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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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


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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 -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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

I, 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 .

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

D, F, 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 .

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

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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O 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 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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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(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 .

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(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 .

(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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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.


( d , e ) GFP : : LGG - 1 punctae were quantified in ( d ) hypodermal seam cells or ( e ) proximal intestinal cells of L3 larvae of WT and glp - 1 ( e2141 ) animals . Animals were fed bacteria expressing control or hlh - 30 dsRNA for two generations at 20 ° C . Eggs were then transferred to plates seeded with the appropriate dsRNA - expressing bacteria at 25 ° C and analysed at the L3 larval stage ( mean ± s . e . m . of ~ 300 seam cells and ~ 25 intestines , * * P0 . 01 , Student ' s t - test ) .

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(d,e) GFP::LGG-1 punctae were quantified in (d) hypodermal seam cells or (e) proximal intestinal cells of L3 larvae of WT and glp-1(e2141) animals. Animals were fed bacteria expressing control or hlh-30 dsRNA for two generations at 20 °C. Eggs were then transferred to plates seeded with the appropriate dsRNA-expressing bacteria at 25 °C and analysed at the L3 larval stage (mean±s.e.m. of ~300 seam cells and ~25 intestines, **P0.01, Student's t-test).


( f , g ) glp - 1 ( e2141 ) animals expressing ( f ) LMP - 1 : : GFP or ( g ) SQST - 1 : : GFP were raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from hatching . Micrographs of the posterior intestine were taken on day 1 of adulthood , and LMP - 1 : : GFP fluorescence ( mean ± s . d . of ~ 10 animals , * * P0 . 01 , Student ' s t - test ) and SQST - 1 : : GFP foci ( mean ± s . d . of ~ 30 animals , * * P0 . 01 , Student ' s t - test ) were quantified . Experiments were performed at least three times with similar results . See Supplementary Fig . S2a , c for images of whole animals and Supplementary Fig . S2b , d for replicates . Magnification , × 200 ; scale bar , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25°C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean±s.d. of ~10 animals, **P0.01, Student's t-test) and SQST-1::GFP foci (mean±s.d. of ~30 animals, **P0.01, Student's t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification, × 200; scale bar, 100 μm.


( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult animals expressing HLH - 30 : : GFP . Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 ° C ( three biological replicates , ~ 50 animals each , mean ± s . d . , * * P0 . 01 , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP. Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 °C (three biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test).


b , c ) Expression of putative autophagy - related and lysosomal target genes was measured by quantitative PCR in day 1 ( b ) WT ( N2 ) and ( c ) hlh - 30 ( tm1978 ) animals fed bacteria expressing control or tor dsRNA from hatching ( 20 ° C ) . Data are mean ± s . d . of biological triplicates . * P0 . 05 , * * P0 . 01 ; Student ' s t - test . csta * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA from hatching (20 °C). Data are mean±s.d. of biological triplicates. *P0.05, **P0.01; Student's t-test. csta* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)).


( d ) Lifespan analysis of WT animals and hlh - 30 ( tm1978 ) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 ° C . See Supplementary Table S3 for details of lifespan analyses and replicate experiments .

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(d) Lifespan analysis of WT animals and hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 °C. See Supplementary Table S3 for details of lifespan analyses and replicate experiments.


Lifespan analyses of ( a ) WT ( N2 ) and ( b ) germline - less glp - 1 ( e2141 ) animals raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood were carried out at 20 ° C . Lifespan analyses of ( c ) dietary - restricted eat - 2 ( ad1116 ) mutants , ( d ) insulin / IGF - 1 receptor daf - 2 ( e1370 ) mutants , ( e ) mitochondrial respiration clk - 1 ( e2519 ) mutants and ( f ) mRNA translation rsks - 1 ( sv31 ) mutants , fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood ( c , d , f ) or larval L4 stage ( e ) , were carried out at 20 ° C . See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments .

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

Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 °C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 °C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 °C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments.


( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult WT , eat - 2 , daf - 2 , clk - 1 and rsks - 1 mutants ( mean ± s . d . of four biological replicates , ~ 50 animals each , * * P0 . 01 , analysis of variance ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 localization of HLH-30 was quantified in day 1 adult WT, eat-2, daf-2, clk-1 and rsks-1 mutants (mean±s.d. of four biological replicates, ~50 animals each, **P0.01, analysis of variance).


( b ) Expression of hlh - 30 was measured by quantitative PCR ( qPCR ) in day 1 adult WT ( N2 ) , eat - 2 ( ad1116 ) ( dietary restriction , DR ) , daf - 2 ( e1370 ) ( insulin / IGF - 1 signaling ) , clk - 1 ( e2519 ) ( mitochondrial respiration ) and rsks - 1 ( sv31 ) ( mRNA translation ) animals ( mean ± s . d . of three biological replicates , * P0 . 05 , Student ' s t - test ) .

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(b) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student's t-test).


( c ) Lifespan analysis of WT and transgenic animals overexpressing HLH - 30 : : GFP , and raised and maintained on OP50 was carried out at 20 ° C . See Supplementary Table S7 for details of lifespan analyses and replicate experiments .

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(c) Lifespan analysis of WT and transgenic animals overexpressing HLH-30::GFP, and raised and maintained on OP50 was carried out at 20 °C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments.


( d ) GFP : : LGG - 1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH - 30 ( n > 300 , ± s . e . m . , * * P0 . 01 , Student ' s t - test ) . The increase in GFP : : LGG - 1 punctae observed in animals overexpressing HLH - 30 could be reversed by atg - 18 RNAi treatment ( data not shown ) .

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(d) GFP::LGG-1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n>300, ±s.e.m., **P0.01, Student's t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment (data not shown).


( e ) TFEB expression was measured by qPCR in the livers of 4 . 5 - month - old female ( F ) and male ( M ) mice fed AL or subjected to DR for 5 . 5 weeks starting at 3 months of age ( mean ± s . e . m . of ~ 20 mice per group , * P0 . 05 , Student ' s t - test ) .

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(e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to DR for 5.5 weeks starting at 3 months of age (mean±s.e.m. of ~20 mice per group, *P0.05, Student's t-test).


( f ) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR . Actin was included as a loading control , see Supplementary Fig . S8 for additional controls . ( g ) TFEB protein levels in Fig . 4f were quantified by densitometry and normalized to actin ( mean ± s . d . , * P0 . 05 , Student ' s t - test ) .

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(f) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein levels in Fig. 4f were quantified by densitometry and normalized to actin (mean±s.d., *P0.05, Student's t-test).


J , Ridge graph comparisons of cyclin expression across the U5 - hNSC defined cell cycle phases . The x - axis is relative expression of the cyclin , and the y - axis is counts of cells per phase . The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot .

J, Ridge graph comparisons of cyclin expression across the U5-hNSC defined cell cycle phases. The x-axis is relative expression of the cyclin, and the y-axis is counts of cells per phase. The cell cycle phase with the peak expression is denoted by an arrowhead at the top of the plot.


C , Number of putative stem - like cells in each ccAF cell cycle phase , and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem - like cells in Bhaduri et al . , 2020 .

C, Number of putative stem-like cells in each ccAF cell cycle phase, and distribution of absolute gene expression across ccAF cell cycle phases for the genes used to identify putative stem-like cells in Bhaduri et al., 2020.


D , Putative stem - like cells and expression of the genes used to identify the putative stem - like cells projected onto Neftel et al . , 2019 . Expression of genes are overlaid as the color of each cell ( white = 0 , dark blue = max expression ) . Quadrants are broken up by dashed grey lines : OPC = upper left ; NPC = upper right ; AC = lower left ; and MES = lower right . Percentages of gene expression greater than 0 are shown in the corners of the plot .

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D, Putative stem-like cells and expression of the genes used to identify the putative stem-like cells projected onto Neftel et al., 2019. Expression of genes are overlaid as the color of each cell (white = 0, dark blue = max expression). Quadrants are broken up by dashed grey lines: OPC = upper left; NPC = upper right; AC = lower left; and MES = lower right. Percentages of gene expression greater than 0 are shown in the corners of the plot.


C , Violin plot showing PLP1 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of PLP1 in single cells is much higher in oligodendrocytes ( Oligo ) than in Neural G0 or oligodendrocyte progenitor cells ( OPC ) . D , Violin plot showing GFAP expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types . E , Violin plot showing AQP4 expression across all cell types from Darmanis et al . , 2017 . Data are log 10 - transformed before plotting . Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit .

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C, Violin plot showing PLP1 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of PLP1 in single cells is much higher in oligodendrocytes (Oligo) than in Neural G0 or oligodendrocyte progenitor cells (OPC). D, Violin plot showing GFAP expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types. E, Violin plot showing AQP4 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit.


B , Representative contour maps of flow cytometry for FUCCI following the loss of NF2 , PTPN14 , TAOK1 , CREBBP , and TP53 .

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B, Representative contour maps of flow cytometry for FUCCI following the loss of NF2, PTPN14, TAOK1, CREBBP, and TP53.


D , G0 / G1 and S / G2 / M transit times using time - lapse microscopy and FUCCI . Differences in G0 / G1 are statistically significant with p < 0 . 001 for targeted U5 - NSCs and p = 0 . 0006 for GSC - 131 compared to NTC .

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D, G0/G1 and S/G2/M transit times using time-lapse microscopy and FUCCI. Differences in G0/G1 are statistically significant with p<0.001 for targeted U5-NSCs and p=0.0006 for GSC-131 compared to NTC.


A ) Ten - fold serial dilutions from overnight cultures of wild - type and cdc14 - 1 cells dropped and grown on solid rich media or media containing MMS at 25 , 28 , 30 or 33ºC . Note that cdc14 - 1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild - type cells .

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A) Ten-fold serial dilutions from overnight cultures of wild-type and cdc14-1 cells dropped and grown on solid rich media or media containing MMS at 25, 28, 30 or 33ºC. Note that cdc14-1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild-type cells.


B ) Ten - fold serial dilution from mid - log phase cultures of wild - type and cdc14 - 1 cells grown on solid rich media or media containing mock DMSO ( as non - treated control ) , 4NQO , HU , phleomycin and benomyl at 30ºC . Note that cdc14 - 1 cells present a market sensitivity to all DNA damaged agents tested .

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B) Ten-fold serial dilution from mid-log phase cultures of wild-type and cdc14-1 cells grown on solid rich media or media containing mock DMSO (as non-treated control), 4NQO, HU, phleomycin and benomyl at 30ºC. Note that cdc14-1 cells present a market sensitivity to all DNA damaged agents tested.


C ) Left panel : Schematic representation showing relevant genomic structure of the strain used to assess intra - chromosomal repair . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of intra - chromosomal repair in wild - type and cdc14 - 1 cultures at the semipermisive temperature . After DSB formation by the expression of the HO , glucose was added to repress it , thus allowing repair with HM donor sequences . Genomic DNA was digested with StyI , separated on agarose gel and blotted . Blots were hybridized with a probe corresponding to the MATa - distal sequence . A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time - point . Immunoblot analysis of Rad53 during mating type switching experiments is shown . Coomassie blue staining is depicted as loading control . Graphs show quantification of gene conversion ( GC ) leading to the re - establishment of MATa or switching to MAT and DSB formation . The data was normalized with the actin loading control . Graphs show the mean ± S . D . from three independent experiments . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Schematic representation showing relevant genomic structure of the strain used to assess intra-chromosomal repair. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of intra-chromosomal repair in wild-type and cdc14-1 cultures at the semipermisive temperature. After DSB formation by the expression of the HO, glucose was added to repress it, thus allowing repair with HM donor sequences. Genomic DNA was digested with StyI, separated on agarose gel and blotted. Blots were hybridized with a probe corresponding to the MATa-distal sequence. A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time-point. Immunoblot analysis of Rad53 during mating type switching experiments is shown. Coomassie blue staining is depicted as loading control. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa or switching to MAT and DSB formation. The data was normalized with the actin loading control. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


A ) Left panel : Schematic diagram displaying relevant genomic structure of the strains used to measure inter - chromosomal DNA repair efficiency . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Note that crossover and non - crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of wild - type and cdc14 - 1 mutant strains carrying the inter - chromosomal gene conversion assay . Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression , thus producing a DSB on chromosome V . Samples were taken at different time - points , genomic DNA was extracted , digested with EcoRI and analysed by Southern blot . Blots were hybridized with a MATa - only DNA sequence and an actin probe was used as loading control . Asterisk denotes an overexposed film to visualize crossover formation . Immunoblot of Rad53 was performed as previously described . Coomassie staining is shown as a control for loading . Graphs show the mean ± S . D . of gene conversion , DSB induction and crossover vs non - crossover ratio from three independent experiments . All data was normalized using actin as loading control . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

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A) Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression, thus producing a DSB on chromosome V. Samples were taken at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa-only DNA sequence and an actin probe was used as loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± S.D. of gene conversion, DSB induction and crossover vs non-crossover ratio from three independent experiments. All data was normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


B ) Left panel : Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice . The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated . Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used . Arrow depicts the localization of the double - strand break . Right panel : Physical analysis of wild - type and cdc14 - 1 cells harbouring the repair pathway choice assay . Cells were grown overnight and HO induction was attained through addition of galactose for 1 . 5 hour at semipermisive temperature . After induction of the HO , glucose was added to repress the HO expression and samples were taken to analyse repair efficiency . DNA was extracted , digested with EcoRV , separated on agarose gels and blotted . Blots were hybridized with a probe corresponding to the MATa - only DNA sequence . A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time - point . Graphs show quantification of mating type switching by gene conversion ( HR ) , restoration of MATa ( NHEJ ) and the kinetics of the DSB induction . All data was normalized with the HIS3 gene . Graphs show the mean ± S . D . from three independent experiments . Replicates were averaged and statistical significance of differences assessed by a 2 - tailed unpaired Student t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 hour at semipermisive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time-point. Graphs show quantification of mating type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data was normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.


A ) Live - cell imaging of Cdc14 - YFP and Cnm67 - RFP asynchronous cells treated with the DNA damage agent phleomycin . Cells were grown overnight and phleomycin was added to a final concentration of 1μM . Samples were taken at different intervals to determine Cdc14 localization . Cnm67 was used as SPB reporter to determine spindle length . Scale bar : 3μm . Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase ( left graph ) and with Cdc14 signal at the nucleoplasm ( right graph ) .

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

A) Live-cell imaging of Cdc14-YFP and Cnm67-RFP asynchronous cells treated with the DNA damage agent phleomycin. Cells were grown overnight and phleomycin was added to a final concentration of 1μM. Samples were taken at different intervals to determine Cdc14 localization. Cnm67 was used as SPB reporter to determine spindle length. Scale bar: 3μm. Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase (left graph) and with Cdc14 signal at the nucleoplasm (right graph).


A ) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown . Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment . Detection of Net1 phosphorylation levels was achieved by using Phos - tag polyacrylamide gels . Coomassie staining is showed as loading control .

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A) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown. Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment. Detection of Net1 phosphorylation levels was achieved by using Phos-tag polyacrylamide gels. Coomassie staining is showed as loading control.


B ) Western blot analysis showing Net1 phosphorylation through the cell cycle is included . The separation of the phospho - bands was achieved by using Phos - tag polyacrylamide gels as in Fig 3A . Coomassie staining is depicted as loading control .

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B) Western blot analysis showing Net1 phosphorylation through the cell cycle is included. The separation of the phospho-bands was achieved by using Phos-tag polyacrylamide gels as in Fig 3A. Coomassie staining is depicted as loading control.


B ) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage . A strain containing Cdc14 - GFP and Net1 - HA was blocked in G1 by using the pheromone alpha - factor and released into the cell cycle . Samples were taken at different time - points to analyse Cdc14 spatial localization and Net1 phosphorylation levels . A representative picture of non - released ( T0 ) , partially released ( T60 ) and fully released ( T75 ) Cdc14 from the nucleolus is shown . Scale bar : 3μm . Graph represents the percentage ± SD from three independent experiments of cells with no released , partially released ( FEAR ) and totally released ( MEN ) Cdc14 from the nucleolus along the cell cycle .

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B) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage. A strain containing Cdc14-GFP and Net1-HA was blocked in G1 by using the pheromone alpha-factor and released into the cell cycle. Samples were taken at different time-points to analyse Cdc14 spatial localization and Net1 phosphorylation levels. A representative picture of non-released (T0), partially released (T60) and fully released (T75) Cdc14 from the nucleolus is shown. Scale bar: 3μm. Graph represents the percentage ± SD from three independent experiments of cells with no released, partially released (FEAR) and totally released (MEN) Cdc14 from the nucleolus along the cell cycle.


C ) Cells expressing Cdc14 - YFP and Cnm67 - RFP were grown overnight and blocked in G2 / M by using nocodazole prior phleomycin treatment ( 1μM ) . Samples were taken every 30 minutes to follow Cdc14 re - localization . The efficiency of the arrest was determined as in Fig 3A . Scale bar : 3μm . Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion ( left graph ) and cells arrested in metaphase ( right graph ) from three independent experiments .

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C) Cells expressing Cdc14-YFP and Cnm67-RFP were grown overnight and blocked in G2/M by using nocodazole prior phleomycin treatment (1μM). Samples were taken every 30 minutes to follow Cdc14 re-localization. The efficiency of the arrest was determined as in Fig 3A. Scale bar: 3μm. Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion (left graph) and cells arrested in metaphase (right graph) from three independent experiments.


D ) Live - cell imaging of Cdc14 localization in the presence of a non - reparable DSB at the MAT locus . Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media , thus producing a DSB on chromosome III . Cdc14 , the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 ( used as DSB reporter ) were labelled with the green , red and cyan fluorescent proteins respectively . Micrographs display the maximum projection of 9 planes showing Cdc14 co - localizing with the DSB and the SPBs in response to DNA damage ( arrows ) . Scale bar : 3μm . Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction ( left graph ) and the percentage of Cdc14 - SPB - DSB co - localization after 5h in galactose ( right graph ) .

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D) Live-cell imaging of Cdc14 localization in the presence of a non-reparable DSB at the MAT locus. Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media, thus producing a DSB on chromosome III. Cdc14, the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 (used as DSB reporter) were labelled with the green, red and cyan fluorescent proteins respectively. Micrographs display the maximum projection of 9 planes showing Cdc14 co-localizing with the DSB and the SPBs in response to DNA damage (arrows). Scale bar: 3μm. Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction (left graph) and the percentage of Cdc14-SPB-DSB co-localization after 5h in galactose (right graph).


E ) Western blot showing Net1 - 6HA phosphorylation state before and after the generation of a DSB induced by the HO expression . A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control . The phospho - bands were resolved by using Phos - tag polyacrylamide gels as in Fig 3A and Fig 3B . Coomassie staining is depicted as loading control .

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E) Western blot showing Net1-6HA phosphorylation state before and after the generation of a DSB induced by the HO expression. A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control. The phospho-bands were resolved by using Phos-tag polyacrylamide gels as in Fig 3A and Fig 3B. Coomassie staining is depicted as loading control.