( E ) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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(E) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( A ) Root phenotypes of 7 - day - old seedlings of indicated genotypes . ( B ) Primary root length of 7 - day - old seedlings . ( C ) Root meristem size in 5 - day - old seedlings .

O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM O

(A) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings.


( D ) Root meristem phenotype of 5 - day - old seedlings , confocal microscopy , propidium iodide - stained ( red ) .

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

(D) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red).


( E ) Auxin activity as visualized by the constitutively expressed inverse DII - NLS - VENUS marker ( green ) in root meristems of 5 - day - old seedlings .

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(E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings.


( F ) Gap cell frequency in brxsingle or brxmakr5 double mutants ( note : gap cells were absent from Col - 0 wild type or makr5single mutants ) . ( G ) Illustration of gap cells ( arrowheads ) in developing protophloem sieve element strands ( asterisks ) of brxsingle or brxmakr5 double mutants .

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

(F) Gap cell frequency in brxsingle or brxmakr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brxsingle or brxmakr5 double mutants.


( A ) Primary root length of 7 - day - old seedlings of indicated genotypes in response to continuous CLE45 treatments .

O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O

(A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments.


( B ) Root meristem phenotypes of ( CLE45 - treated ) 5 - day - old seedlings ( asterisks : protophloem sieve element strands ) . Note absence of protophloem in CLE45 - treated Col - 0 wild type roots .

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

(B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots.


( C ) Transverse sections of ( CLE45 - treated ) 5 - day - old roots , just before the onset of protoxylem ( asterisks ) differentiation . Note failure of protophloem sieve elements ( arrowheads ) to differentiate in CLE45 - treated Col - 0 ( indicated by dense cytoplasm ) .

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

(C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm).


( D ) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5 - day - old Col - 0seedlings . ( E ) Corresponding transverse sections from different positions along the root meristem .

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

(D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings. (E) Corresponding transverse sections from different positions along the root meristem.


( F ) Expression of a MAKR5 - GFP fusion protein under control of the MAKR5 promoter in roots of 5 - day - old seedlings .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-TISSUE O O O O O O B-ORGANISM O

(F) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings.


( G ) Corresponding optical sections illustrating MAKR5 - GFP expression in developing protophloem sieve elements ( asterisk ) and adjacent cell files .

O O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O

(G) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files.


( H ) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements ( CVP2 ) , but not by expression under control of a companion cell - specific promoter ( SUC2 ) ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

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(H) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( I ) Subcellular localization of MAKR5 - GFP ( meristematic zone ) . Note plasma membrane association ( white , green arrowheads ) and dissociation from the cell wall ( red arrowhead ) upon osmosis .

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(I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis.


( B ) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5 - day - old roots ( asterisks : protophloem sieve element strands ) .

O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O O O

(B) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5-day-old roots (asterisks: protophloem sieve element strands).


( C ) Close up of protophloem sieve element strands expressing truncated MAKR5 - CITRINE fusion proteins under control of the UBQ10 promoter .

O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O

(C) Close up of protophloem sieve element strands expressing truncated MAKR5-CITRINE fusion proteins under control of the UBQ10 promoter.


( D ) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant ( 7 - day - old seedlings ; 1 - 2 independent transgenic lines per construct are shown ) .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O

(D) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant (7-day-old seedlings; 1-2 independent transgenic lines per construct are shown).


( E ) Sensitivity of makr5 mutant roots to continuous brassinolide ( BL ) treatment ( 7 - day - old seedlings ) .

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

(E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings).


( F ) Expression of MAKR5 - BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5 - day - old makr5 seedlings .

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(F) Expression of MAKR5-BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5-day-old makr5 seedlings.


( G ) Complementation of CLE45 insensitivity of makr5 roots by BKI1 - MAKR5 , but not MAKR5 - BKI1 fusion protein ( 7 - day - old seedlings ; 3 independent transgenic lines per construct are shown ) .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O

(G) Complementation of CLE45 insensitivity of makr5 roots by BKI1-MAKR5, but not MAKR5-BKI1 fusion protein (7-day-old seedlings; 3 independent transgenic lines per construct are shown).


( A ) Increased expression of BAM3 - CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements ( asterisks ) of 5 - day - old bam3 roots upon CLE45 treatment .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-GENEPROD B-TISSUE O B-GENEPROD O O

(A) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment.


( B ) Expression of BAM3 - CITRINE fusion protein under control of the BAM3 promoter in 5 - day - old Col - 0 roots or makr5 roots .

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

(B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots.


( C ) Expression of MAKR5 - GFP fusion protein under control of the MAKR5 promoter in 5 - day - old Col - 0 or bam3 roots .

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

(C) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots.


( D ) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5 - day - old Col - 0seedlings upon CLE45 treatment .

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

(D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings upon CLE45 treatment.


( E ) Expression of MAKR5 - GFP fusion protein under control of the MAKR5 promoter in 5 - day - old roots upon CLE45 treatment ( 3 h ) . Note accumulation of MAKR5 - GFP in protophloem sieve elements ( magnified ) and its dependence on functional BAM3 and CLE45 . The frequency of representative observations per total observations is indicated .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE 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

(E) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated.


( F ) Close up view of MAKR5 - GFP accumulation in the protophloem cell file upon CLE45 treatment .

O O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O

(F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment.


( G - H ) Accumulation of MAKR5 - BKI1 fusion proteins in protophloem cells upon CLE45 treatment . The frequency of representative observations per total observations is indicated . ( I - J ) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter , showing mean total fluorescence ( I ) and plasma membrane to cytosol signal ratio ( J ) .

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

(G-H) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J).


B . Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs .

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B. Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs.


C . qPCR validations of neutrophil genes . n = 7 DFUs and n = 3 Skin D3 wounds . * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) . Data presented as mean ± SD .

O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O B-DISEASE O O O O B-TISSUE 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 O O O O O

C. qPCR validations of neutrophil genes. n=7 DFUs and n=3 Skin D3 wounds. **P < 0.01 (two-tailed unpaired Student's t-test). Data presented as mean ± SD.


A . Representative images of human neutrophils treated with the FOXM1 inhibitor , FDI - 6 . Vehicle ( DMSO ) served as a control . Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red . Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . Data presented as mean ± SD . ( Scale bar : 100µm ) .

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

A. Representative images of human neutrophils treated with the FOXM1 inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red. Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).


B . Quantification of ROS levels in human neutrophils treated with FDI - 6 or in combination with N - acetylcysteine ( NAC ) . Phorbol 12 - myristate 13 - acetate ( PMA ) treatment served as a positive control . Data presented as mean ± SD . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . C . Quantification of NET formation in human neutrophils treated with FDI - 6 or in combination with NAC . PMA treatment served as a positive control . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . Data presented as mean ± SD . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) .

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B. Quantification of ROS levels in human neutrophils treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). C. Quantification of NET formation in human neutrophils treated with FDI-6 or in combination with NAC. PMA treatment served as a positive control. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).


A . Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response .

O O O O O O O O O B-ORGANISM B-TISSUE B-DISEASE O O O O O O O B-ORGANISM B-DISEASE O O B-CELL_TYPE O O

A. Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response.


A . Representative images of wounded skin after topical treatment with either vehicle ( IgG isotype control ) or ⍺ - TREM1 activator at 0 , 2 , 4 , 6 , and 8 days after wounding . Percent of wound area at each time following vehicle or ⍺ - TREM1 activator treatment relative to the original wound area . Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺ - TREM1 treated wounds were performed with Fiji software . Data presented as mean ± SD . * * P < 0 . 01 , and * * * * P < 0 . 0001 ( two - way ANOVA followed by Tukey ' s post - hoc test ) .

O O O O O O B-TISSUE 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 O O O O O O O B-DISEASE O O O O O O O O O B-GENEPROD O O O O O O B-DISEASE O O O O B-DISEASE O O O O O O O O O B-DISEASE O O B-DISEASE O O O B-GENEPROD 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 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. Representative images of wounded skin after topical treatment with either vehicle (IgG isotype control) or ⍺-TREM1 activator at 0, 2, 4, 6, and 8 days after wounding. Percent of wound area at each time following vehicle or ⍺-TREM1 activator treatment relative to the original wound area. Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺-TREM1 treated wounds were performed with Fiji software. Data presented as mean ± SD. **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey's post-hoc test).


B . Representative pictures of vehicle ( IgG isotype control ) and ⍺ - TREM1 treated diabetic wounds at day 4 show basal keratin marker K5 , and neutrophil marker Ly6G , FOXM1 and citH3 . Treatment of wounds with ⍺ - TREM1 resulted in increased FOXM1 + and decreased citH3 + neutrophils compared to vehicle treated diabetic wounds . ( Scale bar : 50µm ) . Quantification of mean fluorescence intensity was performed with Fiji software . n = 7 diabetic vehicle wounds and 6 diabetic ⍺ - TREM1 wounds . Data presented as mean ± SD . * P < 0 . 05 , * P < 0 . 05 , and * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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

B. Representative pictures of vehicle (IgG isotype control) and ⍺-TREM1 treated diabetic wounds at day 4 show basal keratin marker K5, and neutrophil marker Ly6G, FOXM1 and citH3. Treatment of wounds with ⍺-TREM1 resulted in increased FOXM1+ and decreased citH3+ neutrophils compared to vehicle treated diabetic wounds. (Scale bar: 50µm). Quantification of mean fluorescence intensity was performed with Fiji software. n= 7 diabetic vehicle wounds and 6 diabetic ⍺-TREM1 wounds. Data presented as mean ± SD. *P < 0.05, *P < 0.05, and **P < 0.01 (two-tailed unpaired Student's t-test).


A . qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs ( n = 5 healing and n = 6 nonhealing ) . Data presented as mean ± SD . * P < 0 . 05 . * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O B-DISEASE 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 O 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. qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs (n = 5 healing and n= 6 nonhealing). Data presented as mean ± SD. *P < 0.05. **P < 0.01 (two-tailed unpaired Student's t-test).


B . Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing ( n = 4 ) and nonhealing ( n = 4 ) is shown in the graph . Data presented as mean ± SEM . * P < 0 . 05 ( two - tailed unpaired Student ' s t - test ) . ( Scale bar : 50µm ) .

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B. Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing (n=4) and nonhealing (n=4) is shown in the graph. Data presented as mean ± SEM. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).


C . Representative images of cit - H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs , which was confirmed by corresponding quantification from healing ( n = 3 ) and nonhealing ( n = 3 ) , shown in the graph . Data presented as mean ± SD . * P < 0 . 05 ( two - tailed unpaired Student ' s t - test ) . ( Scale bar : 50µm ) .

O O O O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O B-DISEASE 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 O O O O O O O O O O O O O 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. Representative images of cit-H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs, which was confirmed by corresponding quantification from healing (n=3) and nonhealing (n=3), shown in the graph. Data presented as mean ± SD. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).


A . 3 - OH - C10 : 0 - induced ROS burst is suppressed by A23 and K252a . Two - week - old Col - 0 seedlings were treated with 2μM K252a or 40 μM A23 . One hour later , 1 μM 3 - OH - C10 : 0 was used to trigger the ROS burst , and the photo count was recorded by a luminometer . Mock is solvent control . RLU , relative light unit . Values are means ± SD ( n = 3 biological replicates ) .

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

A. 3-OH-C10:0-induced ROS burst is suppressed by A23 and K252a. Two-week-old Col-0 seedlings were treated with 2μM K252a or 40 μM A23. One hour later, 1 μM 3-OH-C10:0 was used to trigger the ROS burst, and the photo count was recorded by a luminometer. Mock is solvent control. RLU, relative light unit. Values are means ± SD (n=3 biological replicates).


B . A23 and K252a suppress 3 - OH - C10 : 0 - induced PTI marker gene expression . Treatments with 3 - OH - C10 : 0 and inhibitors were same as in ( A ) , and the plant leaves were collected for RT - qPCR 4 hrs later . Values are means ± SD ( n = 3 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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

B. A23 and K252a suppress 3-OH-C10:0-induced PTI marker gene expression. Treatments with 3-OH-C10:0 and inhibitors were same as in (A), and the plant leaves were collected for RT-qPCR 4 hrs later. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).


C . MAPK cascade activation was inhibited by A23 . Treatments with 3 - OH - C10 : 0 and A23 were same as in ( A ) . The plant leaves were collected at 0 , 5 , and 10 min for immunoblotting . Activated MAPKs were detected by immunoblotting with anti - phospho - p44 / 42 MAPK antibody . The corresponding bands are indicated for MPK3 and MPK6 . Ponceau staining of Rubisco was used for estimating equal loading in each lane . The experiment was repeated three times with similar results .

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

C. MAPK cascade activation was inhibited by A23. Treatments with 3-OH-C10:0 and A23 were same as in (A). The plant leaves were collected at 0, 5, and 10 min for immunoblotting. Activated MAPKs were detected by immunoblotting with anti-phospho-p44/42 MAPK antibody. The corresponding bands are indicated for MPK3 and MPK6. Ponceau staining of Rubisco was used for estimating equal loading in each lane. The experiment was repeated three times with similar results.


D . Tyrosine phosphorylation of LORE in vivo . Two - week - old 35S : LORE - FLAG complementation plants were subjected to immunoprecipitation with anti - FLAG agarose beads . A23 ( 40 μM ) was used to inhibit the protein phosphorylation , and the treatments were same as in ( A ) . Protein abundance of precipitated LORE - FLAG was detected by immunoblotting . The tyrosine phosphorylation was probed by anti - pTyrosine ( α - pY ) antibody . Similar results were observed in three biological replicates .

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

D. Tyrosine phosphorylation of LORE in vivo. Two-week-old 35S:LORE-FLAG complementation plants were subjected to immunoprecipitation with anti-FLAG agarose beads. A23 (40 μM) was used to inhibit the protein phosphorylation, and the treatments were same as in (A). Protein abundance of precipitated LORE-FLAG was detected by immunoblotting. The tyrosine phosphorylation was probed by anti-pTyrosine (α-pY) antibody. Similar results were observed in three biological replicates.


E . LORE is an active tyrosine kinase . The α - pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP - tagged LORE - CD ( cytosolic domain ) . The LORE activation loop conserved K516 was mutated to E , and D613 was mutated to V . The bottom gel showing protein abundance stained by Coomassie Brilliant Blue ( CBB ) .

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E. LORE is an active tyrosine kinase. The α-pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP-tagged LORE-CD (cytosolic domain). The LORE activation loop conserved K516 was mutated to E, and D613 was mutated to V. The bottom gel showing protein abundance stained by Coomassie Brilliant Blue (CBB).


A . Phosphorylated Y600 of LORE was identified by LC - MS / MS . MBP - tagged LORE - CD was used for in vitro kinase assays . Trypsin digested recombinant proteins were subjected to LC - MS / MS analysis . The doubly - charged peptide with m / z 476 . 24 matches GLLYLHR of LORE with Y600 phosphorylation . Fragment ions retaining charges in the N and C terminus were denoted by " b " and " y " , respectively . pY600 indicated phosphorylated Y600 .

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

A. Phosphorylated Y600 of LORE was identified by LC-MS/MS. MBP-tagged LORE-CD was used for in vitro kinase assays. Trypsin digested recombinant proteins were subjected to LC-MS/MS analysis. The doubly-charged peptide with m/z 476.24 matches GLLYLHR of LORE with Y600 phosphorylation. Fragment ions retaining charges in the N and C terminus were denoted by "b" and "y", respectively. pY600 indicated phosphorylated Y600.


B . Y600 mutation reduced LORE kinase activity . MBP - tagged LORE - CD and LORE - Y600F - CD ( Y600 tyrosine was substituted with phenylalanine , F ) were subjected to kinase reactions . The kinase dead mutant LOREK516E served as a negative control . Autophosphorylated LORE was visualized by autoradiography following SDS - PAGE gel electrophoresis . Protein abundance was stained by CBB .

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

B. Y600 mutation reduced LORE kinase activity. MBP-tagged LORE-CD and LORE-Y600F-CD (Y600 tyrosine was substituted with phenylalanine, F) were subjected to kinase reactions. The kinase dead mutant LOREK516E served as a negative control. Autophosphorylated LORE was visualized by autoradiography following SDS-PAGE gel electrophoresis. Protein abundance was stained by CBB.


C . LORE Y600 is phosphorylated in plants . Two - week - old pLORE : LORE - FLAG complementation plant leaves were subjected to immunoprecipitation ( IP ) with anti - FLAG agarose beads . Y600 specific phosphorylation antibody was used to detect Y600 phosphorylation in LORE . The experiment was repeated three times with similar results .

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

C. LORE Y600 is phosphorylated in plants. Two-week-old pLORE:LORE-FLAG complementation plant leaves were subjected to immunoprecipitation (IP) with anti-FLAG agarose beads. Y600 specific phosphorylation antibody was used to detect Y600 phosphorylation in LORE. The experiment was repeated three times with similar results.


D . 3 - OH - C10 : 0 induced Y600 phosphorylation . Two - week - old pLORE : LORE - FLAG complementation plants were treated with 1 μM 3 - OH - C10 : 0 at indicated times . Plant leaves were subjected to anti - FLAG IP . Phosphorylation level of Y600 was detected by anti - pY600 antibody . The experiment was repeated three times with similar results .

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

D. 3-OH-C10:0 induced Y600 phosphorylation. Two-week-old pLORE:LORE-FLAG complementation plants were treated with 1 μM 3-OH-C10:0 at indicated times. Plant leaves were subjected to anti-FLAG IP. Phosphorylation level of Y600 was detected by anti-pY600 antibody. The experiment was repeated three times with similar results.


E . Y600 mutation abolished the ROS burst triggered by 3 - OH - C10 : 0 . The transgenic plants were treated with 3 - OH - C10 : 0 and ROS burst was measured . WT ( 6 - 1 ) and WT ( 10 - 3 ) are two independent complementation lines with LORE ; Y600F ( 2 - 6 ) and Y600F ( 7 - 1 ) are two independent lines complemented with LOREY600F ; and kinase - dead LOREK516E was transformed to lore mutant as a negative control . The 2 - 3 and 5 - 2 are two independent lines . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 I-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 B-GENEPROD 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 B-GENEPROD 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 O O O O O O O

E. Y600 mutation abolished the ROS burst triggered by 3-OH-C10:0. The transgenic plants were treated with 3-OH-C10:0 and ROS burst was measured. WT(6-1) and WT(10-3) are two independent complementation lines with LORE; Y600F(2-6) and Y600F(7-1) are two independent lines complemented with LOREY600F; and kinase-dead LOREK516E was transformed to lore mutant as a negative control. The 2-3 and 5-2 are two independent lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


F . Y600 is required for growth inhibition by 3 - OH - C10 : 0 . The Arabidopsis seedlings were grown on ½ MS containing 2 . 5 μM 3 - OH - C10 : 0 for six days , and the root length was measured . The experiment was repeated three times with similar results . WT ( 6 - 1 ) and Y600 ( 2 - 6 ) lines were used for the assay . Scale bar = 0 . 4 cm . Values are means ± SD ( n = 3 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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 I-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM 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 I-SMALL_MOLECULE O O O O O O B-TISSUE 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 O O 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. Y600 is required for growth inhibition by 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. The experiment was repeated three times with similar results. WT(6-1) and Y600 (2-6) lines were used for the assay. Scale bar=0.4 cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).


G . Y600 mutation decreased the disease resistance to Pst DC3000 . Plants were spray - inoculated with Pst DC3000 at a concentration of 1 × 109 cfu / ml , and were subjected to bacterial growth curve analysis at 3 dpi . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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G. Y600 mutation decreased the disease resistance to Pst DC3000. Plants were spray-inoculated with Pst DC3000 at a concentration of 1×109 cfu/ml, and were subjected to bacterial growth curve analysis at 3 dpi. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


A . LORE interacts with specific RLCKs ( PBL34 / 35 / 36 ) in yeast . BD - LORE - CD and AD - PBLs were co - transformed to yeast cells and were screened on synthetic dextrose media lacking leucine and tryptophan ( SD / - Leu - Trp ) . The single yeast colonies were serially diluted onto SD / - Leu - Trp and SD / - Leu - Trp - His ( SD media lacking leucine , tryptophan and histidine ) to observe yeast cell growth . 3 - AT ( 20 mM ) was used to inhibit the autoactivation of LORE . Yeast co - transformed with pGADT7 - T + pGBKT7 - 53 ( + ) served as a positive control , and the yeast co - transformed with pGADT7 - T + pGBKT7 - lam ( - ) and AD - PBL39 + BD - LORE served as the negative controls . EV , empty vector .

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

A. LORE interacts with specific RLCKs (PBL34/35/36) in yeast. BD-LORE-CD and AD-PBLs were co-transformed to yeast cells and were screened on synthetic dextrose media lacking leucine and tryptophan (SD/-Leu-Trp). The single yeast colonies were serially diluted onto SD/-Leu-Trp and SD/-Leu-Trp-His (SD media lacking leucine, tryptophan and histidine) to observe yeast cell growth. 3-AT(20 mM) was used to inhibit the autoactivation of LORE. Yeast co-transformed with pGADT7-T+pGBKT7-53(+) served as a positive control, and the yeast co-transformed with pGADT7-T+pGBKT7-lam(-) and AD-PBL39+BD-LORE served as the negative controls. EV, empty vector.


B . LORE interacts with PBLs by MBP pull - down assays . The recombinant GST - LORE - CD and the MBP - PBLs were subjected to MBP pull - down analysis . MBP served as a negative control . The interacting proteins were revealed by anti - GST immunoblotting . The experiment was repeated three times with similar results .

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

B. LORE interacts with PBLs by MBP pull-down assays. The recombinant GST-LORE-CD and the MBP-PBLs were subjected to MBP pull-down analysis. MBP served as a negative control. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results.


C . Co - immunoprecipitation ( Co - IP ) of LORE with PBLs . 35S promoter - driven LORE - T7 and PBLs - FLAG were transiently co - expressed in N . benthamiana by agroinfiltration . Plant leaves were immunoprecipated with anti - FLAG beads and the proteins were immunoblotted by anti - FLAG antibody . Co - IP proteins were immunoblotted by anti - T7 antibody . The experiment was repeated three times with similar results .

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

C. Co-immunoprecipitation (Co-IP) of LORE with PBLs. 35S promoter-driven LORE-T7 and PBLs-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and the proteins were immunoblotted by anti-FLAG antibody. Co-IP proteins were immunoblotted by anti-T7 antibody. The experiment was repeated three times with similar results.


D . LORE interacts with PBLs , revealed by split - luciferase assays . N . benthamiana leaves were co - infiltrated with Agrobacteria carrying 35S : LORE - nLUC and 35S : cLUC - PBLs . Images of chemiluminescence were obtained by applying 0 . 5 mM luciferin 36 h post infiltration . Similar results were observed in three biological replicates .

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

D. LORE interacts with PBLs, revealed by split-luciferase assays. N. benthamiana leaves were co-infiltrated with Agrobacteria carrying 35S:LORE-nLUC and 35S:cLUC-PBLs. Images of chemiluminescence were obtained by applying 0.5 mM luciferin 36 h post infiltration. Similar results were observed in three biological replicates.


A . ROS burst in pbl34 / 35 / 36 triple mutant , induced by 3 - OH - C10 : 0 . Two - week - old seedlings were treated with 1 μM 3 - OH - C10 : 0 , and the ROS burst was measured . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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

A. ROS burst in pbl34/35/36 triple mutant, induced by 3-OH-C10:0. Two-week-old seedlings were treated with 1 μM 3-OH-C10:0, and the ROS burst was measured. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


B . PTI marker gene expression was reduced in pbl34 / 35 / 36 triple mutant in response to 3 - OH - C10 : 0 . Treatment is same as in A . Samples were collected for RT - qPCR at 4 h post infiltration . Values are means ± SD ( n = 3 biological replicates ) . ( Student ' s t - test , * * P < 0 . 01 ) .

O O O O O O O O O B-GENEPROD 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 I-SMALL_MOLECULE O 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 O O O O O O O O O O O O O O O O O O O O O O O

B. PTI marker gene expression was reduced in pbl34/35/36 triple mutant in response to 3-OH-C10:0. Treatment is same as in A. Samples were collected for RT-qPCR at 4 h post infiltration. Values are means ± SD (n=3 biological replicates). (Student's t-test, **P< 0.01).


C . pbl34 / 35 / 36 triple mutant displayed decreased callose depositions in response to 3 - OH - C10 : 0 treatment . Two - week - old seedlings were sprayed with 1 μM 3 - OH - C10 : 0 . The leaves were sampled for callose deposition assays 12 hrs later . The stained callose was counted for 1 mm2 of the leaves . Mock is solvent control . Scale bar = 100μm . Values are means ± SD ( n = 6 leaves ) . ( Student ' s t - test , * * P < 0 . 01 ) .

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

C. pbl34/35/36 triple mutant displayed decreased callose depositions in response to 3-OH-C10:0 treatment. Two-week-old seedlings were sprayed with 1 μM 3-OH-C10:0. The leaves were sampled for callose deposition assays 12 hrs later. The stained callose was counted for 1 mm2 of the leaves. Mock is solvent control. Scale bar=100μm.Values are means ± SD (n=6 leaves). (Student's t-test, **P< 0.01).


D . Root length of pbl34 / 35 / 36 triple mutant treated with 3 - OH - C10 : 0 . The Arabidopsis seedlings were grown on ½ MS containing 2 . 5 μM 3 - OH - C10 : 0 for six days , and the root length was measured . Scale bar = 0 . 4cm . Values are means ± SD ( n = 3 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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

D. Root length of pbl34/35/36 triple mutant treated with 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. Scale bar=0.4cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).


E . Disease phenotype of pbl34 / 35 double , pbl34 / 35 / 36 triple mutants and PBL34 overexpression plants . 35S : PBL34 - FLAG was introduced to the Col - 0 to generate the overexpression lines . The homozygous transgenic plants were used for the assays . The plants were sprayed with Pst DC3000 at a concentration of 1 × 109 cfu / ml . Plants were subjected to bacterial growth curve analysis at 3 dpi . OE - 3 # and OE - 11 # are two independent transgenic lines . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD 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 B-ORGANISM I-ORGANISM O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Disease phenotype of pbl34/35 double, pbl34/35/36 triple mutants and PBL34 overexpression plants. 35S:PBL34-FLAG was introduced to the Col-0 to generate the overexpression lines. The homozygous transgenic plants were used for the assays. The plants were sprayed with Pst DC3000 at a concentration of 1×109 cfu/ml. Plants were subjected to bacterial growth curve analysis at 3 dpi. OE-3# and OE-11# are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


F . pbl34 / 35 / 36 triple mutant phenotype was rescued by PBL34 . PBL34 - HA was introduced to the triple mutant driven by its native promoter . Four - week - old homozygous seedlings were inoculated with 5 × 104 cfu / ml Pst DC3000 , and the leaves were subjected to bacterial growth curve analysis at 3 dpi . CM2 - 5 and CM7 - 3 are two independent transgenic lines . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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

F. pbl34/35/36 triple mutant phenotype was rescued by PBL34. PBL34-HA was introduced to the triple mutant driven by its native promoter. Four-week-old homozygous seedlings were inoculated with 5×104 cfu/ml Pst DC3000 , and the leaves were subjected to bacterial growth curve analysis at 3 dpi. CM2-5 and CM7-3 are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


A . PBL34 was phosphorylated by LORE in vitro . Purified recombinant proteins were used for in vitro kinase assays . The kinase dead mutant MBP - PBL34 * ( PBL K180 was substituted to E , predicted by https : / / www . uniprot . org / uniprot / ) , was used as the substrate of GST - LORE - CD or its kinase dead variant , GST - LORE - CD * ( LOREK516E ) . MBP served as a negative control . Incorporation of isotope labeled phosphates was visualized by 32P autoradiography . The CBB staining showing protein abundance .

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

A. PBL34 was phosphorylated by LORE in vitro. Purified recombinant proteins were used for in vitro kinase assays. The kinase dead mutant MBP-PBL34* (PBL K180 was substituted to E, predicted by https://www.uniprot.org/uniprot/), was used as the substrate of GST-LORE-CD or its kinase dead variant, GST-LORE-CD* (LOREK516E). MBP served as a negative control. Incorporation of isotope labeled phosphates was visualized by 32P autoradiography. The CBB staining showing protein abundance.


B . Mutation of PBL34 T306 / T310 significantly reduced its phosphorylation by LORE . Recombinant kinase dead PBL34 * and PBL34 * - 2A ( T306 / T310 was mutated to A ) were incubated with LORE - CD . Phosphorylation of PBL34 was visualized by 32P autoradiography . Protein abundance was revealed by CBB staining .

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

B. Mutation of PBL34 T306/T310 significantly reduced its phosphorylation by LORE. Recombinant kinase dead PBL34* and PBL34*-2A (T306/T310 was mutated to A) were incubated with LORE-CD. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining.


C . Mutation at Y600 diminished trans - phosphorylation activity of LORE . Recombinant LORE - CD and LOREY600F - CD were used for the trans - phosphorylation assays . Phosphorylation of PBL34 was visualized by 32P autoradiography . Protein abundance was revealed by CBB staining .

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

C. Mutation at Y600 diminished trans-phosphorylation activity of LORE. Recombinant LORE-CD and LOREY600F-CD were used for the trans-phosphorylation assays. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining.


D . PBL34 phosphorylation is induced by 3 - OH - C10 : 0 . The protoplast of Col - 0 was transformed with 35S : PBL34 - FLAG and 35S : PBL34 - 2A - FLAG , respectively . Sixteen hours later , the protoplasts were treated with1 μM 3 - OH - C10 : 0 . Phosphorylated proteins were immunoprecipitated and detected by anti - pS / T antibody . The experiment was repeated three times with similar results .

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

D. PBL34 phosphorylation is induced by 3-OH-C10:0. The protoplast of Col-0 was transformed with 35S:PBL34-FLAG and 35S:PBL34-2A-FLAG, respectively. Sixteen hours later, the protoplasts were treated with1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results.


E . 3 - OH - C10 : 0 - induced PBL34 phosphorylation depends on LORE . The 35S : PBL34 - FLAG was introduced to Col - 0 and lore , respectively . Four - week - old homozygous transgenic plants were treated with 1 μM 3 - OH - C10 : 0 . Phosphorylated proteins were immunoprecipitated and detected by anti - pS / T antibody . The experiment was repeated three times with similar results .

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

E. 3-OH-C10:0-induced PBL34 phosphorylation depends on LORE. The 35S:PBL34-FLAG was introduced to Col-0 and lore, respectively. Four-week-old homozygous transgenic plants were treated with 1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results.


F . PBL34 dissociated from LORE upon 3 - OH - C10 : 0 activation . The 35S : LORE - FLAG plants were transformed with native promoter driven PBL34 ( pPBL34 : PBL34 - HA ) . 100 nM flg22 or 1 μM 3 - OH - C10 : 0 was sprayed onto plant leaves , and the leaves were sampled for assays15 min later . The experiment was repeated three times with similar results .

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

F. PBL34 dissociated from LORE upon 3-OH-C10:0 activation. The 35S:LORE-FLAG plants were transformed with native promoter driven PBL34 (pPBL34:PBL34-HA). 100 nM flg22 or 1 μM 3-OH-C10:0 was sprayed onto plant leaves, and the leaves were sampled for assays15 min later. The experiment was repeated three times with similar results.


G . Phosphorylation of PBL34K180E did not disassociate from LORE . The 35S : LORE - FLAG protoplasts were transfected with 35S : PBL34K180E - HA . 100nM flg22 or 1 μM 3 - OH - C10 : 0 was used to treat the protoplasts . The protoplasts were sampled 15 min later . The experiment was repeated three times with similar results .

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

G. Phosphorylation of PBL34K180E did not disassociate from LORE. The 35S:LORE-FLAG protoplasts were transfected with 35S:PBL34K180E-HA. 100nM flg22 or 1 μM 3-OH-C10:0 was used to treat the protoplasts. The protoplasts were sampled 15 min later. The experiment was repeated three times with similar results.


H . PBL34 rescues pbl34 / 35 / 36 triple mutant ROS burst in response to 3 - OH - C10 : 0 . Treatment was same as in ( E ) . pPBL34 : PBL34 - HA was introduced to pbl34 / 35 / 36 triple mutant , and the homozygous transgenic line was used for ROS burst assays . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-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 O O B-GENEPROD O O O O O B-GENEPROD 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 O O O O O

H. PBL34 rescues pbl34/35/36 triple mutant ROS burst in response to 3-OH-C10:0. Treatment was same as in (E). pPBL34:PBL34-HA was introduced to pbl34/35/36 triple mutant, and the homozygous transgenic line was used for ROS burst assays. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


I . PTI marker gene expression in PBL34 complementation lines . The PTI marker genes FRK1 and NHL10 expression was examined by RT - qPCR in Col - 0 , pbl34 / 35 / 36 , and CM - PBL34 - 2A lines after 3 - OH - C10 : 0 treatments for 4 hrs . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01 ) .

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

I. PTI marker gene expression in PBL34 complementation lines. The PTI marker genes FRK1 and NHL10 expression was examined by RT-qPCR in Col-0, pbl34/35/36, and CM-PBL34-2A lines after 3-OH-C10:0 treatments for 4 hrs. Values are means ± SD (n=6; ANOVA; p < 0.01).


J . PBL34 phosphorylation at T306 / T310 is essential for disease resistance . pbl34 / 35 / 36 triple mutants were complemented with PBL34 or PBL34 - 2A driven by its native promoter . The homozygous plants were pretreated with 3 - OH - C10 : 0 ( C10 ) 1 d before pathogen inoculation . The plants were inoculated with 2 × 105 cfu / ml Pst DC3000 , and the bacterial titer was analyzed at 3 dpi . Mock is solvent control . Letters indicate the difference from Col - 0 . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01 ) .

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

J. PBL34 phosphorylation at T306/T310 is essential for disease resistance. pbl34/35/36 triple mutants were complemented with PBL34 or PBL34-2A driven by its native promoter. The homozygous plants were pretreated with 3-OH-C10:0(C10) 1 d before pathogen inoculation. The plants were inoculated with 2×105 cfu/ml Pst DC3000, and the bacterial titer was analyzed at 3 dpi. Mock is solvent control. Letters indicate the difference from Col-0. Values are means ± SD (n=6; ANOVA; p < 0.01).


A . LORE interacts with HopAO1 by MBP pull - down assays . The recombinant GST - HopAO1 and the MBP - LORE - CD were subjected to MBP pull - down analysis . MBP and GST proteins served as the negative controls . The interacting proteins were revealed by anti - GST immunoblotting . The experiment was repeated three times with similar results .

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

A. LORE interacts with HopAO1 by MBP pull-down assays. The recombinant GST-HopAO1 and the MBP-LORE-CD were subjected to MBP pull-down analysis. MBP and GST proteins served as the negative controls. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results.


B . LORE interacts with HopAO1 by Co - IP assays . 35S promoter - driven LORE - T7 and HopAO1 - FLAG were transiently co - expressed in N . benthamiana by agroinfiltration . Plant leaves were immunoprecipated with anti - FLAG beads and immunoblotted by anti - FLAG . Coimmunoprecipitated proteins were immunoblotted by anti - T7 . The experiment was repeated three times with similar results .

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

B. LORE interacts with HopAO1 by Co-IP assays. 35S promoter-driven LORE-T7 and HopAO1-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and immunoblotted by anti-FLAG. Coimmunoprecipitated proteins were immunoblotted by anti-T7. The experiment was repeated three times with similar results.


C . HopAO1 dephosphorylates LORE Y600 in vitro . The recombinant GST - HopAO1 / HopAO1CS and GST - LORE - CD were subjected to in vitro phosphatase assays . Phosphorylation level of Y600 was detected by anti - pY600 immunoblotting . The experiment was repeated three times with similar results .

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

C. HopAO1 dephosphorylates LORE Y600 in vitro. The recombinant GST-HopAO1/HopAO1CS and GST-LORE-CD were subjected to in vitro phosphatase assays. Phosphorylation level of Y600 was detected by anti-pY600 immunoblotting. The experiment was repeated three times with similar results.


D . HopAO1 dephosphorylates LORE Y600 in N . benthamiana . LORE - FLAG was transiently co - expressed with GUS - T7 , HopAO1 - T7 or HopAO1CS - T7 in N . benthamiana leaves mediated by Agrobacteria . The protein was immunoprecipitated and probed by anti - pY600 antibody . The experiment was repeated at least three times with similar results .

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

D. HopAO1 dephosphorylates LORE Y600 in N. benthamiana. LORE-FLAG was transiently co-expressed with GUS-T7, HopAO1-T7 or HopAO1CS-T7 in N. benthamiana leaves mediated by Agrobacteria. The protein was immunoprecipitated and probed by anti-pY600 antibody. The experiment was repeated at least three times with similar results.


E . HopAO1 suppresses 3 - OH - C10 : 0 - triggered ROS burst . HopAO1 was ectopically expressed in Col - 0 driven by estradiol - inducible promoter . 100μM estradiol was sprayed on Est : HopAO1 plants , and the leaf disc was used for ROS burst assays 12 hrs later . Values are means ± SD ( n = 6 biological replicates ) .

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

E. HopAO1 suppresses 3-OH-C10:0-triggered ROS burst. HopAO1 was ectopically expressed in Col-0 driven by estradiol-inducible promoter. 100μM estradiol was sprayed on Est:HopAO1 plants, and the leaf disc was used for ROS burst assays 12 hrs later. Values are means ± SD (n=6 biological replicates).


F . HopAO1 inhibits 3 - OH - C10 : 0 - induced disease resistance . The Est : HopAO1 plants were pretreated with 1 μM 3 - OH - C10 : 0 ( C10 ) and100 μM estradiol , and then the plants were inoculated with 5 × 104 cfu / ml Pst DC3000 24 hrs later . The bacterial titer was analyzed at 3 dpi . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01

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

F. HopAO1 inhibits 3-OH-C10:0-induced disease resistance. The Est:HopAO1 plants were pretreated with 1 μM 3-OH-C10:0 (C10) and100 μM estradiol, and then the plants were inoculated with 5×104 cfu/ml Pst DC3000 24 hrs later. The bacterial titer was analyzed at 3 dpi. Values are means ± SD (n=6; ANOVA; p < 0.01


F . HopAO1 inhibits 3 - OH - C10 : 0 - induced disease resistance . The Est : HopAO1 plants were pretreated with 1 μM 3 - OH - C10 : 0 ( C10 ) and100 μM estradiol , and then the plants were inoculated with 5 × 104 cfu / ml Pst DC3000 24 hrs later . The bacterial titer was analyzed at 3 dpi . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 .

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


Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with drugs at the indicated concentrations for six days .

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

Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with drugs at the indicated concentrations for six days.


Human spheroids established from BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) DLD1 cells , were incubated with 1 . 25 µM olaparib or 0 . 5 µM chlorambucil over the indicated period of time .

B-ORGANISM B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O

Human spheroids established from BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) DLD1 cells, were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time.


Dose - dependent viability assays of BRCA1 - proficient ( + BRCA1 ) or - deficient ( - BRCA1 ) human RPE1 - hTERT cells treated with drugs at the indicated concentrations for six days .

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

Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) human RPE1-hTERT cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA1 - proficient ( + BRCA1 ) or - deficient ( - BRCA1 ) TP53 - deleted cells treated with drugs at the indicated concentrations for six days .

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

Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) TP53-deleted cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of Brca1 + / + and Brca1 - / - mouse mammary tumour - derived cell lines treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of Brca1+/+ and Brca1-/- mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA2 - deficient ( Capan - 1 ) or - proficient ( MIA PaCa - 2 ) human pancreatic carcinoma - derived cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-deficient (Capan-1) or -proficient (MIA PaCa-2) human pancreatic carcinoma-derived cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA2 - deficient ( PEO1 ) or - proficient ( C4 - 2 ) human ovarian tumour - derived cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells treated with drugs at the indicated concentrations for six days.


BRCA2 - deficient ( PEO1 ) or - proficient ( C4 - 2 ) human ovarian tumour - derived cells were infected with lentiviruses expressing control or CHD4 shRNAs , followed by selection with puromycin for 72 hours . Dose - dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days .

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BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 hours. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days.


BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells were incubated with 1 µM chlorambucil ( Chl ) . Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown . GAPDH was used as a loading control .

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BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control.


Quantification of chromosome aberrations and chromatid / chromosome break frequencies in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h . Data were obtained from three independent experiments and normalised to untreated controls . A minimum of 60 Giemsa - stained metaphases were analysed for each sample . Cis , cisplatin ; Chl , chlorambucil .

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Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil.


Quantification of G2 / M cell frequency relative to solvent control , using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours . Cis , cisplatin ; Chl , chlorambucil .

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Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil.


Human H1299 cells were treated with control ( CTR ) or indicated siRNAs two days before drugs were added to the media for dose - dependent viability assays . Cell extracts prepared at the time of drug addition were immunoblotted as indicated . SMC1 was used as a loading control .

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Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.


Nude mice ( nu / nu ) were injected subcutaneously with 5 x 106 human DLD1 cells , BRCA2 - proficient ( A ) or BRCA2 - deficient ( B ) . Tumour - bearing mice were treated with 3 mg / kg daily chlorambucil administered intraperitoneally ( i . p . ) for a total of ten days . Tumour weight was determined on the indicated days after initiation of the treatment .

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Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment.


PDTCs derived from breast cancer samples as previously described ( Bruna et al , 2016 ) were treated with chlorambucil at the indicated doses . Cell survival is represented relative to DMSO control . AB521 , ER - negative tumour , no BRCA1 alteration ; STG201 , tumour with BRCA1 promoter methylation and loss of BRCA1 expression ; VHIO179 , tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation ( olaparib - resistant )

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PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant)


CB17 / SCID mice were injected intramuscularly with 5 x 106 human BRCA2 - deficient HCT116 cells . Tumour - bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally ( i . p . ) , or talazoparib administered orally ( o . s . ) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment ( day 1 ) . Scale bar , 40 µm . Each experimental group included n = 5 mice . Error bars represent SEM . Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining .

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CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm. Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining.


Wild type Balb / c mice were injected intra - peritoneally with solvent ( daily ) or 3 mg / kg chlorambucil ( daily for 5 days ) or 3 . 3 mg / kg cisplatin ( daily for 3 days ) . Uptake of the apoptosis tracer 99mTc - Duramycin 2 h after intra - venous injection was quantified in selected organs using SPECT imaging in the indicated organs . Representative maximum intensity partial projections showing tracer distribution are shown .

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Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.


Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A . Scale bar , 25 µm .

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Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A. Scale bar, 25 µm.


( A - D ) Images of a representative wild - type adult eye ( A ) or adult eyes containing mtm mutant clones ( B and C ) . Quantification of relative eye sizes ( n = 10 ) ( D ) .

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(A-D) Images of a representative wild-type adult eye (A) or adult eyes containing mtm mutant clones (B and C). Quantification of relative eye sizes (n=10) (D).


( F - G ) A pupal eye disc containing mtm mutant clones ( marked by the absence of GFP ) was stained for Dlg . Note the increased interommatidial cells in the mtm mutant clone . Quantification of numbers of interommatidial cells per ommatidium ( n = 10 ) ( G ) .

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(F-G) A pupal eye disc containing mtm mutant clones (marked by the absence of GFP) was stained for Dlg. Note the increased interommatidial cells in the mtm mutant clone. Quantification of numbers of interommatidial cells per ommatidium (n=10) (G).


( K ) RNAi of Mtm reduces Yki phosphorylation . Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis . Total cell lysates were probed with anti - phospho - Yki ( p - Yki ) antibody . Note that RNAi of mtm decreased Yki phosphorylation .

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(K) RNAi of Mtm reduces Yki phosphorylation. Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis. Total cell lysates were probed with anti-phospho-Yki (p-Yki) antibody. Note that RNAi of mtm decreased Yki phosphorylation.


PI ( 3 ) P expression levels detected by a 2xFYVE - GFP reporter in a third instar larval wing disc ( A ) and eye disc ( B ) containing mtm mutant clones ( marked by the absence of RFP ) . Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis . Note the strong increases of PI ( 3 ) P levels in the mtm mutant clones .

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PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


Quantification of the mean FYVE - GFP fluorescence intensity of mtm mutant clones and surrounding wild - type ( WT ) cells are shown ( C ) . Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis . Note the strong increases of PI ( 3 ) P levels in the mtm mutant clones .

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Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


( A ) Following transfection of given siRNAs and synchronization in G2 - phase , Hek293 cells were DRB - or mock - treated and then subjected to IFM using the indicated antibodies . Lower panels display a 3 - fold magnification of the boxed area shown above . Scales bars correspond to 5 and 1 μm ,

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(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively


( B ) Hek293 cells were thymidine - arrested for 20h , mock - or OHT - treated to induce DSBs by nuclear accumulation of ER - AsiSI and then subjected to ChIP - multiplex PCR

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(B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR


( C ) ChIP samples from ( B ) were analysed by qPCR . Shown are averages ( bars ) of 3 independent experiments ( dots )

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(C) ChIP samples from (B) were analysed by qPCR. Shown are averages (bars) of 3 independent experiments (dots)


Separase interacts with γH2AX in DSB - containing G2 but not G1 cells . Transgenic Hek293 cells treated with Dox to induce expression of Myc - separase - WT and with OHT to induce nuclear accumulation of ER - AsiSI and infliction of DSBs were synchronized in G1 - or G2 - phase and analysed by IP -

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Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-Western


Separase interacts with γH2AX in DSB - containing G2 but not G1 cells . Transgenic Hek293 cells treated with Dox to induce expression of Myc - separase - WT and with OHT to induce nuclear accumulation of ER - AsiSI and infliction of DSBs were synchronized in G1 - or G2 - phase and analysed b IFM for γH2AX - and Myc - separase - positive foc The quantification of the IFM in ( E ) shows averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 cells

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Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed b IFM for γH2AX- and Myc-separase-positive foc The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) and U2OS EJ5 - GFP ( NHEJ reporter ) cells were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , and then supplemented with OHT in G2 - phase to induce nuclear accumulation of the homing endonuclease . Ethanol - supplemented samples served as negative controls . Two days later , cells were subjected to immunoblotting (

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) and U2OS EJ5 - GFP ( NHEJ reporter ) cells were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , and then supplemented with OHT in G2 - phase to induce nuclear accumulation of the homing endonuclease . Ethanol - supplemented samples served as negative controls . Two days later , cells were subjected t flow cytometry to quantify the percentage of GFP positive cells ( B The GFP quantification in ( B ) displays averages ( bars ) of 3 independent experiments ( dots )

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

Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected t flow cytometry to quantify the percentage of GFP positive cells (B The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots)


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) and U2OS EJ5 - GFP ( NHEJ reporter ) cells were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , and then supplemented with OHT in G2 - phase to induce nuclear accumulation of the homing endonuclease . Ethanol - supplemented samples served as negative controls Two days later PI - stained cellular DNA ( C )

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls Two days later PI-stained cellular DNA (C)


Co - depletion of Wapl and separase has a synergistic effect on HDR . U2OS DR - GFP cells were transfected with the indicated siRNAs , treated as in ( A + B ) and analysed by GFP - flow cytometry ( D Shown in ( D ) are averages ( bars ) of 2 - 3 independent experiments ( dots )

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Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed by GFP-flow cytometry (D Shown in (D) are averages (bars) of 2-3 independent experiments (dots)


Co - depletio of Wapl and separase has a synergistic effect on HDR . U2OS DR - GFP cells were transfected with the indicated siRNAs , treated as in ( A + B ) and analysed b immunoblotting ( E )

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Co-depletio of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed b immunoblotting (E)


( A ) Transgenic Hek293 cells constitutively expressing a separase sensor ( cartoon below ) and inducibly expressing Myc - separase were transiently transfected to express Flag - AsiSI - ER , Dox - and / or OHT - treated in G2 - phase and analysed by ( IP - )

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

(A) Transgenic Hek293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase and analysed by (IP-)Western


( B ) Cells from ( A ) were analysed by IP - Western for cleavage of endogenous Rad21 . As a control , in vitro - translated ( IVT ) Rad21 was incubated with hyperactive separase - SA or a protease - dead ( PD ) varian

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

(B) Cells from (A) were analysed by IP-Western for cleavage of endogenous Rad21. As a control, in vitro-translated (IVT) Rad21 was incubated with hyperactive separase-SA or a protease-dead (PD) varian


( C ) Sensor expressing cells were treated in G2 - phase with DRB and nocodazole for 6 and 2 h , respectively , prior to chromosome spreading and IFM using Hec1 - and γH2AX - antibodies . The separase sensor was detected based on autofluorescence of eGFP and mCherry , while Hec1 - and γH2AX - antibodies were detected with corresponding Cy5 - and marina blue - labeled secondary antibodies , respectively . Note that sizes of spread chromosomes vary greatly with buffer conditions , which is why no scale bar is shown

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

(C) Sensor expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1- and γH2AX-antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1- and γH2AX-antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown


Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX . Hek293 cells were arrested in G2 phase by sequential thymidine - and RO - 3306 treatment , DRB - ( + ) or mock treated ( - ) , and then analysed as indicated Myc - separase - WT or - S1660A expressing cells were subjected to ( IP - ) Western using , amongst others , a pan - specific antibody against phosphorylated serine ( A , pan -

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

Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pS


Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX . Hek293 cells were arrested in G2 phase by sequential thymidine - and RO - 3306 treatment , DRB - ( + ) or mock treated ( - ) , and then analysed as indicated Myc - separase - WT or - S1660A expressing cells were subjected to ( IP - ) Western using , amongst other a separase antibody specific for phosphorylated Ser1660 ( B , pS1660 )

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

Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst other a separase antibody specific for phosphorylated Ser1660 (B, pS1660)