visualize only cold - stable microtubule fibers , stained with anti - tubulin antibody ( green ) . Kinetochores are stained with CREST serum ( red ) and chromosomes with Hoechst ( blue ) . Shown is an overlay of 20 z - sections . Arrows indicate separated sister kinetochores .

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

B) Example of a whole mount oocyte staining, in early anaphase I after cold-treatment to visualize only cold-stable microtubule fibers, stained with anti-tubulin antibody (green). Kinetochores are stained with CREST serum (red) and chromosomes with Hoechst (blue). Shown is an overlay of 20 z-sections. Arrows indicate separated sister kinetochores.


C ) Metaphase I ( 6 hours after GVBD ) and metaphase II ( 16 - 20 hours after GVBD ) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin ( pink ) and the centromere ( green ) , respectively . Chromosomes were stained with Hoechst ( blue ) . Below , schematic representation of a bivalent in metaphase I and a dyad in metaphase II . Enlargement shows the centromere region and a region that we call " chromatid junction " with the corresponding scheme .

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

C) Metaphase I (6 hours after GVBD) and metaphase II (16-20 hours after GVBD) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin (pink) and the centromere (green), respectively. Chromosomes were stained with Hoechst (blue). Below, schematic representation of a bivalent in metaphase I and a dyad in metaphase II. Enlargement shows the centromere region and a region that we call "chromatid junction" with the corresponding scheme.


D ) Quantification of total Sgo2 signal at the centromere and the chromatid junction , normalized to CREST at centromeres , on chromosome spreads from oocytes at the indicated stages of meiotic maturation . Each dot shows mean per oocyte ; the number of oocytes analyzed is indicated . Below : scheme illustrating how Sgo2 signals ( in red ) were measured at the indicated meiotic stages . Ifluor stands for a mean fluorescence intensity of the area within the box ( dotted lines , see Material and Methods for quantification approach ) .

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

D) Quantification of total Sgo2 signal at the centromere and the chromatid junction, normalized to CREST at centromeres, on chromosome spreads from oocytes at the indicated stages of meiotic maturation. Each dot shows mean per oocyte; the number of oocytes analyzed is indicated. Below: scheme illustrating how Sgo2 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines, see Material and Methods for quantification approach).


E ) Chromosome spreads as in A ) , stained for Sgo2 ( red ) , CREST ( green ) , and DNA ( Hoechst , in blue ) . Spreads in early anaphase I and late anaphase I - telophase I were classified , depending on whether dyads of two chromosome sets had been found close to each other as one group , or were scattered apart into two groups . In early anaphase I , Sgo2 is found inbetween sister kinetochores that are already separated . F ) Quantification of E ) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair ( dot plot on the left ) , and distance between sister kinetochores ( dot plot on the right ) at the indicated stages of meiosis I . Meta : metaphase , ana : anaphase , telo : telophase . Each dot represents one kinetochore pair ; the number of kinetochore pairs analysed is indicated . Below : scheme of Sgo2 signal ( in red ) measurements .

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

E) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated. F) Quantification of E) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair (dot plot on the left), and distance between sister kinetochores (dot plot on the right) at the indicated stages of meiosis I. Meta: metaphase, ana: anaphase, telo: telophase. Each dot represents one kinetochore pair; the number of kinetochore pairs analysed is indicated. Below: scheme of Sgo2 signal (in red) measurements.


E ) Chromosome spreads as in A ) , stained for Sgo2 ( red ) , CREST ( green ) , and DNA ( Hoechst , in blue ) . Spreads in early anaphase I and late anaphase I - telophase I were classified , depending on whether dyads of two chromosome sets had been found close to each other as one group , or were scattered apart into two groups . In early anaphase I , Sgo2 is found inbetween sister kinetochores that are already separated .

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

E) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated.


A ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Sgo2 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . On the right , schemes illustrate the chromosome figures observed , and the corresponding Sgo2 staining ( in red ) .

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

A) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Sgo2 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). On the right, schemes illustrate the chromosome figures observed, and the corresponding Sgo2 staining (in red).


B ) Dyads of metaphase II chromosome spreads of control , Bub1KD , Mps1 ∆ N and Reversine - treated oocytes ( from GVBD onwards ) stained with Sgo2 antibody ( red ) , CREST serum ( green ) and Hoechst ( blue ) .

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

B) Dyads of metaphase II chromosome spreads of control, Bub1KD, Mps1∆N and Reversine-treated oocytes (from GVBD onwards) stained with Sgo2 antibody (red), CREST serum (green) and Hoechst (blue).


C ) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards , corresponding to the time of anaphase I onset . PBE : Polar Body Extrusion .

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

C) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards, corresponding to the time of anaphase I onset. PBE: Polar Body Extrusion.


D ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Mps1 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later , respectively . E ) Quantification of D ) showing Mps1 signal relative to CREST per kinetochore pair ( per single kinetochore in anaphase II ) ( left dot plot ) , and Mps1 signal averaged per oocyte ( right dot plot ) at the indicated time points . The number of kinetochore pairs ( kinetochores in anaphase II ) and oocytes analyzed is indicated . The scheme of measurements is shown on the right .

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

D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively. E) Quantification of D) showing Mps1 signal relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Mps1 signal averaged per oocyte (right dot plot) at the indicated time points. The number of kinetochore pairs (kinetochores in anaphase II) and oocytes analyzed is indicated. The scheme of measurements is shown on the right.


D ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Mps1 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later , respectively .

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

D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively.


F ) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst . Where indicated , oocytes were treated with Reversine from GVBD onwards , and treatment was renewed every 8 hours . Early CSF corresponds to 8 hours after GVBD ( Reversine treated oocytes undergo anaphase I on average 4 - 5 hours after GVBD ) , and late CSF corresponds to 36 hours after GVBD ( > 29 hours in CSF arrest ) . PSCS : precocious sister chromatid segregation .

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

F) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst. Where indicated, oocytes were treated with Reversine from GVBD onwards, and treatment was renewed every 8 hours. Early CSF corresponds to 8 hours after GVBD (Reversine treated oocytes undergo anaphase I on average 4-5 hours after GVBD), and late CSF corresponds to 36 hours after GVBD (>29 hours in CSF arrest). PSCS: precocious sister chromatid segregation.


G ) Quantification of total Sgo2 signal on spreads in early or late CSF arrest ( 16 hours or 30 hours after GVBD , respectively ) relative to CREST per kinetochore pair ( per single kinetochore in anaphase II ) ( left dot plot ) , and Sgo2 signal averaged per oocyte ( right dot plot ) at the indicated stages . The number of kinetochore pairs and oocytes analyzed is indicated . The scheme of measurements is shown on the right . Meta : metaphase , ana : anaphase .

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

G) Quantification of total Sgo2 signal on spreads in early or late CSF arrest (16 hours or 30 hours after GVBD, respectively) relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Sgo2 signal averaged per oocyte (right dot plot) at the indicated stages. The number of kinetochore pairs and oocytes analyzed is indicated. The scheme of measurements is shown on the right. Meta: metaphase, ana: anaphase.


( A and D ) Oocytes in meiosis I ( 6 hours after GVBD ) or meiosis II ( left and right panels , respectively ) were treated with STLC for 2 . 5 hours and fixed for whole mount immunofluorescence . Cold stable microtubules were stained with an antibody against tubulin ( green ) , centromeres with CREST ( red ) and DNA with Hoechst ( blue ) . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

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

(A and D) Oocytes in meiosis I (6 hours after GVBD) or meiosis II (left and right panels, respectively) were treated with STLC for 2.5 hours and fixed for whole mount immunofluorescence. Cold stable microtubules were stained with an antibody against tubulin (green), centromeres with CREST (red) and DNA with Hoechst (blue). Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


( B and E ) Chromosome spreads at metaphase and anaphase of meiosis I ( 6 and 9 hours after GVBD ) or meiosis II ( left and right panels , respectively ) , in the presence or absence of STLC ( for 2 . 5 hours ) and Reversine ( + Rev . , for 1 hour ) as indicated . Spreads were stained with CREST serum ( red ) and Hoechst ( grey ) . Single chromosomes are present in anaphase I and single sister chromatids in anaphase II . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

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

(B and E) Chromosome spreads at metaphase and anaphase of meiosis I (6 and 9 hours after GVBD) or meiosis II (left and right panels, respectively), in the presence or absence of STLC (for 2.5 hours) and Reversine (+Rev., for 1 hour) as indicated. Spreads were stained with CREST serum (red) and Hoechst (grey). Single chromosomes are present in anaphase I and single sister chromatids in anaphase II. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


( C and F ) Chromosome spreads exactly as described in B and E ) , except that spreads were additionally stained with an antibody against Rec8 ( green ) . The CREST signal appears in red and DNA in blue . Rev : Reversine . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

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

(C and F) Chromosome spreads exactly as described in B and E), except that spreads were additionally stained with an antibody against Rec8 (green). The CREST signal appears in red and DNA in blue. Rev: Reversine. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


A ) Metaphase II oocytes from mice carrying a conditional , oocyte - specific invalidation of separase ( Separaseflox / flox Zp3 Cre + , or Sep - / - ) and oocytes from litter mates ( Separaseflox / flox , or Sep + / + ) were obtained after superovulation . In vivo matured control ( Sep + / + ) and Sep - / - oocytes are found in oviducts at comparable numbers , enclosed by cumulus cells . Example of cumulus enclosed oocytes from the oviduct is shown below . In ( A ) , n indicates the number of mice and a mean number of oocytes per mouse of each genotype , error bars are + s . e . m .

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

A) Metaphase II oocytes from mice carrying a conditional, oocyte-specific invalidation of separase (Separaseflox/flox Zp3 Cre+, or Sep-/-) and oocytes from litter mates (Separaseflox/flox, or Sep+/+) were obtained after superovulation. In vivo matured control (Sep+/+) and Sep-/- oocytes are found in oviducts at comparable numbers, enclosed by cumulus cells. Example of cumulus enclosed oocytes from the oviduct is shown below. In (A), n indicates the number of mice and a mean number of oocytes per mouse of each genotype, error bars are + s.e.m.


B ) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation . Microtubules were stained with anti - tubulin antibody ( green ) , centromeres with CREST serum ( red ) and DNA with Hoechst ( blue ) . Bivalents are attached in a monopolar fashion . Approximate number of z - sections used for overlays to visualize the whole spindle ( on the left ) or individual chromosomes ( magnifications on the right ) are indicated . C ) Same as B ) , except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards . In ( B and C ) n indicates number of oocytes analyzed .

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

B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated. C) Same as B), except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards. In (B and C) n indicates number of oocytes analyzed.


B ) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation . Microtubules were stained with anti - tubulin antibody ( green ) , centromeres with CREST serum ( red ) and DNA with Hoechst ( blue ) . Bivalents are attached in a monopolar fashion . Approximate number of z - sections used for overlays to visualize the whole spindle ( on the left ) or individual chromosomes ( magnifications on the right ) are indicated .

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

B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated.


D ) Metaphase - to - anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated , in GV . The schemes indicate the chromosome figures observed upon activation . Chromosome spreads are stained with CREST serum ( green ) , and Rec8 antibody ( red ) . DNA was stained with DAPI ( blue ) . In ( D ) n indicates the number of oocytes with the shown phenotype ( e . g . , upon successful activation ) and the total number of oocytes analyzed .

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

D) Metaphase-to-anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated, in GV. The schemes indicate the chromosome figures observed upon activation. Chromosome spreads are stained with CREST serum (green), and Rec8 antibody (red). DNA was stained with DAPI (blue). In (D) n indicates the number of oocytes with the shown phenotype (e.g., upon successful activation) and the total number of oocytes analyzed.


A ) Sep + / + or Sep - / - oocytes were matured in vitro until metaphase II . Sep - / - oocytes were injected with wild - type separase or cleavage mutant separase encoding mRNAs in metaphase II . Oocytes were either fixed in metaphase II or activated and fixed in anaphase II . Spreads were stained with Rec8 antibody ( red ) , CREST serum ( green ) , and DAPI ( blue ) to label DNA . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes analysed

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

A) Sep+/+ or Sep-/- oocytes were matured in vitro until metaphase II. Sep-/- oocytes were injected with wild-type separase or cleavage mutant separase encoding mRNAs in metaphase II. Oocytes were either fixed in metaphase II or activated and fixed in anaphase II. Spreads were stained with Rec8 antibody (red), CREST serum (green), and DAPI (blue) to label DNA. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed


A ) Sep + / + or Sep - / - oocytes were matured in vitro until metaphase II . Sep - / - oocytes were injected with wild - type separase or cleavage mutant separase encoding mRNAs in metaphase II . Oocytes were either fixed in metaphase II or activated and fixed in anaphase II . Spreads were stained with Rec8 antibody ( red ) , CREST serum ( green ) , and DAPI ( blue ) to label DNA . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes

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


B ) Representative image of kinetochore individualization in Sep - / - oocytes injected with wild - type separase in metaphase II and fixed after activation , from A ) . Shown is staining with CREST serum ( green ) , and DAPI ( blue ) to label DNA . Arrows indicate separated sister kinetochores . The scheme on the left illustrates the individualization of sister kinetochores ( green arrows ) at anaphase II . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes analysed Scale bars : 10 µm , except bottom panel in B ) , where the scale bar is 2 μm .

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

B) Representative image of kinetochore individualization in Sep-/- oocytes injected with wild-type separase in metaphase II and fixed after activation, from A). Shown is staining with CREST serum (green), and DAPI (blue) to label DNA. Arrows indicate separated sister kinetochores. The scheme on the left illustrates the individualization of sister kinetochores (green arrows) at anaphase II. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed Scale bars: 10 µm, except bottom panel in B), where the scale bar is 2 μm.


A ) Live imaging movie of GFP - Sgo2 expressing oocytes ( from GV onwards ) undergoing meiosis I . The green channel to visualize GFP - Sgo2 localized to chromosomes is shown . Timepoints were taken every 10min , shown is a montage of selected frames such as indicated ( hours : time after GVBD ) at the metaphase - to - anaphase transition of meiosis I and in metaphase II . Each timepoint is comprised of an overlay of 11 z - sections of 3μm for GFP . The arrow heads indicate bivalents . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed

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

A) Live imaging movie of GFP-Sgo2 expressing oocytes (from GV onwards) undergoing meiosis I. The green channel to visualize GFP-Sgo2 localized to chromosomes is shown. Timepoints were taken every 10min, shown is a montage of selected frames such as indicated (hours: time after GVBD) at the metaphase-to-anaphase transition of meiosis I and in metaphase II. Each timepoint is comprised of an overlay of 11 z-sections of 3μm for GFP. The arrow heads indicate bivalents. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed