Figure 2A , B Expression of pHERK1 : : H2B - TdTomato in mature ovules . White dotted lines delineate the egg cell and synergid cells . Data information : Scale bars = 50 µm . M , micropyle . CC , central cell . EC , egg cell . SC , synergid cell . C , D Expression of pANJ : : H2B - TdTomato in mature ovules . White dotted lines delineate the egg cell and synergid cells . Data information : Scale bars = 50 µm . M , micropyle . CC , central cell . EC , egg cell . SC , synergid cell . E , F Localisation of HERK1 - GFP in the synergid cell from the pFER : : HERK1 - GFP construct in ( F ) and corresponding differential interference contrast ( DIC ) image in ( E ) . White and red dotted lines delineate the egg cell and synergid cells , respectively . Data information : Scale bars = 50 µm . M , micropyle . Arrows , filiform apparatus . G , H Localisation of ANJ - GFP in the synergid cell from the pANJ : : ANJ - GFP construct in ( H ) and corresponding DIC image in ( G ) . White and red dotted lines delineate the egg cell and synergid cells , respectively . Data information : Scale bars = 50 µm . M , micropyle . Arrows , filiform apparatus .

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Figure 2A,B Expression of pHERK1::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.C,D Expression of pANJ::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.E,F Localisation of HERK1-GFP in the synergid cell from the pFER::HERK1-GFP construct in (F) and corresponding differential interference contrast (DIC) image in (E). White and red dotted lines delineate the egg cell and synergid cells, respectively. Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus.G,H Localisation of ANJ-GFP in the synergid cell from the pANJ::ANJ-GFP construct in (H) and corresponding DIC image in (G). White and red dotted lines delineate the egg cell and synergid cells, respectively. Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus.


Figure 3A Localisation of HERK1 , ANJ , LRE , FER and NTA in the synergid cell of wild - type ( Col - 0 ; WT ) , herk1 anj and lre - 5 in unfertilised ovules , as shown by pFER : : HERK1 - GFP , pANJ : : ANJ - GFP , pLRE : : LRE - Citrine , pFER : : FER - GFP and pMYB98 : : NTA - GFP . DIC and fluorescence images are shown , left to right , respectively . White and red dotted lines delineate the egg cell and synergid cells , respectively . Scale bars = 25 µm . B Localisation of NTA in the synergid cell of wild - type and herk1 anj plants before ( upper panels ) and after ( lower panels ) pollen tube arrival . In green , NTA localisation as shown by pMYB98 : : NTA - GFP fluorescence . In magenta , callose of the filiform apparatus and pollen tube stained with SR2200 . From left to right , images shown are DIC , merged fluorescence images , and merged images of DIC and fluorescence . White and red dotted lines delineate the pollen tube and synergid cells , respectively . Scale bars = 25 µm . M , micropyle . Profile of relative fluorescence intensity of NTA - GFP along the synergid cells of wild - type and herk1 anj ovules ( C ) before ( virgin ) and after ( pollinated ) pollen arrival . Data shown are means ± SEM , n = 25 . * * * p < 0 . 001 ( Student ' s t - test ) . FA , filiform apparatus . D Profile of relative fluorescence intensity of NTA - GFP along the synergid cells of wild - type and lre - 5 ovules ( D ) before ( virgin ) and after ( pollinated ) pollen arrival . Data shown are means ± SEM , n = 25 . * * * p < 0 . 001 ( Student ' s t - test ) . FA , filiform apparatus .

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Figure 3A Localisation of HERK1, ANJ, LRE, FER and NTA in the synergid cell of wild-type (Col-0; WT), herk1 anj and lre-5 in unfertilised ovules, as shown by pFER::HERK1-GFP, pANJ::ANJ-GFP, pLRE::LRE-Citrine, pFER::FER-GFP and pMYB98::NTA-GFP. DIC and fluorescence images are shown, left to right, respectively. White and red dotted lines delineate the egg cell and synergid cells, respectively. Scale bars = 25 µm.B Localisation of NTA in the synergid cell of wild-type and herk1 anj plants before (upper panels) and after (lower panels) pollen tube arrival. In green, NTA localisation as shown by pMYB98::NTA-GFP fluorescence. In magenta, callose of the filiform apparatus and pollen tube stained with SR2200. From left to right, images shown are DIC, merged fluorescence images, and merged images of DIC and fluorescence. White and red dotted lines delineate the pollen tube and synergid cells, respectively. Scale bars = 25 µm. M, micropyle.Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and herk1 anj ovules (C) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.D Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and lre-5 ovules (D) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.


Figure 4A Representative images of ovules from wild - type ( Col - 0 ) , herk1 anj , lre - 5 and fer - 4 20 hours after emasculation ( HAE ) displaying the mature female gametophyte structure . Images presented here are maximum intensity projections from confocal microscopy images across several z - planes of ovules stained as per [ 70 ] . Scale bars = 50 µm . B Quantification of H2DCF - DA staining of ROS in ovules from wild - type , herk1 anj , lre - 5 and fer - 4 plants at 20 HAE . Categories are listed in the legend Ovules dissected from at least five siliques per line . * * * p < 0 . 001 ( χ - square tests ) . C Percentage of pollen tubes with normal reception at the female gametophyte ( black bars ) and displaying overgrowth ( grey bars ) in wild - type , herk1 anj , lre - 5 and fer - 4 plants , manually selfed at 20 HAE . Fertilisation events counted from at least three siliques per line . * * * p < 0 . 001 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Representative images of ovules from wild-type (Col-0), herk1 anj, lre-5 and fer-4 20 hours after emasculation (HAE) displaying the mature female gametophyte structure. Images presented here are maximum intensity projections from confocal microscopy images across several z-planes of ovules stained as per [70]. Scale bars = 50 µm.B Quantification of H2DCF-DA staining of ROS in ovules from wild-type, herk1 anj, lre-5 and fer-4 plants at 20 HAE. Categories are listed in the legend Ovules dissected from at least five siliques per line. *** p<0.001 (χ-square tests).C Percentage of pollen tubes with normal reception at the female gametophyte (black bars) and displaying overgrowth (grey bars) in wild-type, herk1 anj, lre-5 and fer-4 plants, manually selfed at 20 HAE. Fertilisation events counted from at least three siliques per line. *** p<0.001 (Student's t-test).


Figure 5A Yeast two hybrid ( Y2H ) assays of the extracellular juxtamembrane domains of HERK1 and ANJ ( HERK1exJM and ANJexJM , respectively ) with LRE ( residues 23 - 138 ; signal peptide and C - terminal domains excluded ) . B Y2H assays with the extracellular domains of HERK1 , ANJ and FER ( HERK1 - ECD , ANJ - ECD and FER - ECD , respectively ) . Ø represents negative controls where no sequence was cloned into the activating domain ( AD ) or DNA - binding domain ( BD ) constructs . - L - W - H , growth medium depleted of leucine ( - L ) , tryptophan ( - W ) and histidine ( - H ) . C Co - immunoprecipitation of HA - LRE with HERK1 - GFP or ANJ - GFP following 2 days of transient expression in N . benthamiana leaves . D Co - immunoprecipitation of FER with HERK1 - GFP in Arabidopsis seedlings expressing pFER : : HERK1 - GFP . Numbers indicate molecular weight marker sizes in kDa . Assays were performed twice with similar results . CBB refers to Coomassie Brilliant Blue staining of total proteins .

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Figure 5A Yeast two hybrid (Y2H) assays of the extracellular juxtamembrane domains of HERK1 and ANJ (HERK1exJM and ANJexJM, respectively) with LRE (residues 23-138; signal peptide and C-terminal domains excluded).B Y2H assays with the extracellular domains of HERK1, ANJ and FER (HERK1-ECD, ANJ-ECD and FER-ECD, respectively). Ø represents negative controls where no sequence was cloned into the activating domain (AD) or DNA-binding domain (BD) constructs. -L-W-H, growth medium depleted of leucine (-L), tryptophan (-W) and histidine (-H).C Co-immunoprecipitation of HA-LRE with HERK1-GFP or ANJ-GFP following 2 days of transient expression in N. benthamiana leaves.D Co-immunoprecipitation of FER with HERK1-GFP in Arabidopsis seedlings expressing pFER::HERK1-GFP. Numbers indicate molecular weight marker sizes in kDa. Assays were performed twice with similar results. CBB refers to Coomassie Brilliant Blue staining of total proteins.


Figure 1Representative WBs of RPE - 1 wild - type ( WT ) cells and the two selected KO clones for AKAP450 ( akap KO ) , PCNT ( pcnt KO ) and CDK5Rap2 ( c5rap2 KO ) probed with the indicated antibodies . GM130 was used as a loading controlConfocal IF images of RPE - 1 WT , single KO ( akap KO , pcnt KO and c5rap2 KO ) and double KO ( pc - ak - 2KO , left panels ; pc - c5 - 2KO , right panels ) cells stained with the indicated antibodies . The yellow dashed line indicates the nucleus contour ( N ) and the arrows point the centrosome ( CTR ) . GA marks the Golgi Apparatus . Scale bars : 5 μm . Representative WB of RPE - 1 WT cells and the two selected PCNT / AKAP450 ( pc - ak - 2KO ) and PCNT / CDK5Rap2 ( pc - c5 - 2KO ) double knockout mutated clones probed with the indicated antibodies . GM130 was used as a loading controlRepresentative WB of RPE - 1 WT cells and the indicated single or double KO cell lines probed with anti - AKAP450 , PCNT and CDK5Rap2 antibodies ( left ) . Quantification of signal intensity from three independent WB experiments is shown at right . # indicates KO proteins . The arrow points the dramatic decrease of CDK5Rap2 levels in pc - ak - 2KO cell line . Hsp70 was used as a loading

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Figure 1Representative WBs of RPE-1 wild-type (WT) cells and the two selected KO clones for AKAP450 (akap KO), PCNT (pcnt KO) and CDK5Rap2 (c5rap2 KO) probed with the indicated antibodies. GM130 was used as a loading controlConfocal IF images of RPE-1 WT, single KO (akap KO, pcnt KO and c5rap2 KO) and double KO (pc-ak-2KO, left panels; pc-c5-2KO, right panels) cells stained with the indicated antibodies. The yellow dashed line indicates the nucleus contour (N) and the arrows point the centrosome (CTR). GA marks the Golgi Apparatus. Scale bars: 5 μm.Representative WB of RPE-1 WT cells and the two selected PCNT/AKAP450 (pc-ak-2KO) and PCNT/CDK5Rap2 (pc-c5-2KO) double knockout mutated clones probed with the indicated antibodies. GM130 was used as a loading controlRepresentative WB of RPE-1 WT cells and the indicated single or double KO cell lines probed with anti-AKAP450, PCNT and CDK5Rap2 antibodies (left). Quantification of signal intensity from three independent WB experiments is shown at right. # indicates KO proteins. The arrow points the dramatic decrease of CDK5Rap2 levels in pc-ak-2KO cell line. Hsp70 was used as a loading control


Figure 2Confocal images of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO and pc - c5 - 2KO cells stained for EB1 ( green ) and the centrosomal protein CAP350 ( red ) . High magnification single - channel images of selected areas are shown at right . Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A . A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets . Horizontal black lines represent the mean . MT regrowth experiments of WT cells mixed with either WT ( as a control ) , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO or pc - c5 - 2KO cells . Samples were fixed 3 min after NZ washout and stained for EB1 ( green ) and the indicated protein in the red channel . Quantification of EB1 signal intensity around the centrosomeWT and pc - ak - 2KO cells were transfected with a combination of three siRNAs against CEP192 , fixed and triple labelled for EB1 , CEP192 and CAP350 , as a centrosomal marker . Scramble siRNA was used as a control . Confocal images of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO and pc - c5 - 2KO cells stained for EB1 ( green ) EB1 comets around the centrosomeThree min MT regrowth experiments in WT and pc - ak - 2KO cells transfected with either scramble or CEP192 siRNAs . Quantification of the EB1 signal intensity around the centrosomeMT regrowth experiments in WT and c5rap2 KO or pcnt KO cells . Cells were stained as indicated . Arrows point the centrosome . Quantification of EB1 signal intensity at Golgi membranes .

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Figure 2Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red). High magnification single-channel images of selected areas are shown at right. Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A. A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets. Horizontal black lines represent the mean.MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel. Quantification of EB1 signal intensity around the centrosomeWT and pc-ak-2KO cells were transfected with a combination of three siRNAs against CEP192, fixed and triple labelled for EB1, CEP192 and CAP350, as a centrosomal marker. Scramble siRNA was used as a control.Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) EB1 comets around the centrosomeThree min MT regrowth experiments in WT and pc-ak-2KO cells transfected with either scramble or CEP192 siRNAs.Quantification of the EB1 signal intensity around the centrosomeMT regrowth experiments in WT and c5rap2 KO or pcnt KO cells. Cells were stained as indicated. Arrows point the centrosome.Quantification of EB1 signal intensity at Golgi membranes.


Figure 3WB of AKAP450 co - immunoprecipitation assays revealed with the indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant . WB of AKAP450 and PCNT ( B ) co - immunoprecipitation assays revealed with the indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant . Centrosomes from WT , single and double KO cells , as indicated , were labelled for AKAP450 and PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for AKAP450 and PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for AKAP450 and PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for CDK5Rap2 Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . Centrosomes from WT , single and double KO cells , as indicated , were labelled for γ - tubulin ( G ) . Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean . WT and pc - ak - 2KO cells were transfected with either scramble or CEP192 siRNAs , fixed and stained for γ - tubulin . Quantification of the fluorescence intensity is show at right . IF images of WT and pcnt KO RPE - 1 cells treated with NZ for 3h and triple stained for AKAP450 ( green ) , CDK5Rap2 ( red ) and GMAP210 as a Golgi marker ( blue ) . At right , quantification of fluorescence co - localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells .

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Figure 3WB of AKAP450 co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.WB of AKAP450 and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for CDK5Rap2 Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.Centrosomes from WT, single and double KO cells, as indicated, were labelled for γ-tubulin (G). Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.WT and pc-ak-2KO cells were transfected with either scramble or CEP192 siRNAs, fixed and stained for γ-tubulin. Quantification of the fluorescence intensity is show at right.IF images of WT and pcnt KO RPE-1 cells treated with NZ for 3h and triple stained for AKAP450 (green), CDK5Rap2 (red) and GMAP210 as a Golgi marker (blue). At right, quantification of fluorescence co-localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells.


Figure 4Control and centrinone - treated cells were subjected to a 3 - min MT regrowth assay , fixed and labelled for EB1 ( green ) and giantin ( red ) . Insets show magnified images of boxed regions . Quantification of the total number of growing MTs per cell ( left ) , the number of MTs nucleated per Golgi fragment ( middle ) and the number of MT - nucleating Golgi fragments ( right ) in both control and centrosome - less RPE - 1 cells . Scatter plots show each individual data point . Horizontal black lines bars represent the mean . RPE - 1 cells were treated wi th centrinone for either 7 days ( left ) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation ( right ) . Representative images of a triple IF staining with EB1 ( green ) , γ - tubulin ( red ) and giantin ( blue ) as a Golgi marker are shown . Insets show MT nucleation from Golgi elements ( left ) or from multiple centrioles ( right ) and are enlarged at right . Arrowheads indicate the position of Golgi elements . Quantification of the percentage of cells exhibiting Golgi - nucleating activity in centrinone - treated cells 48h after the washout of the drug . Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63 . At least 100 cells from two independent experiments were analyzed in each condition .

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Figure 4Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red). Insets show magnified images of boxed regions.Quantification of the total number of growing MTs per cell (left), the number of MTs nucleated per Golgi fragment (middle) and the number of MT-nucleating Golgi fragments (right) in both control and centrosome-less RPE-1 cells. Scatter plots show each individual data point. Horizontal black lines bars represent the mean.RPE-1 cells were treated with centrinone for either 7 days (left) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation (right). Representative images of a triple IF staining with EB1 (green), γ-tubulin (red) and giantin (blue) as a Golgi marker are shown. Insets show MT nucleation from Golgi elements (left) or from multiple centrioles (right) and are enlarged at right. Arrowheads indicate the position of Golgi elements.Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.


Figure 5Control ( top ) and centrinone - treated ( bottom ) RPE - 1 cells double stained for either AKAP450 , CDK5Rap2 , PCNT or CEP192 ( all shown in green ) and giantin ( red ) as a Golgi marker . DNA was counterstained with DAPI and is shown in blue . Single channel ( left , green ) and merged images ( right ) are shown in each case . White arrows indicate the centrosome ( CTR ) . IF images of control and centrinone - treated RPE - 1 cells treated with NZ to induce GA fragmentation and double stained with either anti - AKAP450 , anti - CDK5Rap2 or anti - PCNT ( all shown in green ) and anti - giantin antibodies ( white ) . Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker . The yellow dashed line indicates the contour of the nucleus . Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces . Box - and - whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles , and whiskers represent 90th and 10th percentiles . The median is depicted with a solid line . Individual Golgi elements from at least 14 cells were delineated ( > 500 elements / experiment ) and the intensity of each protein spot associated with each Golgi element was measured . Data were collected from two independent experiments and normalized to WT mean . MT - regrowth experiment after NZ treatment in centrinone - treated cells . High magnification images at the bottom are representative of a triple staining with α - tubulin ( green ) , GM130 ( blue ) and the indicated protein ( red ) and show MTs growing from the surface of the GA . Double labelling without α - tubulin are also shown ( top ) to allow better visualization of PCM - protein spots on Golgi elements acting as MT nucleation sites ( arrowheads ) . Centrinone - treated pcnt KO , c5rap2 KO and akap KO cells , were incubated with NZ for 3h , fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated ( n = 3 ) . MT regrowth assay in centrinone - treated WT , pcnt KO and akap KO RPE - 1 cells stained with antibodies to EB1 and giantin . At right , quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone , as a measure of MT - nucleation from the GA . Data were collected from two independent experiments and normalized to WT mean .

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Figure 5Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean.MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.


Figure 6Representative MT network of a centrosome - less akap KO cell . MT - regrowth experiment after NZ treatment in centrinone - treated akap KO cells . A representative confocal image of a double labelling for EB1 ( green ) and giantin ( blue ) is shown . High magnification at right shows that growing MT asters are not associated to Golgi fragments . Centrinone - treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies . High magnification images of the boxed areas are shown as merge ( top ) or individual green or red labelling ( middle and bottom panels ) . Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 ( top panels ) or PCNT ( bottom panels ) . After 3 - min MT regrowth , cells were fixed and stained with the indicated antibodies . MT regrowth experiments in centrinone - treated akap KO cells transfected with either scramble or PCNT siRNAs . After a 3h NZ treatment , MTs were allowed to polymerize and , at the indicated time points after washout , cells were fixed and labelled for α - tubulin ( green ) , PCNT ( red ) and CAP350 ( blue ) . MT repolymerization experiments in centrinone - treated akap KO cells transfected with either scramble or PCNT siRNAs . After NZ treatment and removal , cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin . Cells were finally fixed and triple stained with EB1 ( green ) , PCNT ( red ) and CAP350 ( blue ) antibodies . Enlarged views of the boxed areas are presented at right . Scale bars , 5 μm .

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Figure 6Representative MT network of a centrosome-less akap KO cell.MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.


Figure 7Confocal images showing MT network in WT , akap KO , c5rap2 KO , pcnt KO and pc - ak - 2KO RPE - 1 cells fixed and stained with antibodies against α - tubulin ( left ) or EB1 ( right ) . The red and yellow lines indicate the contour of cells . Confocal images showing MT network in centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO backgrounds . Cells containing extra centrosomes induced by centrinone washout were also quantified ( bottom panels ) . Quantification of α - tubulin fluorescence intensity ( as a measure of MT mass polymer , C ) , in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean . Quantification of number of EB1 comets per cell ( D ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean . Quantification of α , cell area ( E ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean . Quantification of MT density ( MT mass polymer / cell area ) ( F ) in control and centrinone - treated WT , akap KO , c5rap2 KO and pcnt KO cells . Non - centrinone treated double KO cells and centrinone - treated WT cells after washout were also included . Scatter plots show each individual data point and horizontal black lines bars represent the mean .

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Figure 7Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells.Confocal images showing MT network in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.Quantification of number of EB1 comets per cell (D) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.Quantification of α , cell area (E) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.Quantification of MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


Figure 1A . Chemical structure of Nec - 1 . The schematic diagram was drawn by S . Lee using ChemDraw Professional 15 . 0 . software . B , C . MTT cytotoxicity assay . ( B ) HT22 cell line was treated with various concentrations of Nec - 1 ( 0 - 200 μM ) . Aggregated Aβ ( 1 - 42 ) was used as the positive control for the induction of neural cell death . All data represented the mean ± standard deviation of three independent experiments performed in triplicate . B , C . MTT cytotoxicity assay . ( C ) HT22 , BV2cell lines and primary astrocytes were pre - treated with Nec - 1 ( 50 μM ) 15 minutes prior to application of 10 μM of pre - aggregated Aβ ( 1 - 42 ) for indicated times . Cell proliferation was then measured by MTT assay . All data represented the mean ± standard deviation of three independent experiments performed in triplicate . D , E . Neural cell death levels measured by staining with the Live / Dead Viability / Cytotoxicity Assay Kit ( Molecular Probes ) . Pre - treatment of cells with 50 μM Nec - 1 preceded the application of 10 μM pre - aggregated Aβ ( 1 - 42 ) by 15 minutes . The assay was performed 24 hours after Aβ ( 1 - 42 ) application . ( D ) Representation of the stained cells . Scale bar = 500 μm ( E ) Quantification of dead cells stained by EthD - 1 . F . Internalization of Aβ ( 1 - 42 ) into HT22 and BV2 cell lines in the presence or the absence of Nec - 1 pre - treatment for indicated periods . G . Cellular expression levels of indicated proteins in HT22 , BV2 cell lines , and primary astrocyte stimulated with Aβ ( 1 - 42 ) aggregates in the presence or the absence of Nec - 1 pre - treatment for 24 hours .

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Figure 1A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.B, C. MTT cytotoxicity assay. (C) HT22, BV2cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.


Figure 2A - E . Behavioural tests of APP / PS1mice . ( B - D ) Y - maze and passive avoidance tests on 7 - month - old APP / PS1mice after Nec - 1 administration for 12 weeks . Average alternation ( % ) for each test group ( B ) and total entry number into each arm ( C ) on Y - maze . ( D ) Average latency time in seconds for each test group on passive avoidance test . A - E . Behavioural tests of APP / PS1mice . ( E ) Survival of Wt and APP / PS1mice after injection of Nec - 1 ( 6 . 25 mg / kg ) . F - I . Behavrioural tests of Aβ ( 1 - 42 ) infusion mice . ( H , I ) Y - maze tests on Aβ ( 1 - 42 ) infusion mice model injected with Nec - 1 . Average alternation ( % ) for each test group ( H ) and total entry number into each arm ( I ) on Y - maze .

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Figure 2A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test.A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg).F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.


Figure 3A . Co - immunoprecipitation of RIPK3 with Aβ ( 1 - 42 ) in HT22 , BV2 cell lines , and primary astrocytes in response to Aβ ( 1 - 42 ) aggregates . IP , immunoprecipitation ; IB , immunoblot . B . A close - up view of the interactions between Nec - 1 and surrounding residues in RIPK1 . Nec - 1 is shown in green . The hydrophobic residues around Nec - 1 in RIPK1 kinase domain are shown in sticks . Hydrogen bonds in this and all other figures are represented by yellow dashed lines . Docking score of Nec - 1 in RIPK1 was - 9 . 550 . C . Surface treated view of 12 - mer Aβ fibrils ( 2LMO , VDW radius = 0 . 55 Å ) having hydrophobic pockets plausible for Nec - 1 binding . D . Docked structure of 12 - mer Aβ fibrils ( 2LMO ) with 4 molecules of Nec - 1 . Docking scores for four top - ranked Glide docking poses of Nec - 1 in Aβ plaques ; - 8 . 338 , - 4 . 755 , - 8 . 493 , and - 6 . 754 in clockwise order starting from the top - left corner . E . Surface plasmon resonance sensorgrams of Nec - 1 targeting Aβ ( 1 - 42 ) aggregates ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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Figure 3A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot.B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


Figure 44 - month - old APP / PS1 ( n = 13 , male ) and age - matched wild - type ( Wt , n = 14 , male ) mice were intravenously injected with Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for 12 weeks . After the completion of behavioural tests , the brains of each group of mice were subjected to analysis . A . ThS - stained insoluble Aβplaques in whole brain and the hippocampal region of each group . Scale bars = 1 mM ( upper ) , 200 μM ( lower ) . B - G . Statistics of ThS - positive Aβplaques in the brains of APP / PS1mice . Total numbers and areas of ThS - positive Aβplaques in the whole brains ( B and C ) , cortex ( D and E ) and hippocampus ( F and G ) .

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Figure 44-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).


Figure 5A . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . Diffuse plaques in the brain sections were stained by anti - Aβ antibody ( 6E10 clone , green colour ) and anti - GFAP antibody ( red colour ) . Hoechst 33342 ( blue colour ) was applied for nuclear counterstaining . Scale bars = 200 μM . B - D . Dot blot analysis of total Aβ ( anti - Aβ : 6E10 clone , also recognizes APP ) and protein oligomer ( anti - amyloidogenic protein oligomer A11 ) in the cortical ( B ) and hippocampal ( C ) region of indicated groups . ( D ) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups . Relative intensity of each band was determined by densitometry of dot blots ( B and C ) using ImageJ software and normalized to vehicle - administered APP / PS1mouse group . B - D . Dot blot analysis of total Aβ ( anti - Aβ : 6E10 clone , also recognizes APP ) and protein oligomer ( anti - amyloidogenic protein oligomer A11 ) in the cortical ( B ) and hippocampal ( C ) region of indicated groups . ( D ) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups . Relative intensity of each band was determined by densitometry of dot blots ( B and C ) using ImageJ software and normalized to vehicle - administered APP / PS1mouse group . E . Sandwich ELISA of Aβ - soluble and - insoluble fractions from cortical and hippocampal lysates of APP / PS1mice . F . Western blot analyses of cortical and hippocampal lysates from APP / PS1mice for APP and sAPP expressions . G . ThT assays for the inhibition of Aβ aggregation . Fluorescence intensity was normalized to Aβaggregates ( 100 % , day 5 ) . H . SDS - PAGE analysis of PICUP cross - linked Aβaggregates visualized by silver staining .

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Figure 5A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group.B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group.E. Sandwich ELISA of Aβ-soluble and -insoluble fractions from cortical and hippocampal lysates of APP/PS1mice.F. Western blot analyses of cortical and hippocampal lysates from APP/PS1mice for APP and sAPP expressions.G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβaggregates (100%, day 5).H. SDS-PAGE analysis of PICUP cross-linked Aβaggregates visualized by silver staining.


Figure 6A . Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . The membranes analysed in ( A ) are identical to those in Fig 5F . B . Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild - type ( Wt ) and APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for phosphorylated tau expression levels . Anti - tau ( phospho Ser199 ) antibody ( red colour ) was applied for staining tau phosphorylation on Serine - 199 and Hoechst 33342 ( blue colour ) for nuclear counterstaining in the brain sections . Scale bars = 200 μM . C . Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec - 1 ( lower ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F . C . Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP / PS1mice injected with Nec - 1 ( upper ) . The membranes analysed in ( C ) are identical to those in Fig 1G and 5F . D , E . ThT assays for the inhibition of tau aggregation ( D ) and for disaggregation of tau ( E ) . Fluorescence intensity was normalized to tauaggregates ( 100 % , day 5 ) . F . Surface plasmon resonance sensorgrams of Nec - 1 targeting tau ( left ) , and the corresponding dissociation fitting curve from the saturated region ( right ) under various concentrations of Nec - 1 .

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Figure 6A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tauaggregates (100%, day 5).F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


Figure 1Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and BMI Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression , plasma alanine aminotransferase ( ALAT ) ( B ) Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and liver fat content ( C ) . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * * Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG5 ( E ) mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * * Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG12 mRNA expression . Data were collected in lean subjects ( n = 14 ) , obese subjects ( n = 23 ) and obese subjects with type 2 diabetes ( n = 19 ) . Values are expressed as mean ± SEM ( D ) . * * *

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Figure 1Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and BMI Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).Scatter plot and correlation coefficient (r) of ASK1 mRNA expression , plasma alanine aminotransferase (ALAT) (B) Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and liver fat content (C). Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).ASK1 mRNA expression in patients with different NASH scores Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG5 (E) mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG7 Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG12 mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***


Figure 2Hepatocytes were treated with BSA , Palm or Palm + Rap for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) . Scale bar represents 100 µm . Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ) or non - targeting siRNA control ( siCtrl ) . Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm , Palm + Rap and stained as mentioned above ( n = 4 biological replicates ) . Shown is one representative Western blot from two independent experiments and quantification of LC3 - II / I and p62 protein levels in siCtrl ( n = 4 biological replicates ) or siASK1 ( n = 4 biological replicates ) cells treated with Palm . Colocalization of LC3 - II punctate ( red ; arrow ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in siCtrl or siASK1 cells treated with BSA , Palm or Palm + Rap . Scale bar represents 100 µm . Colocalization of LC3 - II punctate with lipid droplets ( BODIPY 493 / 503 , green ) was quantified in hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ; grey bars ) or non - targeting siRNA control ( siCtrl ; black bars ) and treated with BSA or BSA + Baf for 24 hours ( n = 4 biological replicates ) . Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm or Palm + Baf and stained as mentioned above ( n = 4 biological replicates ) . Values are expressed as mean ± SEM . *

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Hepatocytes were treated with BSA, Palm or Palm+Rap for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). Scale bar represents 100 µm.Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 (siASK1) or non-targeting siRNA control (siCtrl).Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm, Palm+Rap and stained as mentioned above (n=4 biological replicates).Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm.Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm.Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates).Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *


Figure 3Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm . Liver triglyceride ( TG ) content of mice fed a chow ( ASK1F / F n = 6 mice ; ASK1Δhep n = 6 mice ) or HFD ( ASK1F / F n = 14 mice ; ASK1Δhep n = 15 mice ) . Relative mRNA expression of perilipin ( Plin ) and very low - density lipoprotein receptor ( Vldlr ) in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( Vldlr ) or n = 8 mice ( Plin ) ) . Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( F4 / 80 , Mcp1 ) or ASK1Δhep n = 8 mice ( Il - 6 ) ) . Relative mRNA expression of collagen , type I , alpha 1 ( Col1a1 ) ( ASK1F / F n = 11 mice ; ASK1Δhep n = 15 mice ) and transforming growth factor beta 1 ( Tgfβ1 ) ( ASK1F / F n = 6 mice ; ASK1Δhep n = 8 mice ) in liver of HFD - fed mice . Representative images of liver sections stained with Sirius Red ( scale bar represents 100 µm ) quantification of Sirius Red positive area in liver of mice fed a HFD ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) . Representative images of liver sections stained with α - SMA from mice fed a HFD for 20 weeks . Scale bar represents 100 µm . Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) . Liver triglyceride ( TG ) content ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) and quantification of Sirius Red positive area in livers ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) of 15 months old chow - fed mice . Values are expressed as mean ± SEM . #

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Figure 3Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice).Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)).Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)).Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice.Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm)quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #


Figure 4Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) . Representative immunofluorescence images of liver sections stained for LC3 - II punctate ( red ) and nuclei ( DAPI , blue ) in mice . Scale bar represents 100 µm .

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Figure 4Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm.


Figure 5Primary hepatocytes isolated from chow - fed ASK1F / F ( ) and ASK1Δhep ( ) mice were treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) . Colocalization of LC3 - II punctate ( red ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in primary hepatocytes treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf . Scale bar represents 100 µm . Values are expressed as mean ± SEM .

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Figure 5Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates).Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM.


Figure 6Western blots and quantification of respective protein levels in liver of HFD - fed mice treated with or without ASK1 inhibitor ( n = 4 mice per treatment ) . Representative images of liver sections stained with H & E ( scale bar represents 100 µm ) liver triglyceride ( TG ) content of HFD - fed mice treated with or without ASK1 inhibitor ( n = 8 mice per treatment ) .

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Figure 6Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment).Representative images of liver sections stained with H&E (scale bar represents 100 µm)liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).


Figure 7Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm . Liver triglyceride ( TG ) content ( ASK1F / F n = 11 mice ; ASK1 + hep n = 10 mice ) in livers of mice fed a HFD for 20 weeks . Western blots and quantification of protein levels of respective targets in liver of HFD - fed mice ( n = 8 mice per genotype for pJNK and ATG12 , n = 4 mice per genotype for pBCN1 ) . Quantification of Sirius Red positive area ( ASK1F / F n = 11 mice ; ASK1 + hep n = 9 mice ) in livers of CCl4 - injected mice . Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM . # Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks.Western blots and quantification of protein levels of respective targets in liver of HFD-fed mice (n=8 mice per genotype for pJNK and ATG12, n=4 mice per genotype for pBCN1).Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice.Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM.


Figure 1Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) and western blotting . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control . M . W . indicates protein molecular weight marker . Western blotting of Myc - Egg expressed in OSCs . Anti - Myc ( Myc ) and anti - β - Tubulin ( Tub ) antibodies were used . Tub was detected as a loading control . Control : empty vector was used as a negative control . Western blotting of endogenous Egg immunopurified from OSCs . Anti - Egg antibodies were used for immunoprecipitation ( IP ) . Anti - Egg and anti - Ub antibodies were used for western blotting ( WB ) . Input : OSC total lysates used for IP . n . i . : nonimmune IgG used as an IP negative control . Western blotting of Egg immunopurified from total OSC lysates before ( - ) and after ( + ) phosphatase treatment . Anti - Egg antibodies were used for both immunoprecipitation ( IP ) and western blotting . Western blotting of Myc - Egg WT and its K1085R and S215 / T217 ( STAA ) mutants expressed in OSCs . Egg modifications identified in this study are summarized on the right . P : phosphorylation . Ub : monoubiquitination . In vitro histone methyltransferase assays . Top and second from the top : silver staining of purified Myc - Egg and Myc - EGFP . Second from the bottom : CBB staining of histone H3 . 1 . Bottom : 14C autoradiogram . EGFP : Myc - EGFP was used as a negative control . l . c . : light chain of antibody . Time : reaction time ( min ) of individual samples . Rescue assays . Myc - Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting ( WB ) was performed using anti - Egg and anti - Myc antibodies . RT - qPCR was used to quantify Stalker2 transposon expression level . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . n . s . : not significant .

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Figure 1Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP) and western blotting. Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control. M.W. indicates protein molecular weight marker.Western blotting of Myc-Egg expressed in OSCs. Anti-Myc (Myc) and anti-β-Tubulin (Tub) antibodies were used. Tub was detected as a loading control. Control: empty vector was used as a negative control.Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.Western blotting of Egg immunopurified from total OSC lysates before (-) and after (+) phosphatase treatment. Anti-Egg antibodies were used for both immunoprecipitation (IP) and western blotting.Western blotting of Myc-Egg WT and its K1085R and S215/T217 (STAA) mutants expressed in OSCs. Egg modifications identified in this study are summarized on the right. P: phosphorylation. Ub: monoubiquitination.In vitro histone methyltransferase assays. Top and second from the top: silver staining of purified Myc-Egg and Myc-EGFP. Second from the bottom: CBB staining of histone H3.1. Bottom: 14C autoradiogram. EGFP: Myc-EGFP was used as a negative control. l.c.: light chain of antibody. Time: reaction time (min) of individual samples.Rescue assays. Myc-Egg WT and its K1085R and STAA mutants were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting (WB) was performed using anti-Egg and anti-Myc antibodies. RT-qPCR was used to quantify Stalker2 transposon expression level. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test). n.s.: not significant.


Figure 2Immunofluorescence analysis of OSCs using anti - Egg antibodies . Endogenous Egg is shown in red . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting of fractionated OSC materials . Anti - Egg , anti - β - Tubulin ( Tub ) , and anti - histone H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : the whole OSC lysates . Western blotting of Myc - Egg expressed in OSCs before ( siEGFP ) and after ( siUbc2 ) Ubc2 depletion . Anti - Myc - antibody was used for western blotting . The ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 43 ( siEGFP ) and 0 . 78 ( siUbc2 ) . Immunofluorescence analysis using anti - Flag antibody to identify Flag - Ubc2 ( green ) expressed in OSCs . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Immunofluorescence analysis of Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg expressed in OSCs using anti - Myc antibody . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting of Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg expressed in OSCs . Anti - Myc antibody was used . The ratios of mUb - Egg over non - mUb - Egg band signals were 1 . 50 ( WT ) , 2 . 29 ( SV40 - NLS ) , and 0 . 95 ( PKI - NES ) . Rescue assays . Myc - Egg WT , Myc - SV40 - NLS - Egg , and Myc - PKI - NES - Egg were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . Stalker2 transposon expression was quantified using RT - qPCR . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) . In vitro HMT assays . Top : silver staining of purified Myc - Egg WT , Myc - Egg K1085R , Myc - SV40 - NLS - Egg WT , and Myc - SV40 - NLS - Egg K1085R . Middle : CBB staining of histone H3 . 1 . Bottom : 14C autoradiogram . Myc - EGFP was used as a negative control ( EGFP : not shown ) . Rescue assays . Myc - Egg WT , Myc - Egg K1085R , and Myc - SV40 - NLS - K1085R were expressed in OSCs upon endogenous Egg depletion ( siEgg ) . Western blotting was performed using anti - Egg ( left ) and anti - Myc ( right ) antibodies . RT - qPCR quantified the expression level of the Stalker2 transposon . The act5c gene was used for normalization . siEGFP : siRNA for EGFP was used as a negative control . Error bars represent SD from three independent experiments . * p < 0 . 05 ( unpaired Student ' s t - test ) .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Immunofluorescence analysis of OSCs using anti-Egg antibodies. Endogenous Egg is shown in red. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of fractionated OSC materials. Anti-Egg, anti-β-Tubulin (Tub), and anti-histone H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.Western blotting of Myc-Egg expressed in OSCs before (siEGFP) and after (siUbc2) Ubc2 depletion. Anti-Myc-antibody was used for western blotting. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.43 (siEGFP) and 0.78 (siUbc2).Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Immunofluorescence analysis of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs using anti-Myc antibody. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs. Anti-Myc antibody was used. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.50 (WT), 2.29 (SV40-NLS), and 0.95 (PKI-NES).Rescue assays. Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. Stalker2 transposon expression was quantified using RT-qPCR. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).In vitro HMT assays. Top: silver staining of purified Myc-Egg WT, Myc-Egg K1085R, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R. Middle: CBB staining of histone H3.1. Bottom: 14C autoradiogram. Myc-EGFP was used as a negative control (EGFP: not shown).Rescue assays. Myc-Egg WT, Myc-Egg K1085R, and Myc-SV40-NLS-K1085R were expressed in OSCs upon endogenous Egg depletion (siEgg). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of the Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test).


Figure 3Immunofluorescence analysis of endogenous Egg ( red ) in OSCs using anti - Egg antibody before ( siEGFP ) and after ( siWde ) Wde depletion . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting on fractionated materials of OSCs before ( siEGFP ) and after ( siWde ) Wde depletion . Anti - Egg , anti - Wde , anti - Tub , and anti - H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : whole OSC lysates . Immunofluorescence analysis of endogenous Egg ( red ) in OSCs upon Flag - Wde ( green ) co - expression . Anti - Egg and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Immunofluorescence analysis of Myc - Egg WT ( red ) in OSCs upon Wde co - expression ( green ) . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting on fractionated materials of Myc - Egg - expressed OSCs before ( - ) and after Wde WT and Wde ∆ CC co - expression . Anti - Myc , anti - Flag , anti - Tub , and anti - H3 antibodies were used . H3 and Tub were detected as markers for nuclear ( Nuc ) and cytoplasmic ( Cyto ) fractions , respectively . Whole : the whole OSC lysates . Protein - protein interaction assays . Myc - Egg and Flag - Wde WT and its ∆ CC mutant were expressed in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) and anti - Flag ( lower ) antibodies were used for western blotting . Immunofluorescence analysis of Myc - Egg WT ( red ) in OSCs upon Flag - Wde WT and ∆ CC mutant ( green ) co - expression . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm .

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Figure 3Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting on fractionated materials of OSCs before (siEGFP) and after (siWde) Wde depletion. Anti-Egg, anti-Wde, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: whole OSC lysates.Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting on fractionated materials of Myc-Egg-expressed OSCs before (-) and after Wde WT and Wde ∆CC co-expression. Anti-Myc, anti-Flag, anti-Tub, and anti-H3 antibodies were used. H3 and Tub were detected as markers for nuclear (Nuc) and cytoplasmic (Cyto) fractions, respectively. Whole: the whole OSC lysates.Protein-protein interaction assays. Myc-Egg and Flag-Wde WT and its ∆CC mutant were expressed in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.


Figure 4Immunofluorescence analysis of Myc - Egg WT , Myc - Egg K987A , and Myc - Egg R988A mutants ( red ) in OSCs before and after Flag - Wde ( green ) co - expression . Anti - Myc and anti - Flag antibodies were used . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Western blotting of Myc - Egg WT and its K987A and R988A mutants . Flag - Wde : Flag - Wde was co - expressed . SV40 - NLS : SV40 - NLS was added to Myc - Egg . Anti - Myc and anti - Flag antibodies were used . Immunofluorescence analysis of Myc - Egg K987A and Myc - Egg R988A mutants ( red ) in OSCs . Both mutants were fused to SV40 - NLS . Anti - Myc antibody was used for detection . DAPI ( blue ) shows the nuclei . Scale bar : 10 μm . Protein - protein interaction assays . Myc - Egg WT , Myc - SV40 - NLS - Egg WT , and Myc - SV40 - NLS - Egg K1085R mutant were expressed with Flag - Wde in OSCs . Immunoprecipitation was conducted using anti - Flag antibody while anti - Myc ( upper ) and anti - Flag ( lower ) antibodies were used for western blotting .

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Figure 4Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Western blotting of Myc-Egg WT and its K987A and R988A mutants. Flag-Wde: Flag-Wde was co-expressed. SV40-NLS: SV40-NLS was added to Myc-Egg. Anti-Myc and anti-Flag antibodies were used.Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.Protein-protein interaction assays. Myc-Egg WT, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R mutant were expressed with Flag-Wde in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.