A U2OS treated with control siRNA , RBM14 siRNA alone or RBM14 + HsSAS - 6 siRNAs were stained with antibodies against centrin - 2 ( green ) HsSAS - 6 ( magenta ) . is shown in blue . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic with excess centrin foci in indicated conditions . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . n . s . , not significant ( one - t - ) .

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A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test).


B Live imaging of cycling RBM14 - depleted HeLa expressing GFP - centrin1 ( green ) RFP - H2B ( magenta ) . Insets show approximately 1 . 5 - fold magnified of foci . Time is denoted in hh : min , scale bar is 10 μm . Time zero corresponds to onset of excess centrin foci formation . of number total signal of centrin foci in HeLa treated with control or RBM14 siRNA over time . Means ± are shown ( n = 5 ) .

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B Live imaging of cycling RBM14-depleted HeLa cells expressing GFP-centrin1 (green) and RFP-H2B (magenta). Insets show approximately 1.5-fold magnified images of fluorescent foci. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of excess centrin foci formation. Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time. Means ± SEM are shown (n = 5).


C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase . Time - lapse recording across cycle of HeLa expressing GFP - centrin ( green ) RFP - H2B ( magenta ) treated with RBM14 siRNA . Time is denoted in hh : min , scale bar is 10 μm . Time zero corresponds to metaphase onset .

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C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase. Time-lapse recording across the cell cycle of HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the metaphase onset.


D Ectopic formation of centriolar complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment . Insets show threefold magnified of foci . Scale bar , 5 μm . Histograms represent frequency of mitotic with excess centrin foci in indicated conditions . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 , n . s . , not significant ( one - t - ) .

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D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment. Insets show threefold magnified images of fluorescent foci. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).


E Amorphous electron - dense structures containing microtubules are formed in RBM14 - depleted ( see also Supplementary Fig S5I ) . Electron from U2OS depleted of RBM14 . Squares indicate ectopic electron - dense structures , whereas circles indicate pre - existing centrioles . Scale bar , 500 nm 200 nm ( magnified views ) . Average diameter of pre - existing centrioles amorphous electron - dense structures are 225 ± 8 nm ( n = 10 ) 130 + 11 nm ( n = 16 ) , respectively . Insets show magnified views of squares . Note that microtubules ( black arrowheads ) are observed within electron - dense structures ( arrowheads ) .

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E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads).


F Histograms represent frequency of with ectopic foci of GFP - CPAP in indicated conditions . U2OS expressing GFP - CPAP were fixed stained 24 h after RNAi treatment induction of GFP - CPAP . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * * P < 0 . 01 ( one - t - ) .

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F Histograms represent frequency of the cells with ectopic foci of GFP-CPAP in the indicated conditions. U2OS cells expressing GFP-CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP-CPAP. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test).


G U2OS expressing GFP - CPAP ( green ) treated with RBM14 siRNA were analyzed using CLEM . Separate z - plane of with GFP - CPAP foci threefold magnified are aligned from bottom to top along z - axis ( left LM panels ) . broken lines represent shapes , blue broken lines represent shapes . Corresponding regions of EM are shown on right . squares ( a - d ) represent region around GFP - CPAP foci . Note that other than pre - existing centrioles ( a , 1 - 4 ) , ectopic small CPAP foci are recognizable as amorphous electron - dense structures ( b - d ) . One of pre - existing centrioles ( 1 ) is elongated because of exogenous GFP - CPAP expression . Scale bar , 10 μm ( LM ) , 2 μm ( EM , low magnified image ) , 500 nm [ magnified of ( a ) ( d ) ) , 200 nm ( magnified of ( b ) ( c ) ] .

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G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a-d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1-4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b-d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)].


B - E Control U2OS or U2OS treated with siRNA targeting RBM14 were cold - treated for 30 min , followed by 30 - 60 min incubation at 37 ° C stained with antibodies against centrin ( magenta in ( B ) ( D ) ) or γ - tubulin ( magenta in ( E ) ) as well as α - tubulin ( green ) . is shown in blue . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm . Histograms represent percentages of mitotic showing indicated phenotype at each time point . Values are mean percentages ± standard error of mean ( ) from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 , * * P < 0 . 01 ( one - t - ) .

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B-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).


A U2OS treated with RBM14 siRNA stained with antibodies against centrin - 2 ( green ) as well as HsSAS - 6 ( magenta ) . is shown in blue . Insets show approximately twofold magnified views of foci around centrosome . Scale bar , 5 μm . Histograms represent frequency of mitotic with indicated phenotype . Values are mean percentages ± from three independent experiments ( n = 60 for each condition ) .

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A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin-2 (green) as well as HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the frequency of mitotic cells with the indicated phenotype. Values are mean percentages ± SEM from three independent experiments (n = 60 for each condition).


B Control U2OS or U2OS treated with siRNA targeting RBM14 were stained with antibodies against HsSAS - 6 as well as centrin - 2 . Immunofluorescence are shown in Supplementary Fig S6D . When we counted HsSAS - 6 foci in this paper , we only counted before metaphase / anaphase transition considering disappearance of HsSAS - 6 from anaphase in mitosis . Histograms represent frequency of mitotic with excess foci of indicated at spindle poles . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - t - ) .

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B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS-6 as well as centrin-2. Immunofluorescence micrographs are shown in Supplementary Fig S6D. When we counted HsSAS-6 foci in this paper, we only counted cells before metaphase/anaphase transition considering the disappearance of HsSAS-6 from anaphase in mitosis. Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


C U2OS treated with indicated siRNAs were stained with anti - α - tubulin antibodies Hoechst 33258 . Histograms represent frequency of mitotic with multipolar spindle in indicated conditions . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - t - ) .

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C U2OS cells treated with the indicated siRNAs were stained with anti-α-tubulin antibodies and Hoechst 33258. Histograms represent frequency of mitotic cells with the multipolar spindle in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


D RBM14 - depleted HeLa expressing GFP - centrin ( green ) were stained with antibodies against CP110 ( magenta ) HsSAS - 6 ( blue ) . Scale bar , 1 μm . A representative stained procentriole - like structure as well as endogenous centrioles is shown together with a schematic . Histograms represent between centers of foci of indicated . Values are mean percentages ± from seven .

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D RBM14-depleted HeLa cells expressing GFP-centrin (green) were stained with antibodies against CP110 (magenta) and HsSAS-6 (blue). Scale bar, 1 μm. A representative stained procentriole-like structure as well as endogenous centrioles is shown together with a schematic. Histograms represent length between the centers of the foci of the indicated proteins. Values are mean percentages ± SEM from seven individual cells.


E , F Ectopic DsRed - centrin - 2 foci in RBM14 - depleted recruit HsSAS - 6 - GFP . ( E ) Live imaging of RBM14 - depleted U2OS expressing HsSAS - 6 - GFP ( green ) DsRed - centrin - 2 ( magenta ) . Time is denoted in hh : min , scale bar is 10 μm . Time zero corresponds to onset of recording . Pre - existing HsSAS - 6 - GFP foci ( arrowheads ) ectopic HsSAS - 6 - GFP focus ( red arrowhead ) were traced throughout time - lapse recording . broken lines represent shapes , blue broken lines represent shapes . ( F ) Histograms represent percentages of ectopic DsRed - centrin - 2 foci with HsSAS - 6 - GFP recruitment ( n = 69 foci for control , n = 67 foci for RBM14 siRNA ) .

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E, F Ectopic DsRed-centrin-2 foci in RBM14-depleted cells recruit HsSAS-6-GFP. (E) Live imaging of RBM14-depleted U2OS cells expressing HsSAS-6-GFP (green) and DsRed-centrin-2 (magenta). Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of recording. Pre-existing HsSAS-6-GFP foci (white arrowheads) and ectopic HsSAS-6-GFP focus (red arrowhead) were traced throughout the time-lapse recording. White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. (F) Histograms represent the percentages of ectopic DsRed-centrin-2 foci with HsSAS-6-GFP recruitment (n = 69 foci for control, and n = 67 foci for RBM14 siRNA).


G Histograms represent frequency of mitotic with excess foci of HsSAS - 6 in indicated conditions . In this experiment , were treated with double amount of RBM14 siRNA ( 20 pmol ) compared to that in other experiments . Values are mean percentages ± from three independent experiments ( n = 30 for each condition ) . * P < 0 . 05 ( one - t - ) .

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G Histograms represent frequency of mitotic cells with excess foci of HsSAS-6 in the indicated conditions. In this experiment, cells were treated with double amount of RBM14 siRNA (20 pmol) compared to that in the other experiments. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P < 0.05 (one-tailed t-test).


H HeLa expressing GFP - centrin ( green ) RFP - H2B ( magenta ) treated with RBM14 siRNA were analyzed using Live CLEM . Pre - existing GFP - centrin foci ( arrowheads ) ectopic GFP - centrin foci ( red arrowheads ) were traced throughout time - lapse recording . Z - stacked confocal spanning entire height of ( < 30 μm ) are shown ( upper panels ) . Time is denoted in hh : min . Time zero corresponds to onset of ectopic formation of GFP - centrin foci . fixed after live imaging were also analyzed for confirming local correlation between LM EM ( lower panels ) . Red square ( ectopically formed GFP - centrin foci ) represents region corresponding to that of EM acquired from serial sections . Note that ectopic GFP - centrin foci that form a spindle pole include not only electron - dense structures ( 1 , 3 ) but also a morphologically recognizable centriole - like structure ( 2 ) . Scale bars , 10 μm ( live imaging ) 500 nm ( EM ) .

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H HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA were analyzed using Live CLEM. Pre-existing GFP-centrin foci (white arrowheads) and ectopic GFP-centrin foci (red arrowheads) were traced throughout the time-lapse recording. Z-stacked confocal images spanning the entire height of the cells (< 30 μm) are shown (upper panels). Time is denoted in hh:min. Time zero corresponds to the onset of ectopic formation of GFP-centrin foci. The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images (lower panels). Red square (ectopically formed GFP-centrin foci) represents the region corresponding to that of the EM images acquired from serial sections. Note that ectopic GFP-centrin foci that form a spindle pole include not only electron-dense structures (1, 3) but also a morphologically recognizable centriole-like structure (2). Scale bars, 10 μm (live imaging) and 500 nm (EM).


( E ) Confirmation of PARylated with an in vivo PARylation . FLAG - tagged candidate were expressed in protoplasts , immunoprecipitated by α - FLAG antibody after feeding protoplasts with 32P - NAD + . immunoprecipitated were separated in 10 % SDS - PAGE detected by autoradiography ( top panel ) . input of FLAG - tagged is shown in an α - FLAGimmunoblot ( middle panel ) , loading control is shown by Ponceau S for RuBisCo ( RBC ) ( bottom panel ) . experiments were repeated three times with similar results .

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(E) Confirmation of PARylated proteins with an in vivo PARylation assay. FLAG-tagged candidate proteins were expressed in protoplasts, and immunoprecipitated by α-FLAG antibody after feeding protoplasts with 32P-NAD+. The immunoprecipitated proteins were separated in 10% SDS-PAGE and detected by autoradiography (top panel). The input of FLAG-tagged proteins is shown in an α-FLAGimmunoblot (middle panel), and the protein loading control is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). The experiments were repeated three times with similar results.


( B ) DDL transcripts growth phenotype of ddl - 6 ddl - 7 . DDL transcripts were detected in fully expanded leaves of four - week - old by qRT - PCR ( left panel ) . expression of DDL in WT Col - 0 was set as 1 . grown under 12 - hr / 12 - hr condition for 4 are shown ( right panel ) . Bar = 1cm .

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(B) The DDL transcripts and growth phenotype of ddl-6 and ddl-7. DDL transcripts were detected in fully expanded leaves of four-week-old plants by qRT-PCR (left panel). The expression of DDL in WT Col-0 was set as 1. Plants grown under 12-hr light/12-hr dark condition for 4 weeks are shown (right panel). Bar=1cm.


( C ) ddl - 6 ddl - 7 are more susceptible to Pst DC3000 infections . Leaves of four - week - old were inoculated with Pst DC3000 at OD600 = 5x10 - 4 . numbers were counted at 0 3 dpi . Leaf pictures were taken at 3 dpi .

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(C) ddl-6 and ddl-7 are more susceptible to Pst DC3000 infections. Leaves of four-week-old plants were inoculated with Pst DC3000 at OD600=5x10-4. Bacterial numbers were counted at 0 and 3 dpi. Leaf pictures were taken at 3 dpi.


( D ) ddl - 1 mutant in Ws background is more susceptible to Pst DC3000 infection .

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(D) The ddl-1 mutant in the Ws background is more susceptible to Pst DC3000 infection.


( E ) ddl - 6 is more susceptible to Psminfection . Leaves of four - week - old were inoculated with Psmat OD600 = 5x10 - 4 .

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(E) ddl-6 is more susceptible to Psminfection. Leaves of four-week-old plants were inoculated with Psmat OD600=5x10-4.


( F ) Increased growth of Pst DC3000 hrcC in ddl - 6 . Leaves of four - week - old were inoculated with Pst DC3000 hrcC at OD600 = 5x10 - 4 .

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(F) Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of four-week-old plants were inoculated with Pst DC3000 hrcC at OD600=5x10-4.


( G ) Increased growth of Psp in ddl - 6 . Leaves of four - week - old were inoculated with Psp at OD600 = 5x10 - 4 .

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(G) Increased bacterial growth of Psp in ddl-6. Leaves of four-week-old plants were inoculated with Psp at OD600=5x10-4.


( H ) Complementation of DDL in disease resistance . Two independent T3 homozygous lines carrying pDDL : : DDL - FLAG ( W7 W18 ) in ddl - 6 background were assayed for susceptibility to Pst DC3000 .

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(H) Complementation of DDL in disease resistance. Two independent T3 homozygous lines carrying pDDL::DDL-FLAG (W7 and W18) in the ddl-6 background were assayed for the susceptibility to Pst DC3000.


( A ) flg22 - induced callose deposition is reduced in ddl - 6 parp1 , 2 . Leaves of four - week - old were inoculated with 0 . 5 µM flg22 for 20 hr or hrcC at OD600 = 0 . 2 for 24 hr stained with aniline blue . Callose deposits were visualized under UV quantified by Image J . data are shown as mean ± SE ( n = 12 ) . Bar = 100 µm .

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(A) flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of four-week-old plants were inoculated with 0.5 µM flg22 for 20 hr or hrcC at OD600=0.2 for 24 hr and stained with aniline blue. Callose deposits were visualized under UV light and quantified by Image J. The data are shown as mean ± SE (n=12). Bar=100 µm.


( B ) hrcC - mediated induction of some late responsive genes is compromised in ddl - 6 parp1 , 2 mutants . Fully expanded leaves of four - week - old were - inoculated with hrcC ( OD600 = 0 . 5 ) , collected at 0 , 3 24 hpi for qRT - PCR . data are shown as mean ± SE from three independent repeats .

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(B) The hrcC-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of four-week-old plants were hand-inoculated with hrcC(OD600=0.5), and collected at 0, 3 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats.


( C ) Pst DC3000 - induced PR1and PR5transcripts are blocked in ddl - 6 parp1 , 2 . Fully expanded leaves of four - week - old were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , collected at 0 , 8 24 hpi for qRT - PCR . data are shown as mean ± SE from three independent repeats .

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(C) Pst DC3000-induced PR1and PR5transcripts are blocked in ddl-6 and parp1,2. Fully expanded leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, 8 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats.


( D ) flg22 - induced MAPK activation in Col - 0 ddl - 6 . Ten - day - old seedlings were treated with 100 nM flg22 collected at indicted time points . MAPK activation was analyzed by an immunoblot ( IB ) with - pERK antibody ( top panel ) , loading is shown by Ponceau S for RuBisCo ( RBC ) ( bottom panel ) .

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(D) flg22-induced MAPK activation in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 and collected at the indicted time points. MAPK activation was analyzed by an immunoblot (IB) with -pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel).


( E ) flg22 - induced ROS in Col - 0 ddl - 6 . Leaf discs from four - week - old were assayed for ROS production upon 100 nM flg22 treatment over 30 min . data are show as mean ± SE ( n = 24 ) .

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(E) flg22-induced ROS in Col-0 and ddl-6. Leaf discs from four-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean± SE (n=24).


( A ) PARP2 DDL association in Arabidopsis protoplasts . PARP2 - HA DDL - FLAG were transiently expressed in Col - 0protoplasts , immunoprecipitated with α - FLAG antibody ( IP : - FLAG ) , immunoblotted with α - HA ( IB : α - HA ) or α - FLAG antibody ( IB : - FLAG ) ( top two panels ) . inputs are shown with immunoblotting before immunoprecipitation ( bottom two panels ) . Protoplasts were treated without or with 100 nM flg22 for 30 min .

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(A) PARP2 and DDL association in Arabidopsis protoplasts. PARP2-HA and DDL-FLAG were transiently expressed in Col-0protoplasts, immunoprecipitated with α-FLAG antibody (IP: -FLAG), and immunoblotted with α-HA (IB: α-HA) or α-FLAG antibody (IB: -FLAG) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). Protoplasts were treated without or with 100 nM flg22 for 30 min.


( B ) PARP2 DDL association in N . benthamiana . PARP2 - FLAG DDL - HA were transiently co - expressed in leaves of N . benthamiana . samples were collected at 2 dpi for Co - IP with 100nM flg22 treatment for 1 hr .

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(B) PARP2 and DDL association in N. benthamiana. PARP2-FLAG and DDL-HA were transiently co-expressed in leaves of N. benthamiana. The samples were collected at 2 dpi for Co-IP assay with 100nM flg22 treatment for 1 hr.


( C ) PARP2 DDL association with BiFC . Different combinations of constructs were transiently expressed in Arabidopsis Col - 0 protoplasts signals were observed with a confocal microscope . An NLS - RFP construct was co - transfected to show signals . Bar = 10 µm .

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(C) PARP2 and DDL association with BiFC assay. Different combinations of constructs were transiently expressed in Arabidopsis Col-0 protoplasts and the fluorescence signals were observed with a confocal microscope. An NLS-RFP construct was co-transfected to show the nuclear signals. Bar=10 µm.


( D ) PARP2 DDL interaction in vitro . GST - PARP2 immobilized on glutathione beads was used to pull - downHIS - SUMO - DDL - HA ( left panels ) . A PARylation reaction was performed prior to pull - down ( third lane of left panels ) . PARylated PARP2 DDL were detected with α - PAR antibody ( right panels ) .

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(D) PARP2 and DDL interaction in vitro. GST-PARP2 protein immobilized on glutathione beads was used to pull-downHIS-SUMO-DDL-HA proteins (left panels). A PARylation reaction was performed prior to the pull-down assay (the third lane of left panels). The PARylated PARP2 and DDL were detected with α-PAR antibody (right panels).


( E ) In vitro PARylation of DDL by PARP2 . Immunoprecipitated DDL - FLAG from Arabidopsis protoplasts were incubated with GST - PARP2 in a PARylation reaction containing Biotin - NAD + . reactions in lane 4 5 contained 1 mM 3 - AB or GST - PARG1 respectively . PARylated were detected by streptavidin - HRP antibody ( Strep ) , which recognizes Biotin - labelled PAR .

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(E) In vitro PARylation of DDL by PARP2. Immunoprecipitated DDL-FLAG proteins from Arabidopsis protoplasts were incubated with GST-PARP2 in a PARylation reaction containing Biotin-NAD+. The reactions in lane 4 and 5 contained 1 mM 3-AB or GST-PARG1 respectively. PARylated proteins were detected by streptavidin-HRP antibody (Strep), which recognizes Biotin-labelled PAR.


( A ) flg22 stimulates DDL PARylation . DDL - FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P - NAD + . Protoplasts were treated without or with 100 nM flg22 for 30 min . PARylated DDL was immunoprecipitated with α - FLAG affinity beads , separated on 10 % SDS - PAGE visualized by autoradiography . PAR ( DDL ) indicates PARylated DDL . A representative result from three repeats is shown .

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(A) flg22 stimulates DDL PARylation. DDL-FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P-NAD+. Protoplasts were treated without or with 100 nM flg22 for 30 min. PARylated DDL was immunoprecipitated with α-FLAG affinity beads, separated on 10% SDS-PAGE and visualized by autoradiography. PAR(DDL) indicates PARylated DDL proteins. A representative result from three repeats is shown.


( B - C ) DDL PARylation is reduced in parp1 , parp2 parp1 , 2 mutants . Protoplasts isolated from parp1or parp2 single mutants ( B ) , or parp1 , 2 double mutants ( C ) were used for in vivo PARylation with 32P - NAD + . Protoplasts were treated with 100 nM flg22 for 30 min . experiments were repeated twice with similar results .

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(B-C) DDL PARylation is reduced in parp1, parp2 and parp1,2 mutants. Protoplasts isolated from parp1or parp2 single mutants (B), or parp1,2 double mutants (C) were used for in vivo PARylation assay with 32P-NAD+. Protoplasts were treated with 100 nM flg22 for 30 min. The experiments were repeated twice with similar results.


( A ) DDL - N but not DDL - C associates with PARP2 . FLAG - tagged DDL - N or DDL - C was co - expressed with PARP2 - HA in protoplasts for Co - IP . Protoplasts were treated without or with 100 nM flg22 for 30 min .

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(A) DDL-N but not DDL-C associates with PARP2. FLAG-tagged DDL-N or DDL-C was co-expressed with PARP2-HA in protoplasts for Co-IP assay. Protoplasts were treated without or with 100 nM flg22 for 30 min.


( B ) in vivo PARylation of DDL - N or DDL - C in protoplasts fed with 32P - NAD + . PARylation was visualized with autoradiography .

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(B) in vivo PARylation of DDL-N or DDL-C in protoplasts fed with 32P-NAD+. PARylation was visualized with autoradiography.


( C ) In vitro PARylation of DDL - N or DDL - C . FLAG - tagged DDL - N or DDL - C was expressed immunoprecipitated from Arabidopsis protoplasts subjected to in vitro PARylation by GST - PARP2 with Biotin - NAD + . PARylated were detected by streptavidin - HRP antibody .

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(C) In vitro PARylation of DDL-N or DDL-C. FLAG-tagged DDL-N or DDL-C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST-PARP2 with Biotin-NAD+. PARylated proteins were detected by streptavidin-HRP antibody.


( F ) DDL12E mutants substantially diminish its PARylation in vivo . In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P - NAD + .

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(F) DDL12E mutants substantially diminish its PARylation in vivo. In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P-NAD+.


( G ) Reduced association of DDL12E1 with PARP2 in protoplasts . Protoplasts were treated with 100 nm flg22 for 30 min before Co - IP .

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(G) Reduced association of DDL12E1 with PARP2 in protoplasts. Protoplasts were treated with 100 nm flg22 for 30 min before Co-IP assay.


( A ) Phenotype of complemented with WT DDL or DDL12E1 . Four - week - old of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 W18 ) or pDDL : : DDL12E1 - FLAG ( E11 E15 ) . expression of transgene was determined by an α - FLAGimmunoblot ( bottom ) .

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(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15). The expression of transgene was determined by an α-FLAGimmunoblot (bottom).


( A ) Phenotype of complemented with WT DDL or DDL12E1 . Four - week - old of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 W18 ) or pDDL : : DDL12E1 - FLAG ( E11 E15 ) were photographed . Bar = 1cm .

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(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15) were photographed. Bar=1cm.


( B ) Susceptibility of complementation lines to Pst DC3000 infections . Four - week - old transgenic were inoculated with Pst DC3000 ( OD600 = 5x10 - 4 ) number was determined at 0 3 dpi .

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(B) Susceptibility of complementation lines to Pst DC3000 infections. Four-week-old transgenic plants were inoculated with Pst DC3000 (OD600=5x10-4) and the bacterial number was determined at 0 and 3 dpi.


( C ) PR1 PR5 induction in transgenic lines . Leaves of four - week - old were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , collected at 0 , 24 hpi for qRT - PCR . data in B C are shown as mean ± SD ( n = 3 ) from three independent repeats with one - way ANOVA Tukey ( p < 0 . 05 ) . Different letters indicate significant differences .

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(C) PR1 and PR5 induction in transgenic lines. Leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, and 24 hpi for qRT-PCR analysis. The data in B and C are shown as mean ± SD (n=3) from three independent repeats with one-way ANOVA analysis and Tukey test (p<0.05). Different letters indicate significant differences.


( D ) flg22 - induced callose deposition in transgenic lines . Leaves of four - week - old were inoculated with 500 nM flg22 , callose deposits were stained with aniline blue at 20 hpi . Number of callose deposits was quantified with Image J ( mean ± SE , n = 10 ) . experiments were repeated three times with similar results .

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(D) flg22-induced callose deposition in transgenic lines. Leaves of four-week-old plants were inoculated with 500 nM flg22, and callose deposits were stained with aniline blue at 20 hpi. Number of callose deposits was quantified with Image J (mean ± SE, n=10). All experiments were repeated three times with similar results.


F , G Immunoblot of ST3GAL5 in ( F ) NMuMG ( G ) A549 - VIM - RFP treated with vehicle control or TGF - β for indicated time . Vinculin GAPDH : loading controls .

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F, G Immunoblot analysis of ST3GAL5 in (F) NMuMG and (G) A549-VIM-RFP cells treated with vehicle control or TGF-β for the indicated time. Vinculin and GAPDH: loading controls.


Immunoblot of p - SMAD2 total SMAD2 ( t - SMAD2 ) in NMuMG with or without Ugcg deficiency treated with vehicle control or TGF - β for 1 h . Tubulin : loading control .

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Immunoblot analysis of p-SMAD2 and total SMAD2 (t-SMAD2) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF-β for 1 h. Tubulin: loading control.


Immunoblot of E - cadherin N - cadherin in Ugcg KO NMuMG or control NMuMG treated with vehicle control or TGF - β for 48 h . Tubulin : loading control .

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Immunoblot analysis of E-cadherin and N-cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF-β for 48 h. Tubulin: loading control.


Immunoblot of p - SMAD t - SMAD2 in A549 - VIM - RFP pretreated with eliglustat for 4 or 6 days then treated with vehicle control or TGF - β for 1 h . GAPDH : loading control . of p - SMAD2 level in A549 - VIM - RFP as shown in ( A ) . were normalized to that of GAPDH , fold changes were then further normalized to level of p - SMAD2 in control without TGF - β treatment .

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Immunoblot analysis of p-SMAD and t-SMAD2 in A549-VIM-RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells as shown in (A). The levels were normalized to that of GAPDH, and fold changes were then further normalized to the level of p-SMAD2 in control cells without TGF-β treatment.


Immunoblot of epithelial marker E - cadherin mesenchymal markers N - cadherin vimentin in A549 - VIM - RFP treated with eliglustat for 4 or 6 days / or with vehicle control / or TGF - β for 2 days . Tubulin : loading control .

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Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers N-cadherin and vimentin in A549-VIM-RFP cells treated with eliglustat for 4 or 6 days and/or with vehicle control and/or TGF-β for 2 days. Tubulin: loading control.


mCherry - labeled A549 were pretreated with eliglustat ( 2 μM ) for 4 days were then injected into of Cuvier of zebrafish . Representative with magnified regions ( outlined with dotted squares ) of extravasated were acquired 4 days after injection by confocal microscopy . SB group zebrafish were treated with inhibitor SB505124 ( 1 μM ) in egg water together with eliglustat for 4 days after injection with daily refreshment of treatments .

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mCherry-labeled A549 cells were pretreated with eliglustat (2 μM) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. SB group zebrafish were treated with the inhibitor SB505124 (1 μM) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments.


of number of extravasated clusters from 28 per group .

Quantification of the number of extravasated cell clusters from 28 embryos per group.


Lysates from NMuMG control ( Cas9 - expressing ) Ugcg KO were subjected to sucrose density gradient ultracentrifugation . expression of flotillin - 1 , β1 - integrin , EEA1 , TβRI in sucrose gradient fractions were analyzed by immunoblotting . Fractions 3 , 4 , 5 contained lipid rafts , whereas fractions 10 - 12 corresponded to non - lipid raft fractions . of TβRI percentages in lipid raft non - lipid raft fractions from Ugcg KO NMuMG control . Isolation of lipid rafts from other components in A549 - VIM - RFP treated with UGCG inhibitor eliglustat ( 2 μM ) for 6 days using sucrose density gradient ultracentrifugation measurement of flotillin - 1 , β1 - integrin , EEA1 , TβRI in sucrose gradient fractions using immunoblot . Fractions 3 - 6 contained lipid rafts , whereas fractions 10 - 12 corresponded to non - lipid raft fractions . of TβRI percentages in lipid raft non - lipid raft fractions from A549 - VIM - RFP treated with eliglustat ( 2 μM ) for 6 days .

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Lysates from NMuMG control cells (Cas9-expressing cells) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation. The expression levels of flotillin-1, β1-integrin, EEA1, and TβRI in the sucrose gradient fractions were analyzed by immunoblotting. Fractions 3, 4, and 5 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from Ugcg KO NMuMG and control cells. Isolation of lipid rafts from other cellular components in A549-VIM-RFP cells treated with the UGCG inhibitor eliglustat (2 μM) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin-1, β1-integrin, EEA1, and TβRI levels in the sucrose gradient fractions using immunoblot analysis. Fractions 3-6 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from A549-VIM-RFP cells treated with eliglustat (2 μM) for 6 days.


Ubiquitination of TβRI was detected by immunoprecipitation ( IP ) of Myc - tagged caTβRI from HA - Ub - transfected HEK293T with or without eliglustat ( 2 μM ) treatment for 6 days . groups were treated with MG132 ( 5 μM ) for 6 h .

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Ubiquitination of TβRI was detected by immunoprecipitation (IP) of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without eliglustat (2 μM) treatment for 6 days. All groups were treated with MG132 (5 μM) for 6 h.


Immunoblot of TβRI expression in control eliglustat ( 2 μM ) - treated A549 - VIM - RFP treated with 50 μg / mL CHX for indicated times . Vinculin : loading control . of TβRI expression with normalization to t = 0 controls .

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Immunoblot analysis of TβRI expression levels in control and eliglustat (2 μM)-treated A549-VIM-RFP cells treated with 50 μg/mL CHX for the indicated times. Vinculin: loading control.   Quantification of TβRI expression levels with normalization to the t=0 controls.


Immunoblot of p - SMAD2 , t - SMAD2 , ST3GAL5 in A549 - VIM - RFP with siRNA - mediated ST3GAL5 knockdown or transfection of nontargeting siRNA treated with vehicle control or TGF - β for 1 h . Vinculin : loading control . of p - SMAD2 level in A549 - VIM - RFP transfected with nontargeting or ST3GAL5 siRNA treated with TGF - β as shown in ( B ) .

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Immunoblot analysis of p-SMAD2, t-SMAD2, and ST3GAL5 in A549-VIM-RFP cells with siRNA-mediated ST3GAL5 knockdown or transfection of nontargeting siRNA and treated with vehicle control or TGF-β for 1 h. Vinculin: loading control.   Quantification of the p-SMAD2 level in A549-VIM-RFP cells transfected with nontargeting or ST3GAL5 siRNA and treated with TGF-β as shown in (B).


Expression of ST3GAL5 , epithelial marker E - cadherin , mesenchymal markers , including N - cadherin , vimentin , SNAIL , in siRNA ST3GAL5 - depleted nontargeting siRNA - transfected A549 - VIM - RFP treated with vehicle control or TGF - β for 48 h . Vinculin : loading control .

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Expression levels of ST3GAL5, the epithelial marker E-cadherin, and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in siRNA ST3GAL5-depleted and nontargeting siRNA-transfected A549-VIM-RFP cells treated with vehicle control or TGF-β for 48 h. Vinculin: loading control.


Immunoblot of p - SMAD2 t - SMAD2 in A549 - VIM - RFP transduced with empty vector ( pLV - EV ) or ST3GAL5 expression construct stimulated with vehicle control or TGF - β for 1 h . GAPDH : loading control . of p - SMAD2 level in A549 - VIM - RFP transduced with pLV - EV or ST3GAL5 overexpression construct treated with TGF - β , as shown in ( F ) .

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

Immunoblot analysis of p-SMAD2 and t-SMAD2 in A549-VIM-RFP cells transduced with empty vector (pLV-EV) or the ST3GAL5 expression construct and stimulated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells transduced with pLV-EV or the ST3GAL5 overexpression construct and treated with TGF-β, as shown in (F).


Immunoblot of epithelial marker E - cadherin mesenchymal markers , including N - cadherin , vimentin , SNAIL , in A549 - VIM - RFP transduced with pLV - EV control or ST3GAL5 expression construct treated with vehicle control or TGF - β for 48 h . GAPDH : loading control .

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

Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with vehicle control or TGF-β for 48 h. GAPDH: loading control.


HEK293T transfected with Myc - tagged caTβRI , ( HA - Ub , nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti - Myc antibody immunoblot . groups were treated with MG132 ( 5 μM ) for 6 h .

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HEK293T cells transfected with Myc-tagged caTβRI, (HA-Ub, and nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti-Myc antibody and immunoblot analysis. All groups were treated with MG132 (5 μM) for 6 h.


Ubiquitination of TβRI was detected by IP of Myc - tagged caTβRI from HA - Ub - transfected HEK293T with or without ST3GAL5 overexpression . groups were treated with MG132 ( 5 μM ) for 6 h .

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Ubiquitination of TβRI was detected by IP of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without ST3GAL5 overexpression. All groups were treated with MG132 (5 μM) for 6 h.


Western blot of TβRI expression in A549 - VIM - RFP transduced with pLV - EV control or ST3GAL5 expression construct treated with CHX for indicated times . Vinculin : loading control .

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Western blot analysis of TβRI expression in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with CHX for the indicated times. Vinculin: loading control.


of TβRI expression in pLV - EV control - ST3GAL5 - expressing A549 - VIM - RFP . results were normalized to t = 0 controls .

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Quantification of TβRI expression in pLV-EV control- and ST3GAL5-expressing A549-VIM-RFP cells. The results were normalized to the t=0 controls.


In vivo zebrafish extravasation experiments with mCherry - labeled A549 with or without ectopic expression of ST3GAL5 . Representative with magnified regions ( outlined with dotted squares ) of extravasated were acquired 4 days after injection by confocal microscopy . number of extravasated clusters was quantified in 30 injected with A549 transduced with pLV - EV control or ST3GAL5 expression construct .

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In vivo zebrafish extravasation experiments with mCherry-labeled A549 cells with or without ectopic expression of ST3GAL5. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. The number of extravasated cell clusters was quantified in 30 embryos injected with A549 cells transduced with the pLV-EV control or ST3GAL5 expression construct.


Kaplan - Meier survival curves showing first progression survival of lung cancer in a publicly available lung cancer dataset according to ST3GAL5 expression ( n = 982 ) .

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Kaplan-Meier survival curves showing the first progression survival of lung cancer patients in a publicly available lung cancer dataset according to ST3GAL5 expression (n=982).


Box plots of ST3GAL5 gene expression in lung cancer tissues normal tissues in Bhattacharjee Lung database . central bands indicate medium expression values of ST3GAL5 , boxes indicate expression level ranges of ST3GAL5 . Data are presented as means ± SDs from indicated number of tissues .

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Box plots of ST3GAL5 gene expression levels in lung cancer tissues and normal tissues in the Bhattacharjee Lung database. The central bands indicate the medium expression values of ST3GAL5, boxes indicate the expression level ranges of ST3GAL5. Data are presented as the means ± SDs from the indicated number of tissues.


Representative of ST3GAL5 immunohistochemistry in a human lung tissue microarray including normal cancer tissues . Wide field magnified ( outlined with dotted squares ) are shown .

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Representative images of ST3GAL5 immunohistochemistry in a human lung tissue microarray including normal and cancer tissues. Wide field and magnified images (outlined with dotted squares) are shown.


Scatter plot showing expression of ST3GAL5 in normal cancerous lung tissues . Each point represents H - score of a single tissue sample with ST3GAL5 ranging from completely absent ( H - score 0 ) to very strong ( H - score 300 ) . H - scores are presented as means ± SDs ; normal tissues , n = 48 ; cancer tissues , n = 97 ; * * * * , P < 0 . 0001 on unpaired Student ' s t .

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Scatter plot showing the expression of ST3GAL5 in normal and cancerous lung tissues. Each point represents the H-score of a single tissue sample with ST3GAL5 staining ranging from completely absent (H-score 0) to very strong (H-score 300). H-scores are presented as the means ± SDs; normal tissues, n=48; cancer tissues, n=97; ****, P < 0.0001 based on unpaired Student's t test.


emerging timeline for highly pathogenic proposed Disease X . b . Schematic representation of SARS - CoV - 2 S . Its S1 subunit contains NTD ( 14 - 305 aa ) , RBD ( 319 - 541 aa ) , RBM ( 437 - 508 aa ) . Its S2 subunit contains FP ( 788 - 806 aa ) , HR1 ( 912 - 984 aa ) , HR2 ( 1163 - 1213 aa ) , TM ( 1214 - 1237 aa ) CP ( 1238 - 1273 aa ) . c . formation of syncytium in Huh - 7 24 h after SARS - CoV - 2 infection , with scale bar of 200 μm .

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The emerging timeline for highly pathogenic viruses and the proposed Disease X. b. Schematic representation of SARS-CoV-2 S protein. Its S1 subunit contains NTD (14-305 aa), RBD (319-541 aa), and RBM (437-508 aa). Its S2 subunit contains FP (788-806 aa), HR1 (912-984 aa), HR2 (1163-1213 aa), TM (1214-1237 aa) and CP (1238-1273 aa). c. The formation of syncytium in Huh-7 cells 24 h after SARS-CoV-2 infection, with scale bar of 200 μm.


d . of SARS - CoV - SARS - CoV - 2 S - mediated - fusion on 293T / ACE2 at 2 h ( left ) 24 h ( right ) .

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d. Images of SARS-CoV- and SARS-CoV-2 S-mediated cell-cell fusion on 293T/ACE2 cells at 2 h (left) and 24 h (right).


e . SARS - CoV ( I - II ) SARS - CoV - 2 ( III - IV ) S - mediated syncytium formation on 293T / ACE2 at 48 h . f . SARS - CoV ( I - II ) SARS - CoV - 2 ( III - IV ) S - mediated syncytium formation on Huh - 7 at 48 h . Scale bar equals 400 μm in d - f .

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e. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on 293T/ACE2 cells at 48 h. f. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on Huh-7 cells at 48 h. Scale bar equals 400 μm in d-f.


b . SARS - CoV - 2 S - mediated - fusion in presence of EK1 - scramble ( I ) , EK1 ( II ) , EK1C ( III ) , EK1P ( IV ) at 2 . 5 μM ( scale bar : 400 μm ) .

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b. SARS-CoV-2 S protein-mediated cell-cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 μm).


c . Inhibitory activity of EK1 - scramble , EK1 , EK1C EK1P against SARS - CoV - 2 S - mediated - fusion .

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c. Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell-cell fusion.


e . Inhibitory activity of EK1 - lipopeptides on SARS - CoV - 2 S - mediated - fusion .

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e. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell-cell fusion.


f . Inhibitory activity of EK1 - lipopeptides on SARS - CoV - 2 PsV infection . Experiments were repeated twice , data are expressed as means ± SD ( error bar ) .

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f. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of SARS - CoV ( a )

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HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SARS-CoV (a)


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of MERS - CoV ( b )

HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of MERS-CoV (b)


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of HCoV - OC43 ( c )

HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-OC43 (c)


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of HCoV - 229E ( d )

HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-229E (d)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of HCoV - NL63 ( e )

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to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-NL63 (e)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of WIV1 ( f )

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to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of WIV1 (f)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of Rs3367 ( g )

to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of Rs3367 (g)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of SHC014 ( h ) .

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to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SHC014 (h).


i to o . Inhibitory activity of EK1C4 in PsV infection assays against SARS - CoV ( i )

i to o. Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i)


to o . Inhibitory activity of EK1C4 in PsV infection assays against MERS - CoV ( j )

to o. Inhibitory activity of EK1C4 in PsV infection assays against MERS-CoV (j)


Inhibitory activity of EK1C4 in PsV infection assays against HCoV - OC43 ( k )

Inhibitory activity of EK1C4 in PsV infection assays against HCoV-OC43(k)


Inhibitory activity of EK1C4 in PsV infection assays against HCoV - 229E ( l )

Inhibitory activity of EK1C4 in PsV infection assays against HCoV-229E (l)


Inhibitory activity of EK1C4 in PsV infection assays against , NL63 ( m )

Inhibitory activity of EK1C4 in PsV infection assays against , NL63 (m)


Inhibitory activity of EK1C4 in PsV infection assays against WIV1 ( n )

Inhibitory activity of EK1C4 in PsV infection assays against WIV1 (n)


Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 ( o ) .

Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 (o).


Inhibitory activity of EK1 on live HCoV replication for SARS - CoV - 2 ( a ) , MERS - CoV ( b ) , HCoV - OC43 ( c ) , HCoV - 229E ( d ) , HCoV - NL63 ( e ) .

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Inhibitory activity of EK1 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e).


In vivo prophylactic efficacy of EK1C4 against HCoV - OC43 infection in mice . change ( f ) survival curves ( g ) of mice challenged with HCoV - OC43 . h - i . In vivo therapeutic efficacy of EK1C4 against HCoV - OC43 infection in mice . change ( h ) survival curves ( i ) of mice challenged with HCoV - OC43 . Experiments were repeated twice , data are expressed as means ± SD .

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In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h-i. In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD.


A , Representative immunofluorescence of normal , , residual 3D organoids grown from mammary of a preclinical mouse model of breast cancer ( Methods ) . Similar of normal residual organoids shown on left with polarity markers ITGA6 ( red ) , ZO - 1 ( green ) , GM - 130 ( magenta ) ; DAPI ( blue ) . Right : human MYC oncoprotein ( green ) is expressed only in , CDH1 ( red ) . Scale bar : 25 μm . Data information : Number of replicates corresponds to mice used to derive organoids .

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A, Representative immunofluorescence staining of normal, tumor, and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130 (magenta); DAPI (blue). Right: human MYC oncoprotein (green) is expressed only in tumor cells, CDH1 (red). Scale bar: 25 μm. Data information: Number of replicates corresponds to individual mice used to derive organoids.


, Selective secreted metabolites with significant change linked to urea cycle components from healthy ( n = 3 ; blue ) regressed ( n = 3 ; green ) mammary . Values represent metabolite abundance as quantified by under curve ( AUC ) of marker fragment ions / transitions for each metabolite . Values are plotted on log2 scale . Statistics were calculated using limma package n R with significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ata information : umbers of replicates correspond to mice or humans . ox plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . umbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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, Selective secreted metabolites with significant change linked to urea cycle components from healthy (n=3; blue) and regressed (n=3; green) mammary glands. Values represent metabolite abundance levels as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Values are plotted on the log2 scale. Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). ata information: umbers of replicates correspond to individual mice or humans. ox plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. umbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


C , Left , of expressing ARG1 ( top ) , an enzyme converting arginine to urea ornithine , of ARG1 ( bottom ) in normal from healthy ( n = 5 , 2921 analyzed ; blue ) residual from regressed ( n = 5 , 2241 analyzed ; green ) mammary tissue sections . Statistical differences were calculated with Mann - Whitney U ( Wilcoxon rank - sum ) . Right , Representative of immunofluorescence in normal ( top ) residual ( bottom ) stained for ARG1 ( green ) , CDH1 ( red ) , DAPI ( blue ) . Scale bar : 20 μm . Data information : Numbers of replicates correspond to mice or humans . Box plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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C, Left, Quantification of cells expressing ARG1 (top), an enzyme converting arginine to urea and ornithine, and intensity of ARG1 (bottom) in normal cells from healthy (n=5, 2921 cells analyzed; blue) and residual cells from regressed (n=5, 2241 cells analyzed; green) mammary gland tissue sections. Statistical differences were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test). Right, Representative images of immunofluorescence staining in normal (top) and residual (bottom) duct stained for ARG1 (green), CDH1 (red), and DAPI (blue). Scale bar: 20 μm. Data information: Numbers of replicates correspond to individual mice or humans. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


D , Nitric oxide synthase ( NOS ) activity , an enzyme involved in arginine metabolism , in healthy ( n = 3 ; blue ) in residual ( n = 3 ; green ) mouse mammary . difference is statistically significant by unpaired two - samples t - . E , Fractional labeling of lactate after cultivation of isolated regressed ( n = 3 ; green ) healthy ( n = 3 ; blue ) mouse mammary in growth medium supplemented with [ U - 13C ] glucose for 8 h ( Methods ) . three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted shows enrichment in residual of regressed mammary . difference is statistically significant by unpaired two - samples t - . Data information : Numbers of replicates correspond to mice or humans . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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D, Nitric oxide synthase (NOS) activity, an enzyme involved in arginine metabolism, in healthy (n=3; blue) and in residual (n=3; green) mouse mammary glands. The difference is statistically significant by unpaired two-samples t-test. E, Fractional labeling of lactate after cultivation of isolated regressed (n=3; green) and healthy (n=3; blue) mouse mammary glands in cell growth medium supplemented with [U-13C] glucose for 8 h (Methods). The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted and shows enrichment in the residual cells of the regressed mammary glands. The difference is statistically significant by unpaired two-samples t-test. Data information: Numbers of replicates correspond to individual mice or humans. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


A , ( Top ) Experimental design ( bottom ) of ( Methods ) of normal , , residual after 72 - h treatment with 3 - BP at indicated doses ( n = 5 mice per condition ) . Two - way ANOVA with Tukey ' s HSD multiple comparison testing was utilized to calculate statistical significance . Dox , doxycycline . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers , minimum to maximum . Number of replicates corresponds to mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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A, (Top) Experimental design and (bottom) quantification of cell death (Methods) of normal, tumor, and residual cells after 72-h treatment with 3-BP at the indicated doses (n=5 mice per condition). Two-way ANOVA with Tukey's HSD multiple comparison testing was utilized to calculate statistical significance. Dox, doxycycline. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers , minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


B , Representative bright - field of normal ( left ) , ( middle ) , residual ( right ) organoids , treated with vehicle ( top ) or with 50 μM 3 - BP ( bottom ) . Scale bar , 100 μm .

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B, Representative bright-field images of normal (left), tumor (middle), and residual (right) organoids, treated with vehicle (top) or with 50 μM 3-BP (bottom). Scale bar, 100 μm.


C , ( Top ) Experimental design ( bottom ) - of passaged residual normal after 48 h of treatment with 3 - BP ( n = 5 mice per condition ) . Statistics were calculated with multiple t - . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers minimum to maximum . Number of replicates corresponds to mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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C, (Top) Experimental design and (bottom) cell-death quantification of passaged residual and normal cells after 48 h of treatment with 3-BP (n=5 mice per condition). Statistics were calculated with multiple t-tests. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


D , ( Main ) Extracellular glucose abundance changes upon treatment with 3 - BP in three populations ( n = 4 mice per condition ) . Values represent of glucose abundance in 3 - BP - treated versus untreated . Statistics were calculated using limma package in R with significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ( Insert ) Fractional labeling of lactate in untreated 3 - BP treated normal ( n = 2 ) residual ( n = 2 ) following cultivation in growth medium supplemented with [ U - 13C ] glucose . three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted . Statistics were calculated with unpaired two - samples t - . Data information : , Box plots : midline , median ; box , 25 - 75th percentile minimum to maximum . Number of replicates corresponds to mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, (Main) Extracellular glucose abundance changes upon treatment with 3-BP in all three populations (n=4 mice per condition). Values represent the ratio of glucose abundance in 3-BP-treated versus untreated cells. Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). (Insert) Fractional labeling of lactate in untreated and 3-BP treated normal (n=2) and residual (n=2) cells following cultivation in growth medium supplemented with [U-13C] glucose. The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted. Statistics were calculated with unpaired two-samples t-tests. Data information: , Box plots: midline, median; box, 25-75th percentile minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptotene


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in zygotene Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in zygotene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in , early pachytene Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in , early pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in mid pachytene Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in mid pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in diplotene Arrows in ( I - L ) indicate position of some chromocenters . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated . DAPI of ( blue ) is shown for some spermatocytes .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diplotene Arrows in (I-L) indicate the position of some chromocenters. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in diakinesis Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diakinesis Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


O , P Double immunolabeling of PDS5A ( green ) kinetochores revealed with an ACA serum ( red ) in a metaphase I spermatocyte . Data information : DAPI of ( blue ) is shown for some spermatocytes .

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O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Q , R Double immunolabeling of PDS5A ( green ) REC8 ( pseudocolored in purple ) in a metaphase I spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated . DAPI of ( blue ) is shown for some spermatocytes .

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Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.


S , T Double immunolabeling of PDS5A ( green ) kinetochores ( ACA , red ) in a metaphase II spermatocyte . Data information : DAPI of ( blue ) is shown for some spermatocytes .

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S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) in spread spermatocytes at indicated stages .

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Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) in spread spermatocytes at indicated stages . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) REC8 ( pseudocolored in blue onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Immunolabeling of PDS5B (green) and REC8 (pseudocolored in blue onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) SYCP3 ( red ) TRF1 ( pseudocolored in yellow onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Immunolabeling of PDS5B (green) and SYCP3 (red) and TRF1 (pseudocolored in yellow onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) ACA ( pseudocolored in purple onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Immunolabeling of PDS5B (green) and ACA (pseudocolored in purple onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) on diplotene spread nuclei . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5B (green) and SYCP3 (red) on diplotene spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) on diakinesis spread nuclei . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

Double immunolabeling of PDS5B (green) and SYCP3 (red) on diakinesis spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


A - F Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) in a metaphase I spermatocyte ( A - B ) selected bivalents ( C - F ) . sex bivalent ( XY ) is indicated .

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A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A-B) and selected bivalents (C-F). The sex bivalent (XY) is indicated.


G - J Double immunolabeling of PDS5B ( green ) kinetochores ( ACA , red ) , of ( DAPI ) in a metaphase II spermatocyte .

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G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.


Double immunolabeling of PDS5A ( green , A - E ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at indicated stages .

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Double immunolabeling of PDS5A (green, A-E) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green , F - J ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at indicated stages .

Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


K - M Preparations from same mice were immunolabeled with testis - specific histone variant H1t ( green ) SYCP3 ( red ) .

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K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).


N Scatter dot - plot graph of total SC measured in autosomal pairs of 33 early pachytene from three different individuals of each genotype . Bars indicate mean standard error . Statistical significance was assessed using an unpaired two - T - function ( * * * * , p < 0 . 0001 ) .

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N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).


A - H Double immunolabeling of SYCP1 ( green ) SYCP3 ( red ) in wild - type ( A - D ) Pds5AB cKO ( E - H ) spread spermatocytes at indicated stages . I - L Double immunolabeling of HORMAD2 ( pseudocolored in ) SYCP3 ( red ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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A-H Double immunolabeling of SYCP1 (green) and SYCP3 (red) in wild-type (A-D) and Pds5AB cKO (E-H) spread spermatocytes at the indicated stages. I-L Double immunolabeling of HORMAD2 (pseudocolored in white) and SYCP3 (red). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin subunits SMC3 ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin subunits REC8 ( green in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and the cohesin subunits REC8 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin subunits SMC1β ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC1β (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin cofactors Sororin ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors Sororin (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin cofactors WAPL ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) either γH2AX ( pseudocolored in blue in Pds5AB cKO spread spermatocytes at indicated stages .

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Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of SYCP3 ( red ) RAD51 ( green in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and RAD51 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Scatter dot - plot graph of of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype . Bars indicate mean standard error statistical significance was assessed with an unpaired two - T - function ( * * * * , p < 0 . 0001 ) .

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I Scatter dot-plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype. Bars indicate mean and standard error and statistical significance was assessed with an unpaired two-tailed T-test function (****, p < 0.0001).


J - L Double immunolabeling of MLH1 ( pseudocolored in ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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J-L Double immunolabeling of MLH1 (pseudocolored in white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) TRF1 ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and TRF1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) RAP1 ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Selected zygotene - like pachytene - like bivalents presenting altered TRF1 distribution as ( from left to right ) ( i ) telomere stretches , ( ii ) multiple telomere , ( iii ) distant telomeres ( iv ) telomere - less . Arrowheads in ( I ) indicate position of altered telomeres . J of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at indicated stages ( n = 1646 telomeres in zygotene - like spermatocytes 738 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres. J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


K , L As in ( I , J ) , respectively , but for RAP1 ( n = 1376 telomeres in zygotene - like spermatocytes 328 in pachytene - like ones ) . Arrowheads in ( K ) indicate position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


M - P Double immunolabeling of SUN1 ( green ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at indicated stages . 0 , 1 2 in ( N O ) indicate number of chromosome ends labelled by SUN1 in a given bivalent . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Q As in ( I ) , but for SUN1 . Arrowheads in ( Q ) indicate position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


R Selected zygotene - like early pachytene - like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 ( red ) SUN1 ( green ) , their respective of appearance . S of telomeres with a regular or altered disposition of SUN1 ( n = 715 telomeres in zygotene - like spermatocytes 410 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance. S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


A - C Immunolabeling of SYCP3 ( red ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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A-C Immunolabeling of SYCP3 (red) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


D 300 % magnification of selected zygotene - like pachytene - like bivalents presenting regular ( left ) or altered ( i - iv ) telomere FISH signals .

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D 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.


Double immunolabeling of SYCP3 ( red ) RAP1 ( blue ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread

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Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes