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 ) .

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

A 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 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 ) .

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

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 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 .

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

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 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 ) .

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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 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 ) .

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

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 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 ) .

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

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 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 ) ] .

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

G 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 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 ) .

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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 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 ) .

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

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 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 ) .

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

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 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 ) .

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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 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 .

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

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 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 ) .

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

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 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 ) .

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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 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 ) .

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

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 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 .

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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 ) 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 .

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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 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 .

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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 ) The ddl - 1 mutant in the 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 plants 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 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 .

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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 bacterial growth of Psp in ddl - 6 . Leaves of four - week - old plants 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 and W18 ) in the ddl - 6 background were assayed for the 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 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 and visualized by autoradiography . PAR ( DDL ) indicates PARylated DDL proteins . 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 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 .

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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 assay . 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 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 .

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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 assay .

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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 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 ) .

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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 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 .

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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 plants were inoculated with Pst DC3000 ( OD600 = 5x10 - 4 ) and the bacterial number was determined at 0 and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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.


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

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


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 .

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

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 cells with or without eliglustat ( 2 μM ) treatment for 6 days . All groups were treated with MG132 ( 5 μM ) for 6 h .

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

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 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 .

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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 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 ) .

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

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 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 .

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

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 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 ) .

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

B-EXP_ASSAY O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

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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 cells with or without ST3GAL5 overexpression . All groups were treated with MG132 ( 5 μM ) for 6 h .

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

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 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 .

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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.


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

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

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 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 .

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

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

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

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 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 .

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

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 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 .

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

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 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 .

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-DISEASE B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 .

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

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 . 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 ) .

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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 ) 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 .

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

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 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 ) .

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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 and EK1P against SARS - CoV - 2 S - mediated cell - cell 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 cell - cell fusion .

O O O O O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

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 , and the data are expressed as means ± SD ( error bar ) .

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

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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins 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 cell - cell fusion mediated by the S proteins of SHC014 ( h ) .

O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O B-ORGANISM O O O O

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 )

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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 )

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

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 )

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

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 )

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

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


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

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

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


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

O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O 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 ) , and HCoV - NL63 ( e ) .

O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O

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 . 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 .

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

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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O B-DISEASE I-DISEASE O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

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 ) 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 ) .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

, 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 , 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 ) .

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

C, Left, 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 ) 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 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE I-TISSUE 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-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

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

A, (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 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 .

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

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 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 ) .

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

C, (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 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 ) .

O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O 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 ) and SYCP3 ( red ) in mouse spread spermatocytes in

O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM O B-CELL_TYPE O

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


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 .

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

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

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

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

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

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 ) 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 .

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

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 ) and SYCP3 ( red ) in mouse spread spermatocytes in diakinesis Data information : Sex chromosomes ( X , Y ) and 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 ) 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 .

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

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 ) 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 .

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

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

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

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 ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages .

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

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


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 .

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

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

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

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

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

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

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

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

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

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 ) and SYCP3 ( red ) on diakinesis spread nuclei . Data information : Sex chromosomes ( X , Y ) and 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 ) and SYCP3 ( red ) in a metaphase I spermatocyte ( A - B ) and selected bivalents ( C - F ) . The sex bivalent ( XY ) is indicated .

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

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.