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 .

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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 .

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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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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

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

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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 ) .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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

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 .

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

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

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

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

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

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


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

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


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

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


B ) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP - Sgo2 expressing oocyte . Spreads were stained with Rec8 antibody ( pink ) , CREST serum ( green ) , Hoechst for DNA ( blue ) , GFP - Sgo2 was visualized by GFP fluorescence ( grey ) . The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed

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

B) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP-Sgo2 expressing oocyte. Spreads were stained with Rec8 antibody (pink), CREST serum (green), Hoechst for DNA (blue), GFP-Sgo2 was visualized by GFP fluorescence (grey). The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed


B ) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP - Sgo2 expressing oocyte . Spreads were stained with Rec8 antibody ( pink ) , CREST serum ( green ) , Hoechst for DNA ( blue ) , GFP - Sgo2 was visualized by GFP fluorescence ( grey ) . The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes

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


C ) Metaphase I ( 4hrs after GVBD ) and II spreads after GFP - Sgo2 expression ( from GV onwards ) . Spreads were stained with CREST serum ( red ) , Hoechst for DNA ( grey ) , GFP - Sgo2 was visualized by GFP fluorescence ( green ) . Arrowheads mark separated sister kinetochores . On the right , graph showing percentage of metaphase II spreads containing bivalent chromosomes . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed The number of analysed oocytes in C )

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

C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes. In (A-C) n indicates the number of control non-injected and GFP -Sgo2-expressing oocytes analyzed The number of analysed oocytes in C)


C ) Metaphase I ( 4hrs after GVBD ) and II spreads after GFP - Sgo2 expression ( from GV onwards ) . Spreads were stained with CREST serum ( red ) , Hoechst for DNA ( grey ) , GFP - Sgo2 was visualized by GFP fluorescence ( green ) . Arrowheads mark separated sister kinetochores . On the right , graph showing percentage of metaphase II spreads containing bivalent chromosomes . In ( A - C ) n indicates the number of control non - injected and

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

C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes. In (A-C) n indicates the number of control non-injected and GFP


D ) Chromosome spreads of oocytes expressing GFP - Sgo2 ( from GV onwards ) in metaphase II , and upon activation . Spreads were labeled with Rec8 antibody ( shown in red , far - red secondary antibody was used ) , CREST serum ( pseudo - colored in green ) , and Hoechst ( blue ) . GFP direct fluorescence was imaged for GFP - Sgo2 detection . Below the corresponding chromosome figures are represented in the schemes . E ) Quantification of Rec8 signal found around and in between CREST signals that were close to each other . Below : scheme illustrating how Rec8 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 ) . Meta : metaphase , ana : anaphase . D ) , the number of oocytes with the shown phenotype and the total number of oocytes analyzed . On each graph , mean is shown , error bars are + s . e . m . in C ) and and ± SD in E ) , asterisks indicate significant difference ( * * * * = p < 0 , 0001 ) according to Mann - Whitney U test . AU : arbitrary units . The number of analysed kinetochore pairs in E ) is indicated .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes. E) Quantification of Rec8 signal found around and in between CREST signals that were close to each other. Below: scheme illustrating how Rec8 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). Meta: metaphase, ana: anaphase. D), the number of oocytes with the shown phenotype and the total number of oocytes analyzed. On each graph, mean is shown, error bars are +s.e.m. in C) and and ± SD in E), asterisks indicate significant difference (**** = p<0,0001) according to Mann-Whitney U test. AU: arbitrary units. The number of analysed kinetochore pairs in E) is indicated.


D ) Chromosome spreads of oocytes expressing GFP - Sgo2 ( from GV onwards ) in metaphase II , and upon activation . Spreads were labeled with Rec8 antibody ( shown in red , far - red secondary antibody was used ) , CREST serum ( pseudo - colored in green ) , and Hoechst ( blue ) . GFP direct fluorescence was imaged for GFP - Sgo2 detection . Below the corresponding chromosome figures are represented in the schemes .

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

D) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes.


G , Fluorescent in situ hybridization for cd74a , tnfa , and ctsc at 7 dpi , respectively . mpeg1 was used to label all MC cells . The blue boxed region is highlighted in the zoom - in image to the right . Scale bar = 50 μm . A stands for arium , V stand for ventricle and OFT stands for outflow tract . White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane .

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

G, Fluorescent in situ hybridization for cd74a, tnfa, and ctsc at 7 dpi, respectively. mpeg1 was used to label all MC cells. The blue boxed region is highlighted in the zoom-in image to the right. Scale bar = 50 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane.


F , In situ hybridization showing temporal spatial expression patterns of postnb . The white boxed region is shown in zoom - in images at the bottom . Scale bar = 25 μm . White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane . A stands for arium , V stand for ventricle and OFT stands for outflow tract .

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

F, In situ hybridization showing temporal spatial expression patterns of postnb. The white boxed region is shown in zoom-in images at the bottom. Scale bar = 25 μm. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. A stands for arium, V stand for ventricle and OFT stands for outflow tract.


H , Fluorescent in situ hybridization for postnb , tnfα , and tnfrsf1a in the injury area at 7 dpf , respectively . The white boxed regions are shown in their respective zoom - in images to the right . White dashed lines outline cardiac apex and the yellow dashed lines indicate approximate resection plane . Scale bar = 25 μm .

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

H, Fluorescent in situ hybridization for postnb, tnfα, and tnfrsf1a in the injury area at 7 dpf, respectively. The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines outline cardiac apex and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm.


J , Violin plots showing expression of cxcl18b , sele , atf3 , and fosl1a in each eEC subpopulation .

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

J, Violin plots showing expression of cxcl18b, sele, atf3, and fosl1a in each eEC subpopulation.


K , Fluorescent in situ hybridization for fosl1a at 7 dpi . The white boxed region is shown in a higher magnification image to the right for the injury area . cdh5 was used to label all EC cells and the red boxed region is shown in a higher magnification image to highlight a pan - EC cdh5 expression in green . White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane . Scale bar = 25 μm . A stands for arium , V stand for ventricle and OFT stands for outflow tract .

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

K, Fluorescent in situ hybridization for fosl1a at 7 dpi. The white boxed region is shown in a higher magnification image to the right for the injury area. cdh5 was used to label all EC cells and the red boxed region is shown in a higher magnification image to highlight a pan-EC cdh5 expression in green. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract.


A , Wildtype or kit mutant stained with AFOG at 14 days and 30 dpi . Sham - operated zebrafish hearts serve as control . Scale bar = 100 μm .

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

A, Wildtype or kit mutant stained with AFOG at 14 days and 30 dpi. Sham-operated zebrafish hearts serve as control. Scale bar = 100 μm.


C , PCNA / Mef2 double staining showing the proliferating CMs in wildtype and mutant hearts at 7 dpi . The white boxed regions are shown in zoom - in images to their right . Blue dashed lines indicate approximate resection plane . White arrows point to the PCNA / Mef2 double positive nuclei . Scale bar = 50 μm .

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

C, PCNA/Mef2 double staining showing the proliferating CMs in wildtype and mutant hearts at 7 dpi. The white boxed regions are shown in zoom-in images to their right. Blue dashed lines indicate approximate resection plane. White arrows point to the PCNA/Mef2 double positive nuclei. Scale bar = 50 μm.


I , Immunostaining for MC marker IB4 in WT ( left ) and mutant hearts ( middle ) at 7 dpi . Quantification of IB4 + cell number per unit area ( right ) . The white boxed regions are shown in their respective zoom - in images to the right . White dashed lines indicate approximate resection plane .

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I, Immunostaining for MC marker IB4 in WT (left) and mutant hearts (middle) at 7 dpi. Quantification of IB4+ cell number per unit area (right). The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines indicate approximate resection plane.


D , F , Violin plots showing the expression of il1b in MC1 ( D ) , and ctsd expression in MC ( F ) . E , G , Expression of il1b ( E ) and ctsd ( G ) in WT and kit mutant hearts determined by qRT - PCR .

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D, F, Violin plots showing the expression of il1b in MC1 (D), and ctsd expression in MC (F). E, G, Expression of il1b (E) and ctsd (G) in WT and kit mutant hearts determined by qRT-PCR.


O , Violin plots showing expression of junba in eEC . P , Expression of junba in WT and kit mutant hearts determined by qRT - PCR .

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O, Violin plots showing expression of junba in eEC. P, Expression of junba in WT and kit mutant hearts determined by qRT-PCR.


Q , Representative image of hearts from 7 dpi tcf21 : nucGFP transgenic fish treated by PBS liposome , Clodronate liposome and Dex , respectively . The red boxes mark the peripheral area , and the white boxes mark the injury area and are shown in their respective zoom - in images to the right . . White dashed lines indicate approximate resection plane .

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Q, Representative image of hearts from 7 dpi tcf21:nucGFP transgenic fish treated by PBS liposome, Clodronate liposome and Dex, respectively. The red boxes mark the peripheral area, and the white boxes mark the injury area and are shown in their respective zoom-in images to the right.. White dashed lines indicate approximate resection plane.


( a ) Atg7 + / − mice were generated as described in the Methods . PCR was done employing genomic DNA and primers flanking the floxed Atg7 regions . Arrows indicate wild - type Atg7 band in Atg7 + / + mice , increased size of PCR band due to inserted flox sequence in Atg7F / + mice and decreased size of PCR band due to Cre - mediated deletion in Atg7 + / − mice , respectively .

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

(a) Atg7+/− mice were generated as described in the Methods. PCR was done employing genomic DNA and primers flanking the floxed Atg7 regions. Arrows indicate wild-type Atg7 band in Atg7+/+ mice, increased size of PCR band due to inserted flox sequence in Atg7F/+ mice and decreased size of PCR band due to Cre-mediated deletion in Atg7+/− mice, respectively.


( b ) RT - PCR ( left ) and real - time RT - PCR ( right ) were performed using total RNA prepared from tissues of Atg7 + / − or control Atg7 + / + mice , and primers specific for Atg7 or β - actin sequence . Fold changes of the RT - PCR band intensities are shown ( left ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , n = 3 .

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(b) RT-PCR (left) and real-time RT-PCR (right) were performed using total RNA prepared from tissues of Atg7+/− or control Atg7+/+mice, and primers specific for Atg7 or β-actin sequence. Fold changes of the RT-PCR band intensities are shown (left). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3.


( c ) Tissue lysate from fasted 12 - week - old Atg7 + / − and control Atg7 + / + mice was subjected to immunoblot analysis using anti - LC3 or - p62 antibody . Fold changes of the immunoblot band intensities are shown ( middle and right ) . * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , n = 3 .

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(c) Tissue lysate from fasted 12-week-old Atg7+/− and control Atg7+/+mice was subjected to immunoblot analysis using anti-LC3 or -p62 antibody. Fold changes of the immunoblot band intensities are shown (middle and right). **P0.01, ***P0.001; Student's t-test, n=3.


( d ) Cell lysate of Atg7 + / + , Atg7 + / − and Atg7 − / − MEFs was subjected to immunoblot analysis using antibodies specific for LC3 and p62 after rapamycin ( Rap ) or control solvent ( dimethyl sulfoxide , DMSO ) treatment for 3 h . Fold changes of the immunoblot band intensities are shown ( middle and right ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , n = 3 .

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(d) Cell lysate of Atg7+/+, Atg7+/− and Atg7−/− MEFs was subjected to immunoblot analysis using antibodies specific for LC3 and p62 after rapamycin (Rap) or control solvent (dimethyl sulfoxide, DMSO) treatment for 3 h. Fold changes of the immunoblot band intensities are shown (middle and right). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3.


( e ) Sections of the liver and muscle tissues from fasted 12 - week - old GFP - LC3 + - Atg7 + / + and GFP - LC3 + - Atg7 + / − mice were subjected to fluorescent microscopy to examine GFP - LC3 puncta ( left ) . The number of GFP - LC3 puncta was counted ( right ) . Scale bars , 10 μm . * P0 . 05 ; Student ' s t - test , n = 5 .

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(e) Sections of the liver and muscle tissues from fasted 12-week-old GFP-LC3+-Atg7+/+ and GFP-LC3+-Atg7+/− mice were subjected to fluorescent microscopy to examine GFP-LC3 puncta (left). The number of GFP-LC3 puncta was counted (right). Scale bars, 10 μm. *P0.05; Student's t-test, n=5.


( f ) Tissue lysates were prepared from the liver of fasted Atg7 + / + and Atg7 + / − mice 4 h after leupeptin administration , and immunoblotting was done . Fold changes of the immunoblot band intensities are shown ( right ) . * P0 . 05 ; Student ' s t - test , n = 3 .

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(f) Tissue lysates were prepared from the liver of fasted Atg7+/+ and Atg7+/− mice 4 h after leupeptin administration, and immunoblotting was done. Fold changes of the immunoblot band intensities are shown (right). *P0.05; Student's t-test, n=3.


( a ) Non - fasting blood glucose level was monitored weekly in Atg7 + / + , Atg7 + / − , Atg7 + / + - ob / ob and Atg7 + / − - ob / ob mice . * * * P0 . 001 ; two - way ANOVA .

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(a) Non-fasting blood glucose level was monitored weekly in Atg7+/+, Atg7+/−, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice. ***P0.001; two-way ANOVA.


( b ) IPGTT was done by glucose injection at 16 weeks of age as described in the Methods ( left ) , and AUC calculated ( right ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test ( left ) and one - way ANOVA ( right ) .

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(b) IPGTT was done by glucose injection at 16 weeks of age as described in the Methods (left), and AUC calculated (right). *P0.05, **P0.01, ***P0.001; Student's t-test (left) and one-way ANOVA (right).


( c ) HOMA - IR index was calculated as described in Methods . * P0 . 05 ; one - way ANOVA .

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(c) HOMA-IR index was calculated as described in Methods. *P0.05; one-way ANOVA.


( d ) ITT was performed by insulin injection at 16 weeks of age as described in the Methods . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test .

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(d) ITT was performed by insulin injection at 16 weeks of age as described in the Methods. *P0.05, **P0.01, ***P0.001; Student's t-test.


( e ) Tissue lysates from Atg7 + / + - ob / w , Atg7 + / − - ob / w , Atg7 + / + - ob / ob and Atg7 + / − - ob / ob mice were prepared before and 7 min after injection of 5 U kg − 1 regular insulin ( Ins ) into the tail vein , and subjected to immunoblot analysis using antibodies specific for phospho - Akt S473 and total Akt . Numbers below immunoblot bands indicate the fold changes normalized to control bands .

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

(e) Tissue lysates from Atg7+/+-ob/w, Atg7+/−-ob/w, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice were prepared before and 7 min after injection of 5 U kg−1 regular insulin (Ins) into the tail vein, and subjected to immunoblot analysis using antibodies specific for phospho-Akt S473 and total Akt. Numbers below immunoblot bands indicate the fold changes normalized to control bands.


( a ) Tissue lysates from each type of mouse were subjected to immunoblot analysis using antibodies specific for total JNK , phospho - JNK , total IRS - 1 and phospho - IRS - 1 S307 .

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

(a) Tissue lysates from each type of mouse were subjected to immunoblot analysis using antibodies specific for total JNK, phospho-JNK, total IRS-1 and phospho-IRS-1 S307.


( b ) Liver sections from each type of mouse were subjected to immunohistochemistry using anti - nitrotyrosine antibody . Scale bars , 100 μm .

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(b) Liver sections from each type of mouse were subjected to immunohistochemistry using anti-nitrotyrosine antibody. Scale bars, 100 μm.


( c ) Tissue lysates were prepared from 18 - week - old mice of each genotype , and subjected to immunoblot analysis using a kit detecting carbonylated proteins .

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(c) Tissue lysates were prepared from 18-week-old mice of each genotype, and subjected to immunoblot analysis using a kit detecting carbonylated proteins.


( d ) Immunoblot analysis was performed using anti - phospho - p53 and - p53 antibodies . p21 and β - actin mRNA expression was evaluated by RT - PCR . Numbers below immunoblot bands indicate the fold changes normalized to control bands .

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

(d) Immunoblot analysis was performed using anti-phospho-p53 and -p53 antibodies. p21 and β-actin mRNA expression was evaluated by RT-PCR. Numbers below immunoblot bands indicate the fold changes normalized to control bands.


( a ) GFP - LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state , and the numbers of GFP - LC3 puncta counted . Scale bars , 20 μm . * P0 . 05 , * * P0 . 01 ; Student ' s t - test .

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(a) GFP-LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state, and the numbers of GFP-LC3 puncta counted. Scale bars, 20 μm. *P0.05, **P0.01; Student's t-test.


( b ) Tissues of ob / w and ob / ob mice were subjected to EM , and the number of autophagosomes was counted . * * * P0 . 001 ; Student ' s t - test .

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(b) Tissues of ob/w and ob/ob mice were subjected to EM, and the number of autophagosomes was counted. ***P0.001; Student's t-test.


( c ) Immunoblotting using anti - GFP or - p62 antibody .

(c) Immunoblotting using anti-GFP or -p62 antibody.


( d ) Tissue lysates were prepared from the liver of fed ob / w or ob / ob mice 4 h after leupeptin administration , and immunoblotting was performed . Numbers below immunoblot bands indicate the fold changes normalized to control bands .

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(d) Tissue lysates were prepared from the liver of fed ob/w or ob/ob mice 4 h after leupeptin administration, and immunoblotting was performed. Numbers below immunoblot bands indicate the fold changes normalized to control bands.


( e ) After labelling SK - Hep1 cells with C14 - leucine , cells were treated with FFAs ( 600 μM PA or 1 , 200 μM OA ) . Percent release of radioactivity was measured for 3 - 6 h as described in the Methods . The difference between the percent proteolysis without E64d / pepstatin A / NH4Cl and that with E64d / pepstatin A / NH4Cl was regarded as lysosomal proteolysis . * P0 . 05 , * * P0 . 01 ; one - way ANOVA , n = 3 .

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(e) After labelling SK-Hep1 cells with C14-leucine, cells were treated with FFAs (600 μM PA or 1,200 μM OA). Percent release of radioactivity was measured for 3-6 h as described in the Methods. The difference between the percent proteolysis without E64d/pepstatin A/NH4Cl and that with E64d/pepstatin A/NH4Cl was regarded as lysosomal proteolysis. *P0.05, **P0.01; one-way ANOVA, n=3.


( f ) Atg7 + / + and Atg7 + / − primary MEFs were loaded with a mixture of the indicated concentrations of PA and OA for 48 h , and TG content was estimated by ORO staining . * * P0 . 01 , * * * P0 . 001 ; two - way ANOVA , n = 4 .

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(f) Atg7+/+ and Atg7+/− primary MEFs were loaded with a mixture of the indicated concentrations of PA and OA for 48 h, and TG content was estimated by ORO staining. **P0.01, ***P0.001; two-way ANOVA, n=4.


( a ) The number of CLSs was counted in WAT of each type of mouse as described in the Methods . * P0 . 05 ; one - way ANOVA , n = 3 .

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(a) The number of CLSs was counted in WAT of each type of mouse as described in the Methods. *P0.05; one-way ANOVA, n=3.


( b ) Total mRNA was extracted from each tissue , and RT - PCR was done using primers specific for Tnfa , Il6 , F4 / 80 or pro - Il1b . The expression of pro - Il1b was determined using SVF fraction of WAT tissue .

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(b) Total mRNA was extracted from each tissue, and RT-PCR was done using primers specific for Tnfa, Il6, F4/80 or pro-Il1b. The expression of pro-Il1b was determined using SVF fraction of WAT tissue.


( c , d ) Lysate of SVF was subjected to immunoblot analysis using anti - IL - 1β ( c ) or anti - caspase - 1 antibody ( d ) .

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(c,d) Lysate of SVF was subjected to immunoblot analysis using anti-IL-1β (c) or anti-caspase-1 antibody (d).


( e ) Primary peritoneal macrophages were isolated , and treated with PA of the indicated concentrations in the presence or absence of LPS . ELISA was conducted to determine IL - 1β concentrations in the culture supernatant . ( BSA , bovine serum albumin ) * * * P0 . 001 ; two - way ANOVA , n = 4 .

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(e) Primary peritoneal macrophages were isolated, and treated with PA of the indicated concentrations in the presence or absence of LPS. ELISA was conducted to determine IL-1β concentrations in the culture supernatant. (BSA, bovine serum albumin) ***P0.001; two-way ANOVA, n=4.


( f ) After incubation of primary peritoneal macrophages with PA of the indicated concentrations with or without LPS for 16 h , NAD + / NADH ratio was determined using a commercial kit . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; two - way ANOVA , n = 5 .

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(f) After incubation of primary peritoneal macrophages with PA of the indicated concentrations with or without LPS for 16 h, NAD+/NADH ratio was determined using a commercial kit. *P0.05, **P0.01, ***P0.001; two-way ANOVA, n=5.


( g ) After the same treatment of peritoneal macrophages as in f , cells were incubated with MitoSOX to measure mitochondrial ROS by fluorescence - activated cell sorting ( FACS ) analysis .

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(g) After the same treatment of peritoneal macrophages as in f, cells were incubated with MitoSOX to measure mitochondrial ROS by fluorescence-activated cell sorting (FACS) analysis.


( h ) After the same treatment of peritoneal macrophages as in f , cells were stained with MitoTracker Red and MitoTracker Green for FACS analysis to determine mitochondrial potential . The numbers in g , h indicate the percentages of cells in the designated gate .

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(h) After the same treatment of peritoneal macrophages as in f, cells were stained with MitoTracker Red and MitoTracker Green for FACS analysis to determine mitochondrial potential. The numbers in g,h indicate the percentages of cells in the designated gate.


( a ) Atg7 + / − and control Atg7 + / + mice were fed HFD or normal chow diet ( NCD ) , and non - fasting blood glucose levels were monitored . * P0 . 05 , * * P0 . 01 ; Student ' s t - test .

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(a) Atg7+/− and control Atg7+/+ mice were fed HFD or normal chow diet (NCD), and non-fasting blood glucose levels were monitored. *P0.05, **P0.01; Student's t-test.


( b ) Fasting bloodglucose levels were determined after 18 weeks of HFD . * P0 . 05 ; one - way ANOVA .

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(b) Fasting bloodglucose levels were determined after 18 weeks of HFD. *P0.05; one-way ANOVA.


( c ) IPGTT was performed after 18 weeks of HFD , ( left ) and AUC calculated ( right ) . * P0 . 05 ; Student ' s t - test ( left ) and one - way ANOVA ( right ) .

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(c) IPGTT was performed after 18 weeks of HFD, (left) and AUC calculated (right). *P0.05; Student's t-test (left) and one-way ANOVA (right).


( d ) HOMA - IR was calculated after 18 weeks of HFD . * * * P0 . 001 ; one - way ANOVA .

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(d) HOMA-IR was calculated after 18 weeks of HFD. ***P0.001; one-way ANOVA.


( e ) ITT was performed after 18 weeks of HFD ( left ) , and KITT calculated ( right ) . * P0 . 05 ; Student ' s t - test ( left ) and one - way ANOVA ( right ) .

(e) ITT was performed after 18 weeks of HFD (left), and KITT calculated (right). *P0.05; Student's t-test (left) and one-way ANOVA (right).


( a ) One hour after intraperitoneal injection of 30 mg kg − 1 leupeptin to C57BL / 6 mice , 25 mg kg − 1 of imatinib ( Ima ) was injected intraperitoneally . Three hours later , tissue lysate was prepared from the liver and subjected to immunoblot analysis .

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(a) One hour after intraperitoneal injection of 30 mg kg−1 leupeptin to C57BL/6 mice, 25 mg kg−1 of imatinib (Ima) was injected intraperitoneally. Three hours later, tissue lysate was prepared from the liver and subjected to immunoblot analysis.


( b ) Imatinib ( 25 mg kg − 1 ) , trehalose ( 2 g kg − 1 ) or PBS was injected intraperitoneally into 12 - week - old diabetic Atg7 + / − - ob / ob mice 3 times a week , and the blood glucose level was monitored . * * * P0 . 001 , # # # P0 . 001 ; two - way ANOVA .

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(b) Imatinib (25 mg kg−1), trehalose (2 g kg−1) or PBS was injected intraperitoneally into 12-week-old diabetic Atg7+/−-ob/ob mice 3 times a week, and the blood glucose level was monitored. ***P0.001, ###P0.001; two-way ANOVA.


( c ) IPGTT were performed after treatment of Atg7 + / − - ob / ob mice with imatinib or trehalose for 8 weeks . # P0 . 05 ; # # P or * * P0 . 01 ; # # # P or * * * P0 . 001 ; Student ' s t - test .

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(c) IPGTT were performed after treatment of Atg7+/−-ob/ob mice with imatinib or trehalose for 8 weeks. #P0.05; ##P or **P0.01; ###P or ***P0.001; Student's t-test.


( d ) ITT were performed after treatment of Atg7 + / − - ob / ob mice with imatinib or trehalose for 8 weeks . # P0 . 05 ; # # P or * * P0 . 01 ; # # # P or * * * P0 . 001 ; Student ' s t - test .

(d) ITT were performed after treatment of Atg7+/−-ob/ob mice with imatinib or trehalose for 8 weeks. #P0.05; ##P or **P0.01; ###P or ***P0.001; Student's t-test.


( e ) Regular insulin ( Ins ) was injected into the tail vein of Atg7 + / − - ob / ob mice that were treated with imatinib or PBS for 8 weeks . Seven minutes later , tissue lysates were prepared and subjected to immunoblotting .

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(e) Regular insulin (Ins) was injected into the tail vein of Atg7+/−-ob/ob mice that were treated with imatinib or PBS for 8 weeks. Seven minutes later, tissue lysates were prepared and subjected to immunoblotting.


( f ) Tissue lysates prepared from the same mice without insulin injection were subjected to immunoblotting . Numbers below immunoblot bands indicate the fold changes normalized to control bands . ( ' * ' indicates comparison between imatinib and control : ' # ' indicates comparison between trehalose and control ) .

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(f) Tissue lysates prepared from the same mice without insulin injection were subjected to immunoblotting. Numbers below immunoblot bands indicate the fold changes normalized to control bands. ('*' indicates comparison between imatinib and control: '#' indicates comparison between trehalose and control).


B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in ( A ) . 1cs , 1 - cell stage ; 2cs , 2 - cell stage ; st6 , 32 - cell stage ; st8 . 5 , blastula stage . Blots are representative of 3 independent experiments . C Densitometry analysis of Hwa bands from the western blot in ( B ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 by unpaired t test .

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B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in (A). 1cs, 1-cell stage; 2cs, 2-cell stage; st6, 32-cell stage; st8.5, blastula stage. Blots are representative of 3 independent experiments. C Densitometry analysis of Hwa bands from the western blot in (B) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, *p < 0.05, **p < 0.01 by unpaired t test.


D Western blot analysis of exogenous Hwa in HEK293T cells . Treatment of cells with lysosome inhibitors bafilomycin A1 ( Baf - A1 ) and NH4Cl for 24 hours , DMSO was used as control . Dosage of Baf - A1 : 10 nM , 50 nM , 100 nM ; Dosage of NH4Cl : 2 . 5 mM , 5 mM , 10 mM . Hwa with modification is indicated by white arrow ; Degraded Hwa is indicated by black arrow . Blots are representative of 3 independent experiments . E Densitometry analysis of Hwa bands from the western blot in ( D ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , * * p < 0 . 01 by unpaired t test .

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D Western blot analysis of exogenous Hwa in HEK293T cells. Treatment of cells with lysosome inhibitors bafilomycin A1 (Baf-A1) and NH4Cl for 24 hours, DMSO was used as control. Dosage of Baf-A1: 10 nM, 50 nM, 100 nM; Dosage of NH4Cl: 2.5 mM, 5 mM, 10 mM. Hwa with modification is indicated by white arrow; Degraded Hwa is indicated by black arrow. Blots are representative of 3 independent experiments. E Densitometry analysis of Hwa bands from the western blot in (D) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, **p < 0.01 by unpaired t test.


C Co - IP with HEK293T cells co - transfected with plasmids encoding Hwa - Flag and ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . Blots are representative of 3 independent experiments . D Co - IP with HEK293T cells co - transfected with plasmids encoding Hwa - Flag and ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - HA were immunoblotted with Flag and HA antibodies . Blots are representative of 3 independent experiments .

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C Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments. D Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-HA were immunoblotted with Flag and HA antibodies. Blots are representative of 3 independent experiments.


E Colocalization of Hwa - Flag ( red ) and ZNRF3 - HA ( green ) or ZNRF3ΔRING - HA ( green ) in Hela cells is indicated by yellow dots . Baf - A1 , 50 nM , 12 hrs . Magnification : 100 × ; scale bar : 10 μm .

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E Colocalization of Hwa-Flag (red) and ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm.


B Whole mount in situ hybridization of Spemann organizer markers chrd , gsc , and xnr3 in gastrula embryos ( stage 10 . 5 ) . Oocytes were injected with AS or mRNAs as indicated , Dosage : znrf3 AS ( 3 ng AS4 + 3 ng AS9 ) , znrf3 mRNA 100 pg , znrf3ΔRING mRNA 50 pg . embryos were obtained through Host transfer technology . Scale bars : 1 mm .

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B Whole mount in situ hybridization of Spemann organizer markers chrd, gsc, and xnr3 in gastrula embryos (stage 10.5). Oocytes were injected with AS or mRNAs as indicated, Dosage: znrf3 AS (3 ng AS4 + 3 ng AS9), znrf3 mRNA 100 pg, znrf3ΔRING mRNA 50 pg. embryos were obtained through Host transfer technology. Scale bars: 1 mm.


E Relative levels of β - catenin ( left ) and Axin ( right ) in ( D ) normalized to GAPDH . n = 3 independent experiments , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by unpaired t test .

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E Relative levels of β-catenin (left) and Axin (right) in (D) normalized to GAPDH. n = 3 independent experiments, mean ± SEM, *p < 0.05, ***p < 0.005, ****p < 0.0001 by unpaired t test.


H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos ( stage 10 . 5 ) . Embryos were injected and obtained as descripted in ( G ) . Scale bars : 1 mm .

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H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos (stage 10.5). Embryos were injected and obtained as descripted in (G). Scale bars: 1 mm.


I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl ( gfp mRNA ) , znrf3FL , and znrf3 truncations as indicated at 4 - cell stage . Duplicated axes ( red arrowhead ) . znrf3FL , full - length znrf3 ; znrf3ΔRING , znrf3 lacking the RING domain ; znrf31 - 320 , amino acids 1 - 320 of znrf3 ; znrf3320 - 606 , amino acids 320 - 606 of znrf3 ; znrf3606 - 854 , amino acids 606 - 854 of znrf3 . Scale bars : 1 mm .

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I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl (gfp mRNA), znrf3FL, and znrf3 truncations as indicated at 4-cell stage. Duplicated axes (red arrowhead). znrf3FL, full-length znrf3; znrf3ΔRING, znrf3 lacking the RING domain; znrf31-320, amino acids 1-320 of znrf3; znrf3320-606, amino acids 320-606 of znrf3; znrf3606-854, amino acids 606-854 of znrf3. Scale bars: 1 mm.


B Hwa ubiquitination was analyzed under denaturing conditions . HEK293T cells co - transfected with wildtype His - HA - Ubiquitin ( His - HA - Ub WT ) or lysine - less mutant ( K0 ) , Hwa - Flag , and ZNRF3 - Myc . The lysates were immunoblotted with Myc , Flag , and α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . K0 , all lysine sites on ubiquitin were mutated . Blots are representative of 3 independent experiments .

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B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and ZNRF3-Myc. The lysates were immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. K0, all lysine sites on ubiquitin were mutated. Blots are representative of 3 independent experiments.


D Screen of ZNRF3 - mediated Hwa ubiquitination sites under denaturing conditions . HEK293T cells were co - transfected with His - HA - Ub , Hwa - Flag or Hwa10KR - Flag , and ZNRF3 or ZNRF3ΔRING . The lysate was immunoblotted with Myc , Flag , and α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . Hwa10KR - Flag mutant , all lysine sites in Hwa sequence were replaced with arginine . Denaturing conditions : before immunoprecipitation , lysates were denatured by adding SDS to a final concentration at 1 % and boiling at 95 ℃ for 15 min . Blots are representative of 3 independent experiments .

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D Screen of ZNRF3-mediated Hwa ubiquitination sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or Hwa10KR-Flag, and ZNRF3 or ZNRF3ΔRING. The lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa sequence were replaced with arginine. Denaturing conditions: before immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots are representative of 3 independent experiments.


A Western blot analysis of Hwa in HEK293T cells co - transfected with Hwa - Flag , and ZNRF3 - HA ( 5 μg ) or ZNRF3ΔRING ( 2 . 5 μg ) . Lysate was immunoblotted with Flag , HA , and GAPDH antibodies . Blots are representative of 3 independent experiments . B Densitometry analysis of Hwa bands from the western blot in ( A ) for corresponding lanes normalized to GAPDH . n = 3 independent experiments , mean ± SEM , ns p > 0 . 05 , * * p < 0 . 01 by unpaired t test .

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A Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag, and ZNRF3-HA (5 μg) or ZNRF3ΔRING (2.5 μg). Lysate was immunoblotted with Flag, HA, and GAPDH antibodies. Blots are representative of 3 independent experiments. B Densitometry analysis of Hwa bands from the western blot in (A) for corresponding lanes normalized to GAPDH. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01 by unpaired t test.


C Western blot analysis of Hwa in HEK293T cells co - transfected with Hwa - Flag and ZNRF3 - HA . Cells were treated with or without Baf - A1 ( 50 nM ) or MG132 ( 20 μM ) for about 12 hours . Lysates were immunoblotted with Flag , HA , and α - tub ( α - tubulin ) antibodies . Blots are representative of 3 independent experiments . D Densitometry analysis of Hwa bands from the western blot in ( C ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , ns p > 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by unpaired t test .

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C Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag and ZNRF3-HA. Cells were treated with or without Baf-A1 (50 nM) or MG132 (20 μM) for about 12 hours. Lysates were immunoblotted with Flag, HA, and α-tub (α-tubulin) antibodies. Blots are representative of 3 independent experiments. D Densitometry analysis of Hwa bands from the western blot in (C) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test.


F Colocalization by immunofluorescence of Hwa ( green signals ) with lysosome ( LAMP1 , red signals ) in Hela cells . Cells were co - transfected with Hwa - Flag , and ZNRF3 - HA or ZNRF3ΔRING - HA . Magnification , 100 × ; scale bars : 10 μm .

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F Colocalization by immunofluorescence of Hwa (green signals) with lysosome (LAMP1, red signals) in Hela cells. Cells were co-transfected with Hwa-Flag, and ZNRF3-HA or ZNRF3ΔRING-HA. Magnification, 100 ×; scale bars: 10 μm.


I Surface biotinylation and streptavidin pulldown of Hwa - Myc - Flag . HEK293T cells were co - transfected with Hwa - MF ( Hwa - Myc - Flag ) , and ZNRF3 - Myc or ZNRF3ΔRING - Myc . Lysate was immunoblotted with Myc , Flag , and GAPDH antibodies . Lysates immunoprecipitated with anti - Flag ( 2nd elution ) were immunoblotted with Flag and HA antibodies .

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I Surface biotinylation and streptavidin pulldown of Hwa-Myc-Flag. HEK293T cells were co-transfected with Hwa-MF (Hwa-Myc-Flag), and ZNRF3-Myc or ZNRF3ΔRING-Myc. Lysate was immunoblotted with Myc, Flag, and GAPDH antibodies. Lysates immunoprecipitated with anti-Flag (2nd elution) were immunoblotted with Flag and HA antibodies.


C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos . Embryos were obtained as descripted in ( B ) . Scale bars : 1mm .

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C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos. Embryos were obtained as descripted in (B). Scale bars: 1mm.


E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage . 1 - cell stage embryos were co - injected with hwa ( 100 pg ) or hwa10KR ( 100 pg ) and znrf3 ( 1 ng ) or znrf3ΔRING ( 100 pg ) as indicated . Lysates were immunoblotted with HA , Flag , and α - tub ( α - tubulin ) .

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E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage. 1-cell stage embryos were co-injected with hwa (100 pg) or hwa10KR (100 pg) and znrf3 (1 ng) or znrf3ΔRING (100 pg) as indicated. Lysates were immunoblotted with HA, Flag, and α-tub (α-tubulin).


( a ) Nuclear localization of HLH - 30 was visualized by fluorescence microscopy in day 1 adult wild - type ( WT ) ( upper panel ) and glp - 1 ( e2141 ) ( lower panel ) animals expressing HLH - 30 : : GFP raised at the non - permissive temperature ( 25 ° C ) . Inserts show enlarged intestinal cells . Graph shows percentage of animals with HLH - 30 in the nuclei of intestinal cells ( four biological replicates , ~ 50 animals each , mean ± s . d . , * * P0 . 01 , Student ' s t - test ) . Magnification × 100 ; scale bar , 100 μm .

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(a) Nuclear localization of HLH-30 was visualized by fluorescence microscopy in day 1 adult wild-type (WT) (upper panel) and glp-1(e2141) (lower panel) animals expressing HLH-30::GFP raised at the non-permissive temperature (25°C). Inserts show enlarged intestinal cells. Graph shows percentage of animals with HLH-30 in the nuclei of intestinal cells (four biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test). Magnification × 100; scale bar, 100 μm.


( b ) Expression of hlh - 30 and putative autophagy - related and lysosomal target genes was measured by quantitative PCR ( qPCR ) in day 1 adult WT and glp - 1 ( e2141 ) animals raised at 25 ° C ( mean ± s . d . of three biological replicates , * P0 . 05 , * * P0 . 01 , Student ' s t - test ) .

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(b) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by quantitative PCR (qPCR) in day 1 adult WT and glp-1(e2141) animals raised at 25°C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student's t-test).


( c ) Expression of hlh - 30 and putative autophagy - related and lysosomal target genes was measured by qPCR in day 1 adult glp - 1 ( e2141 ) and glp - 1 ( e2141 ) ; hlh - 30 ( tm1978 ) animals raised at 25 ° C ( mean ± s . d . of three biological replicates , * P0 . 05 , * * P0 . 01 , Student ' s t - test ) . ctsa * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) . See Supplementary Fig . 1 for qPCR analyses of hlh - 30 ( tm1978 ) ( control for c ) and WT and glp - 1 ( e2141 ) animals fed bacteria expressing hlh - 30 dsRNA .

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(c) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by qPCR in day 1 adult glp-1(e2141) and glp-1(e2141); hlh-30(tm1978) animals raised at 25°C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student's t-test). ctsa* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). See Supplementary Fig. 1 for qPCR analyses of hlh-30(tm1978) (control for c) and WT and glp-1(e2141) animals fed bacteria expressing hlh-30 dsRNA.