figf2 ( a ) AMPK phosphorylates the Ulk1 S / T domain in vitro . Top : schematic representation of Ulk1 domain structure and deletion constructs used to map phosphorylation sites . The mouse Ulk1 protein consists of an N - terminal kinase domain ( KD ; 1 - 278 ) , serine / threonine - rich domain ( S / T domain , 279 - 828 ) , and C - terminal domain ( CTD , 829 - 1051 ) . Bottom : the indicated Flag - Ulk1 deletion mutants were immunopurified from transfected HEK293 cells and were used for in vitro AMPK assay as a substrate . Phosphorylation was examined by 32P - autoradiogram and protein level was determined by western blot . ( b ) Determination of AMPK phosphorylation sites in Ulk1 . The indicated recombinant GST - Ulk1 mutants were expressed and purified from Escherichia coli , and used as substrates for in vitro phosphorylation by AMPK . Deletion analyses indicated that two Ulk1 fragments in the S / T domain , 279 - 425 and 769 - 782 , were highly phosphorylated by AMPK in vitro . Mutation of Ser 317 abolished the majority of phosphorylation in the Ulk1 fragment 279 - 425 . Within the fragment 769 - 782 , mutations of five serine residues ( Ser 774 , Ser 777 , Ser 778 , Ser 779 and Ser 780 ) to alanine , denoted as ( 769 - 782 ) 5SA , completely abolished the phosphorylation by AMPK . Reconstitution of Ser 777 in this mutation background , ( 769 - 782 ) 4SA - S777 , but not any of the other four residues , restored the phosphorylation by AMPK . GST and GST - TSC2F ( TSC2 fragment 1300 - 1367 containing AMPK phosphorylation site at Ser 1345 ) were used as negative and positive controls for AMPK reaction , respectively . Phosphorylation was determined by 32P - autoradiograph and the protein levels were detected by Coomassie staining . ( c ) Ser 317 / Ser 777 are required for glucose - starvation induced Ulk1 phosphorylation in vivo . HA - Ulk1 and mutants were transfected into HEK293 cells . Cells were starved for glucose for 4 h as indicated . HA - Ulk1 was immunoprecipitated and examined by western blot for mobility . ( d ) Phosphorylation of Ulk1 Ser 317 and Ser 777 are induced by AMPK . Wild - type HA - Ulk1 or S317 / 777A mutant were co - transfected with AMPK into HEK293 cells as indicated . HA - Ulk1 was immunoprecipitated ( IP ) and phosphorylation of Ser 317 and Ser 777 were determined by western blotting . Uncropped images of blots are shown in Supplementary Fig . S5 .

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

figf2(a) AMPK phosphorylates the Ulk1 S/T domain in vitro. Top: schematic representation of Ulk1 domain structure and deletion constructs used to map phosphorylation sites. The mouse Ulk1 protein consists of an N-terminal kinase domain (KD; 1-278), serine/threonine-rich domain (S/T domain, 279-828), and C-terminal domain (CTD, 829-1051). Bottom: the indicated Flag-Ulk1 deletion mutants were immunopurified from transfected HEK293 cells and were used for in vitro AMPK assay as a substrate. Phosphorylation was examined by 32P-autoradiogram and protein level was determined by western blot.(b) Determination of AMPK phosphorylation sites in Ulk1. The indicated recombinant GST-Ulk1 mutants were expressed and purified from Escherichia coli, and used as substrates for in vitro phosphorylation by AMPK. Deletion analyses indicated that two Ulk1 fragments in the S/T domain, 279-425 and 769-782, were highly phosphorylated by AMPK in vitro. Mutation of Ser 317 abolished the majority of phosphorylation in the Ulk1 fragment 279-425. Within the fragment 769-782, mutations of five serine residues (Ser 774, Ser 777, Ser 778, Ser 779 and Ser 780) to alanine, denoted as (769-782) 5SA, completely abolished the phosphorylation by AMPK. Reconstitution of Ser 777 in this mutation background, (769-782) 4SA-S777, but not any of the other four residues, restored the phosphorylation by AMPK. GST and GST-TSC2F (TSC2 fragment 1300-1367 containing AMPK phosphorylation site at Ser 1345) were used as negative and positive controls for AMPK reaction, respectively. Phosphorylation was determined by 32P-autoradiograph and the protein levels were detected by Coomassie staining.(c) Ser 317/Ser 777 are required for glucose-starvation induced Ulk1 phosphorylation in vivo. HA-Ulk1 and mutants were transfected into HEK293 cells. Cells were starved for glucose for 4 h as indicated. HA-Ulk1 was immunoprecipitated and examined by western blot for mobility.(d) Phosphorylation of Ulk1 Ser 317 and Ser 777 are induced by AMPK. Wild-type HA-Ulk1 or S317/777A mutant were co-transfected with AMPK into HEK293 cells as indicated. HA-Ulk1 was immunoprecipitated (IP) and phosphorylation of Ser 317 and Ser 777 were determined by western blotting. Uncropped images of blots are shown in Supplementary Fig. S5.


figf3 ( a ) AMPK wild - type or DKO MEFs were starved of glucose ( 4 h ) as indicated . Total cell lysates were probed for Ulk1 protein and phosphorylation . ( b ) Time course of Ulk1 Ser 317 and Ser 777 phosphorylation in response to glucose starvation / re - addition . MEFs were starved of glucose ( - Glu ) for the indicated times . After 3 h starvation , the culture was switched to glucose - containing ( 25 mM ) medium and samples were harvested ( Re - Glu ) . In parallel , cells were treated with amino - acid - free ( - A . A ) medium or 50 nM rapamycin ( Rapa ) for 3 h . ( c ) Phosphorylation of Ulk1 Ser 317 and Ser 777 correlates with AMPK activity . MEFs were starved of glucose ( 4 h ) as indicated in the presence or absence of 20 μM compound C ( C . C ) . In parallel , cells were treated with 2 mM Metformin ( Met , 2 h ) in glucose - rich medium . Phosphorylation of ACC S79 was tested as a positive control for AMPK activation . ( d ) Ulk1 is highly phosphorylated at Ser 317 and Ser 777 by glucose starvation in vivo . To determine the Ulk1 phosphorylation level in vivo , immunopurified HA - Ulk1 protein was phosphorylated by AMPK in vitro ( 100 % represents full phosphorylation of Ulk1 by AMPK ) . In vitro phosphorylated HA - Ulk1 was diluted as indicated , and was immunoblotted along with the immunoprecipitated HA - Ulk1 from cells grown in either glucose - rich ( + Glu ) or glucose - free ( - Glu , 4 h ) medium . The density of the bands was then quantified . By this measurement , approximately 50 % of Ulk1 isolated from glucose - starved cells was phosphorylated on Ser 317 and Ser 777 . ( e ) The indicated HA - Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high - glucose medium , and then incubated with AMPK in the presence of cold ATP for 15 minin vitro . After the reaction , AMPK was removed by extensive washing , the resulting Ulk1 immuno - complexes were assayed for kinase activity in the presence of 32P - ATP . ( f ) HA - Ulk1 proteins ( wild type or S317 / 777A mutant ) were immunoprecipitated from the transfected HEK293 cells , which were incubated with or without glucose ( 4 h ) before lysis . An in vitro kinase reaction was performed in the presence of GST - ATG13 and FIP200 . Uncropped images of blots are shown in Supplementary Fig . S5 .

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

figf3(a) AMPK wild-type or DKO MEFs were starved of glucose (4 h) as indicated. Total cell lysates were probed for Ulk1 protein and phosphorylation.(b) Time course of Ulk1 Ser 317 and Ser 777 phosphorylation in response to glucose starvation/re-addition. MEFs were starved of glucose (-Glu) for the indicated times. After 3 h starvation, the culture was switched to glucose-containing (25 mM) medium and samples were harvested (Re-Glu). In parallel, cells were treated with amino-acid-free (-A.A) medium or 50 nM rapamycin (Rapa) for 3 h.(c) Phosphorylation of Ulk1 Ser 317 and Ser 777 correlates with AMPK activity. MEFs were starved of glucose (4 h) as indicated in the presence or absence of 20 μM compound C (C.C). In parallel, cells were treated with 2 mM Metformin (Met, 2 h) in glucose-rich medium. Phosphorylation of ACC S79 was tested as a positive control for AMPK activation.(d) Ulk1 is highly phosphorylated at Ser 317 and Ser 777 by glucose starvation in vivo. To determine the Ulk1 phosphorylation level in vivo, immunopurified HA-Ulk1 protein was phosphorylated by AMPK in vitro (100% represents full phosphorylation of Ulk1 by AMPK). In vitro phosphorylated HA-Ulk1 was diluted as indicated, and was immunoblotted along with the immunoprecipitated HA-Ulk1 from cells grown in either glucose-rich (+ Glu) or glucose-free (- Glu, 4 h) medium. The density of the bands was then quantified. By this measurement, approximately 50% of Ulk1 isolated from glucose-starved cells was phosphorylated on Ser 317 and Ser 777.(e) The indicated HA-Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high-glucose medium, and then incubated with AMPK in the presence of cold ATP for 15 minin vitro. After the reaction, AMPK was removed by extensive washing, the resulting Ulk1 immuno-complexes were assayed for kinase activity in the presence of 32P-ATP.(f) HA-Ulk1 proteins (wild type or S317/777A mutant) were immunoprecipitated from the transfected HEK293 cells, which were incubated with or without glucose (4 h) before lysis. An in vitro kinase reaction was performed in the presence of GST-ATG13 and FIP200. Uncropped images of blots are shown in Supplementary Fig. S5.


figf4 ( a ) AMPK interacts with Ulk1 . HEK293 cells were transfected with the various Flag - Ulk1 deletion mutants together with AMPK α / β / γ , Atg13 and FIP200 . Flag - Ulk1 protein ( indicated by white arrows ) was immunoprecipitated and co - immunoprecipitation of AMPK α / β / γ , Atg13 and FIP200 were examined by western blots . ( b ) Deletion analysis of Ulk1 regions responsible for AMPK interaction . The indicated Flag - Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 cells co - expressing AMPK complex ( α / β / γ ) . Co - immunoprecipitation of AMPK subunits was determined by western blots . ( c ) Rheb inhibits the Ulk1 - AMPK interaction . HA - AMPKα , Flag - Ulk1 and Myc - Rheb were co - transfected into HEK293 cells as indicated . Cells were treated with or without rapamycin ( 50 nM Rapa ) for 1 h before lysis . Flag - Ulk1 was immunoprecipitated and co - immunoprecipitates of AMPKα were determined by western blot . ( d ) Rapamycin treatment enhances the interaction of endogenous Ulk1 and AMPK . Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild - type and knockout ( single - knockout ; KO or double - knockout ; DKO ) MEFs . Treatment with 50 nM rapamycin for 1 h is indicated ( Rapa ) . Co - immunoprecipitation of endogenous AMPKα protein was determined by western blot . The arrow indicates AMPKα protein . ( e ) Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro . CBP / SBP - Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1 - bead complex was incubated with mTORC1 , which was prepared by Raptor immunoprecipitation , in the presence of cold ATP , as indicated . The resulting Ulk1 complex was incubated with the cell lysates containing AMPK , then extensively washed . The Ulk1 and associated AMPKα were detected by western blot . Uncropped images of blots are shown in Supplementary Fig . S5 .

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

figf4(a) AMPK interacts with Ulk1. HEK293 cells were transfected with the various Flag-Ulk1 deletion mutants together with AMPK α/β/γ, Atg13 and FIP200. Flag-Ulk1 protein (indicated by white arrows) was immunoprecipitated and co-immunoprecipitation of AMPK α/β/γ, Atg13 and FIP200 were examined by western blots.(b) Deletion analysis of Ulk1 regions responsible for AMPK interaction. The indicated Flag-Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 cells co-expressing AMPK complex (α/β/γ). Co-immunoprecipitation of AMPK subunits was determined by western blots.(c) Rheb inhibits the Ulk1-AMPK interaction. HA-AMPKα, Flag-Ulk1 and Myc-Rheb were co-transfected into HEK293 cells as indicated. Cells were treated with or without rapamycin (50 nM Rapa) for 1 h before lysis. Flag-Ulk1 was immunoprecipitated and co-immunoprecipitates of AMPKα were determined by western blot.(d) Rapamycin treatment enhances the interaction of endogenous Ulk1 and AMPK. Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild-type and knockout (single-knockout; KO or double-knockout; DKO) MEFs. Treatment with 50 nM rapamycin for 1 h is indicated (Rapa). Co-immunoprecipitation of endogenous AMPKα protein was determined by western blot. The arrow indicates AMPKα protein.(e) Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro. CBP/SBP-Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1-bead complex was incubated with mTORC1, which was prepared by Raptor immunoprecipitation, in the presence of cold ATP, as indicated. The resulting Ulk1 complex was incubated with the cell lysates containing AMPK, then extensively washed. The Ulk1 and associated AMPKα were detected by western blot. Uncropped images of blots are shown in Supplementary Fig. S5.


figf5 ( a ) mTORC1 phosphorylates the Ulk1 S / T domain . Ulk1 deletion mutants were prepared from the transfected HEK293 cells and used for in vitro mTORC1 assay . Phosphorylation was examined by 32P - autoradiogram ( top ) and protein level was determined by western blot ( bottom ) . ( b ) Ser 757 is phosphorylated by mTORC1 . Left : the indicated recombinant GST - mUlk1 mutants were purified from E . coli and used for in vitro mTORC1 assay as substrates . Deletion analyses isolated the fragment ( 753 - 771 ) as a target for mTORC1 . The Ulk1 ( 753 - 771 ) fragment contains five conserved serine / threonine residues , Thr 754 , Ser 757 , Ser 760 , Thr 763 and Thr 770 . Right : mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro . GST was used as negative control for mTORC1 phosphorylation reaction . Phosphorylation was determined by 32P - autoradiograph ( top ) , whereas protein levels were detected by Coomassie staining ( bottom ) . ( c ) Rheb increases Ulk1 Ser 757 phosphorylation . HA - Ulk1 wild type and the S757A mutant were immunoprecipitated from transfected HEK293 cells . Co - transfection with Rheb and rapamycin ( Rapa ) treatment are indicated . Ulk1 Ser 757 phosphorylation was determined by western blot . ( d ) Rheb induces a mobility shift in wild - type Ulk1 , but not the Ulk1S757A mutant . HA - Ulk1 was transfected with or without Rheb into HEK293 cells . HA - Ulk1 was immunoprecipitated from the cells under nutrient - rich medium and Ulk1 mobility was examined by western blot . ( e ) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1 − / − Tsc1 . Tsc1 + / + ( WT ) and Tsc1 − / − ( KO ) MEFs were starved of glucose ( 4 h ) , or treated with 50 nM rapamycin ( Rapa , 1 h ) . Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho - Ulk1 Ser 757 antibody . Uncropped images of blots are shown in Supplementary Fig . S5 .

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

figf5(a) mTORC1 phosphorylates the Ulk1 S/T domain. Ulk1 deletion mutants were prepared from the transfected HEK293 cells and used for in vitro mTORC1 assay. Phosphorylation was examined by 32P-autoradiogram (top) and protein level was determined by western blot (bottom).(b) Ser 757 is phosphorylated by mTORC1. Left: the indicated recombinant GST-mUlk1 mutants were purified from E. coli and used for in vitro mTORC1 assay as substrates. Deletion analyses isolated the fragment (753-771) as a target for mTORC1. The Ulk1 (753-771) fragment contains five conserved serine/threonine residues, Thr 754, Ser 757, Ser 760, Thr 763 and Thr 770. Right: mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro. GST was used as negative control for mTORC1 phosphorylation reaction. Phosphorylation was determined by 32P-autoradiograph (top), whereas protein levels were detected by Coomassie staining (bottom).(c) Rheb increases Ulk1 Ser 757 phosphorylation. HA-Ulk1 wild type and the S757A mutant were immunoprecipitated from transfected HEK293 cells. Co-transfection with Rheb and rapamycin (Rapa) treatment are indicated. Ulk1 Ser 757 phosphorylation was determined by western blot.(d) Rheb induces a mobility shift in wild-type Ulk1, but not the Ulk1S757A mutant. HA-Ulk1 was transfected with or without Rheb into HEK293 cells. HA-Ulk1 was immunoprecipitated from the cells under nutrient-rich medium and Ulk1 mobility was examined by western blot.(e) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho-Ulk1 Ser 757 antibody. Uncropped images of blots are shown in Supplementary Fig. S5.


figf6 ( a ) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo . CBP / SBP tagged Ulk1 ( wild type or S757C ) was co - transfected with HA - AMPKα and Rheb into HEK293 cells as indicated . Ulk1 was purified by streptavidin beads and the co - precipitatedHA - AMPKα was examined by western blot ( Rapa , 50 nM rapamycin treatment for 1 h before cell lysis ) . ( b ) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1 - AMPK interaction in vitro . CBP / SBP Ulk1 proteins ( wild type or S757C ) were prepared from transfected HEK293 cells , which were pre - incubated with 50 nM rapamycin ( Rapa , 1h ) as indicated . The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1 - bead was incubated with the bacterial purified AMPKα / β / γ complex . AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody . L . E . ; long exposure . ( c ) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1 − / − Tsc1 . Tsc1 + / + ( WT ) and Tsc1 − / − ( KO ) MEFs were starved of glucose ( 4 h ) , or treated with 50 nM rapamycin ( Rapa , 1 h ) . Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777 . ( d ) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1 . HA - Ulk1 , AMPKα kinase - dead mutant ( DN ) , and Myc - Rheb were co - transfected into HEK293 cells as indicated . The cells were incubated with glucose - free medium ( - Glu , glucose ) , in which either 20 μM compound C ( C . C . ) or 50 nM Rapamycin ( Rapa ) was added . Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317 , Ser 777 , Ser 757 , and HA , as indicated . ( e ) Rheb inhibits glucose starvation - induced Ulk1 activation . HA - Ulk1 and Myc - Rheb was transfected into HEK293 cells , which were incubated with glucose - free ( - Glu ) , amino - acid - free ( - A . A ) medium , or 50 nM rapamycin ( Rapa ) for 4 h before lysis . HA - Ulk1 was immunoprecipitated and kinase assays were performed . Ulk1 activity was measured by 32P - autoradiogram and the protein level of HA - Ulk1 and GST - Atg13 used in the assay was determined by western blot and by Coomassie staining , respectively . Uncropped images of blots are shown in Supplementary Fig . S5 .

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

figf6(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis).(b) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1-AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1-bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure.(c) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777.(d) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA-Ulk1, AMPKα kinase-dead mutant (DN), and Myc-Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (-Glu, glucose), in which either 20 μM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated.(e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA-Ulk1 and Myc-Rheb was transfected into HEK293 cells, which were incubated with glucose-free (-Glu), amino-acid-free (-A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA-Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA-Ulk1 and GST-Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.


figf7 ( a ) Ser 317 / Ser 777 is required for Ulk1 to protect cells from glucose starvation . Viability ( 24 h , mean ± s . d . , n = 4 ; top ) and PARP cleavage ( 8 h ; western blot , middle ; quantification , n = 2 , bottom ) was examined in Ulk1 + / + ( WT ) , Ulk1 − / − ( KO ) , Ulk1 − / − re - expressing wild - type Ulk1 ( KO - WT ) , and Ulk1 − / − re - expressing Ulk1 S317 / 777A mutant ( KO - S317 / 777A ) MEFs . Arrows in western blots indicate non - cleaved and cleaved PARP . ( b ) The Ulk1 S317 / 777A mutant is compromised in LC3 lipidation in response to glucose starvation . ULK1MEFs were cultured in MEFs - free medium for the indicated times . LC3 - II level was determined by western blotting and the LC3 - II accumulation was normalized by α - tubulin and quantified ( bottom , n = 3 , mean ± s . d . ) . A representative western blot was shown . The LC3 antibody used in this experiment seemed to preferentially recognise the lipid - modified form of LC3 - II , which migrated faster on the gel . ( c ) The Ulk1 S317 / 777A mutant is defective in autophagosome formation . The indicated MEFs were starved of glucose ( 4 h ) and the formation of GFP - LC3 - positive autophagosomes was examined by confocal microscopy . GFP - LC3 ; green and DAPI ; blue . Scale bar , 20 μm . ( d ) Autophagy vacuole analysis by electron microscopy . Low - magnification images of Ulk1 − / − ( KO , upper left panel ) , Ulk1 − / − reconstituted with wild - type Ulk1 ( KO - WT , two middle panels with accompanying higher magnification images ) , and Ulk1 − / − reconstituted with Ulk1 S317 / 777A ( KO - S317 / 777A , lower left panel ) are shown . High - magnification images of autophagosomes from KO - WT are shown in upper right and lower right panels . Autophagosome / autolysosome - like structures indicated by arrowheads on the lower - magnification images and arrows in higher - magnification images . Scale bars ; lower - magnification , 1 μm ; higher - magnification , 200 nm .

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figf7(a) Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation. Viability (24 h, mean ± s.d., n = 4; top) and PARP cleavage (8 h; western blot, middle; quantification, n = 2, bottom) was examined in Ulk1+/+ (WT), Ulk1−/− (KO), Ulk1−/− re-expressing wild-type Ulk1 (KO-WT), and Ulk1−/− re-expressing Ulk1 S317/777A mutant (KO-S317/777A) MEFs. Arrows in western blots indicate non-cleaved and cleaved PARP.(b) The Ulk1 S317/777A mutant is compromised in LC3 lipidation in response to glucose starvation. ULK1MEFs were cultured in MEFs-free medium for the indicated times. LC3-II level was determined by western blotting and the LC3-II accumulation was normalized by α-tubulin and quantified (bottom, n = 3, mean ± s.d.). A representative western blot was shown. The LC3 antibody used in this experiment seemed to preferentially recognise the lipid-modified form of LC3-II, which migrated faster on the gel.(c) The Ulk1 S317/777A mutant is defective in autophagosome formation. The indicated MEFs were starved of glucose (4 h) and the formation of GFP-LC3-positive autophagosomes was examined by confocal microscopy. GFP-LC3; green and DAPI; blue. Scale bar, 20 μm.(d) Autophagy vacuole analysis by electron microscopy. Low-magnification images of Ulk1−/− (KO, upper left panel), Ulk1−/− reconstituted with wild-type Ulk1 (KO-WT, two middle panels with accompanying higher magnification images), and Ulk1−/− reconstituted with Ulk1 S317/777A (KO-S317/777A, lower left panel) are shown. High-magnification images of autophagosomes from KO-WT are shown in upper right and lower right panels. Autophagosome/autolysosome-like structures indicated by arrowheads on the lower-magnification images and arrows in higher-magnification images. Scale bars; lower-magnification, 1 μm; higher-magnification, 200 nm.


Figure 1A Immunostaining of HeLa cells probed for FITC ‐ labeled streptavidin and DNA and the merge streptavidin ( red ) and DNA ( blue ) . Cell surface was labeled using amine biotinylation . Scale bar , 10 μm . B Western blot analysis of cell surface preparation . The surface ‐ biotinylated cells were lysed using a detergent containing lysis buffer , incubated with and then eluted from streptavidin beads ( i . e . , cell sur . fraction ) . Cell surface fraction and the whole ‐ cell lysate were blotted against a cell surface protein , EGFR , and a cytoplasmic protein , actin .

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Figure 1A Immunostaining of HeLa cells probed for FITC‐labeled streptavidin and DNA and the merge streptavidin (red) and DNA (blue). Cell surface was labeled using amine biotinylation. Scale bar, 10 μm.B Western blot analysis of cell surface preparation. The surface‐biotinylated cells were lysed using a detergent containing lysis buffer, incubated with and then eluted from streptavidin beads (i.e., cell sur. fraction). Cell surface fraction and the whole‐cell lysate were blotted against a cell surface protein, EGFR, and a cytoplasmic protein, actin.


Figure 2B Western blot confirmation of the mitosis ‐ selective cell surface localization of PCDH7 . Western blotting analysis of cell surface protein preparation from mitosis and interphase cells . The whole ‐ cell lysate and elution from streptavidin beads were blotted against TFRC , EGFR , actin , and phospho ‐ Histone H3 . TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation , while PCDH7 protein was enriched in mitotic cell surface preparation . C Western blot confirmation of the mitosis ‐ selective cell surface localization of PCDH1 . Whole ‐ cell lysate samples were subjected to SDS - PAGE and blotted with the anti ‐ PCDH1 , anti ‐ β ‐ actin , and anti ‐ phospho ‐ Histone H3 antibodies , as indicated on the left . Cell surface fraction samples were blotted with the anti ‐ PCDH1 and anti ‐ EGFR antibodies .

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Figure 2B Western blot confirmation of the mitosis‐selective cell surface localization of PCDH7. Western blotting analysis of cell surface protein preparation from mitosis and interphase cells. The whole‐cell lysate and elution from streptavidin beads were blotted against TFRC, EGFR, actin, and phospho‐Histone H3. TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation, while PCDH7 protein was enriched in mitotic cell surface preparation.C Western blot confirmation of the mitosis‐selective cell surface localization of PCDH1. Whole‐cell lysate samples were subjected to SDS-PAGE and blotted with the anti‐PCDH1, anti‐β‐actin, and anti‐phospho‐Histone H3 antibodies, as indicated on the left. Cell surface fraction samples were blotted with the anti‐PCDH1 and anti‐ EGFR antibodies.


Figure 3A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells : tubulin , green ; PCDH7 , red ; DNA , blue . PCDH7 localizes to the mitotic cell surface ( arrows ) . PCDH7 staining is barely detectable in the surrounding interphase cells . Scale bar , 5 μm . B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis ( n = 6 , red ) and interphase ( n = 7 , blue ) . Bars show mean ± SEM . C PCDH7 ‐ EGFP ‐ transfected unsynchronized U2OS cells , stably expressing mCherry ‐ tubulin , at mitosis ( top ) and interphase ( bottom ) . PCDH7 , green ; tubulin , red ; DNA , blue . Scale bar , 10 μm . D Immunostaining of HeLa Kyoto cells stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes ( green ) probed for tubulin ( red ) and DNA ( blue ) . Top panel shows mitotic cell those were treated with 5 μM STC for 10 h , and bottom panel shows interphase cells . Scale bar , 10 μm .

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Figure 3A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue. PCDH7 localizes to the mitotic cell surface (arrows). PCDH7 staining is barely detectable in the surrounding interphase cells. Scale bar, 5 μm.B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis (n = 6, red) and interphase (n = 7, blue). Bars show mean ± SEM.C PCDH7‐EGFP‐transfected unsynchronized U2OS cells, stably expressing mCherry‐tubulin, at mitosis (top) and interphase (bottom). PCDH7, green; tubulin, red; DNA, blue. Scale bar, 10 μm.D Immunostaining of HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes (green) probed for tubulin (red) and DNA (blue). Top panel shows mitotic cell those were treated with 5 μM STC for 10 h, and bottom panel shows interphase cells. Scale bar, 10 μm.


Figure 4A Confocal microscopy images representing the localization of PCDH7 . U2OS cells expressing full ‐ length ( WT ) PCDH7 at mitosis ( top ) and interphase ( middle ) . Cytoplasmic deleted ( CytΔ ) PCDH7 ‐ expressing cells at mitosis ( middle ) and interphase ( bottom ) . PCDH7 , green ; DNA , blue . Scale bar , 20 μm . B Quantification of PCDH7 : : GFP ( WT and CytΔ ) protein localization on the cell surface in mitosis [ WT ( n = 11 ) , CytΔ ( n = 6 ) , red ] and interphase [ WT ( n = 25 ) , CytΔ ( n = 20 ) , blue ] . Interphase cytΔ PCDH7 localizes to the cell surface more than WT ( full length ) PCDH7 ( insets ) . Bars show mean ± SEM . C In interphase co ‐ localization between PCDH7 and ER marker KDEL . HeLa cells were transiently transfected with PCDH7 : : GFP ( green ) ( ectopic ) , fixed and stained for ER marker KDEL ( red ) ( interphase , top panel ) . Interphase ( middle panel ) and mitotic ( bottom panel ) HeLa cells were fixed and stained against ( endogenous ) PCDH7 and KDEL . Scale bar , 5 μm . D PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction . Whole ‐ cell lysate , cytoplasmic , endoplasmic , and mitochondrial fractions were blotted against an endoplasmic reticulum protein , KDEL , a cytoplasmic protein , actin , a mitochondrial protein Cox ‐ 2 and PCDH7 , PCDH1 .

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Figure 4A Confocal microscopy images representing the localization of PCDH7. U2OS cells expressing full‐length (WT) PCDH7 at mitosis (top) and interphase (middle). Cytoplasmic deleted (CytΔ) PCDH7‐expressing cells at mitosis (middle) and interphase (bottom). PCDH7, green; DNA, blue. Scale bar, 20 μm.B Quantification of PCDH7::GFP (WT and CytΔ) protein localization on the cell surface in mitosis [WT (n = 11), CytΔ (n = 6), red] and interphase [WT (n = 25), CytΔ (n = 20), blue]. Interphase cytΔ PCDH7 localizes to the cell surface more than WT (full length) PCDH7 (insets). Bars show mean ± SEM.C In interphase co‐localization between PCDH7 and ER marker KDEL. HeLa cells were transiently transfected with PCDH7::GFP (green) (ectopic), fixed and stained for ER marker KDEL (red) (interphase, top panel). Interphase (middle panel) and mitotic (bottom panel) HeLa cells were fixed and stained against (endogenous) PCDH7 and KDEL. Scale bar, 5 μm.D PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction. Whole‐cell lysate, cytoplasmic, endoplasmic, and mitochondrial fractions were blotted against an endoplasmic reticulum protein, KDEL, a cytoplasmic protein, actin, a mitochondrial protein Cox‐2 and PCDH7, PCDH1.


Figure 5A PCDH7 antibody detects a single band of ˜ 110 kDa in luciferase RNAi ( luc ) , PCDH1 RNAi HeLa cell lysates , but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells . α ‐ tubulin was used as a loading control ( left ) . HeLa Kyoto cells stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes treated with luciferase RNAi ( control ) and 150 nM ( 1 × ) and 300 nM ( 2 × ) PCDH1 esiRNA and blotted against anti ‐ GFP , anti ‐ PCDH1 , and anti ‐ actin antibodies . PCDH1 bands were reduced in PCDH1 RNAi cells , whereas mouse GFP : : PCDH1 was not affected by the esiRNA treatment ( right ) . B Rounding pressure plots of MYH9 ( positive control , n = 7 ) , luc ( negative control , n = 12 ) , PCDH7 ( n = 11 ) , PCDH1 ( n = 12 ) , and PCDH1 and PCDH7 ( n = 11 ) RNAi cells . Bars show mean ± SEM ( left ) . Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes treated with MYH9 ( positive control , n = 7 ) , luc ( negative control , n = 14 ) , PCDH1 ( n = 13 ) esiRNA . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; ns , P > 0 . 05 ( non ‐ significant ) .

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Figure 5A PCDH7 antibody detects a single band of ˜110 kDa in luciferase RNAi (luc), PCDH1 RNAi HeLa cell lysates, but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells. α‐tubulin was used as a loading control (left). HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with luciferase RNAi (control) and 150 nM (1×) and 300 nM (2×) PCDH1 esiRNA and blotted against anti‐GFP, anti‐PCDH1, and anti‐actin antibodies. PCDH1 bands were reduced in PCDH1 RNAi cells, whereas mouse GFP::PCDH1 was not affected by the esiRNA treatment (right).B Rounding pressure plots of MYH9 (positive control, n = 7), luc (negative control, n = 12), PCDH7 (n = 11), PCDH1 (n = 12), and PCDH1 and PCDH7 (n = 11) RNAi cells. Bars show mean ± SEM (left). Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with MYH9 (positive control, n = 7), luc (negative control, n = 14), PCDH1 (n = 13) esiRNA. *P 0.05; **P 0.01; ***P 0.001; ns, P > 0.05 (non‐significant).


Figure 6B Flow cytometry analysis of HeLa cells treated with anti ‐ PCDH7 antibody , fixed and stained for phospho ‐ Histone H3 and DNA . Forty ‐ two percent of unsorted HeLa cella are in G2 / M phase . When cells are sorted based on their phospho ‐ Histone H3 ( middle ) expression , phospho ‐ Histone H3 + ( high expression ) cells are preferentially mitotic ( 87 . 3 % in G2 / M phase ) and 42 % of those are PCDH7 + . Similarly , PCDH7 + ( high expression ) cells are preferentially mitotic ( 82 . 1 % in G2 / M phase ) and 79 % of those are phospho ‐ Histone H3 + ( bottom ) .

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Figure 6B Flow cytometry analysis of HeLa cells treated with anti‐PCDH7 antibody, fixed and stained for phospho‐Histone H3 and DNA. Forty‐two percent of unsorted HeLa cella are in G2/M phase. When cells are sorted based on their phospho‐Histone H3 (middle) expression, phospho‐Histone H3+ (high expression) cells are preferentially mitotic (87.3% in G2/M phase) and 42% of those are PCDH7+. Similarly, PCDH7+ (high expression) cells are preferentially mitotic (82.1% in G2/M phase) and 79% of those are phospho‐Histone H3+ (bottom).


Figure 1 ( C ) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A ( ant . A ) . ( D , F ) Mean FRET efficiency measured over time in the ( D ) AP boundary ( APB ) or ( F ) DV boundary ( DVB ) and non - organizer regions ( NO ) . Shaded regions indicate standard deviation ( SD ) ; dots indicate mean per timepoint , and solid lines indicate the fit to the mean . Black arrows indicate the addition of the drug .

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Figure 1(C) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A (ant.A).(D, F) Mean FRET efficiency measured over time in the (D) AP boundary (APB) or (F) DV boundary (DVB) and non-organizer regions (NO). Shaded regions indicate standard deviation (SD); dots indicate mean per timepoint, and solid lines indicate the fit to the mean. Black arrows indicate the addition of the drug.


Figure 2 ( A ) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A ( ant . A ) , 100 μM 3 - Bromopyruvate ( 3BP ) , and 50 mM 2 - deoxy - D - glucose ( 2DG ) . ( B ) Mean FRET efficiency of organizer ( ORG ) regions and non - organizer ( NO ) regions analyzed over time . Shaded regions indicate SD ; dots indicate mean per timepoint , and solid lines indicate the fit to the mean . ( C ) Fit parameters of individual time traces for organizer and non - organizer regions . Each dot represents data from one disc , and lines connect the corresponding regions of the same disc . Box plots summarize the data : boxes encompass the 2nd - 3rd quartiles , with whiskers indicating the 1st and 4th quartiles and the red line indicating the median . * = p - value < 0 . 05 , ns = not significant p - value using a Kruskal - Wallis test ( n = 26 discs ) . ( D ) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition . ( E ) Mean FRET efficiency time trace of the entire wing pouch ; shaded region indicates SD ; black arrows indicate the addition of the drugs . Brackets indicate the mean FRET values before and 2h after 2DG addition ( 1 - 10 min and 121 - 130 min , respectively ) . The distribution of mean values within these brackets are shown as violin plots in ( F ) ( solid line indicates median , dotted indicate quartiles ) . These were compared using an unpaired t - test ( ns = not significant p - value , n = 17 discs ) including ( G ) Welch ' s correction and estimation plot ( n = 17 discs for each group ) . In ( G ) , horizontal dotted lines run through the mean values for the two samples ( before 2DG and 2hr after 2DG ) ; on the right , the difference between the two means is plotted on a separate axis ( right ) , where the bars indicate the upper and lower 95 % confidence intervals .

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Figure 2(A) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A (ant.A), 100 μM 3-Bromopyruvate (3BP), and 50 mM 2-deoxy-D-glucose (2DG). (B) Mean FRET efficiency of organizer (ORG) regions and non-organizer (NO) regions analyzed over time. Shaded regions indicate SD; dots indicate mean per timepoint, and solid lines indicate the fit to the mean. (C) Fit parameters of individual time traces for organizer and non-organizer regions. Each dot represents data from one disc, and lines connect the corresponding regions of the same disc. Box plots summarize the data: boxes encompass the 2nd-3rd quartiles, with whiskers indicating the 1st and 4th quartiles and the red line indicating the median. * = p-value < 0.05, ns = not significant p-value using a Kruskal-Wallis test (n=26 discs).(D) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition. (E) Mean FRET efficiency time trace of the entire wing pouch; shaded region indicates SD; black arrows indicate the addition of the drugs. Brackets indicate the mean FRET values before and 2h after 2DG addition (1-10 min and 121-130 min, respectively). The distribution of mean values within these brackets are shown as violin plots in (F) (solid line indicates median, dotted indicate quartiles). These were compared using an unpaired t-test (ns = not significant p-value, n = 17 discs) including (G) Welch's correction and estimation plot (n=17 discs for each group). In (G), horizontal dotted lines run through the mean values for the two samples (before 2DG and 2hr after 2DG); on the right, the difference between the two means is plotted on a separate axis (right), where the bars indicate the upper and lower 95% confidence intervals.


Figure 3 ( C ) Timelapse of ATP sensor FRET efficiency in the apGalts > PtcRNAi wing pouch after 10 μM antimycin A ( ant . A ) addition . Red dashed line indicates the position of the AP boundary . ( D , F ) Mean FRET efficiency analyzed over time in the ( D ) dorsal anterior ( DA ) and ventral anterior ( VA ) sub - compartments or the ( F ) dorsal anterior ( DA ) and dorsal posterior ( DP ) sub - compartments of apGalts > PtcRNAi wing discs . Shaded regions indicate SD ; dots represent the mean per timepoint ; solid lines illustrate a fit of the mean data . ( I ) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition . ( J ) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings . Shaded regions indicate SD ; dots represent the average per timepoint ; solid lines illustrate a fit of the mean data .

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Figure 3(C) Timelapse of ATP sensor FRET efficiency in the apGalts>PtcRNAi wing pouch after 10 μM antimycin A (ant.A) addition. Red dashed line indicates the position of the AP boundary.(D, F) Mean FRET efficiency analyzed over time in the (D) dorsal anterior (DA) and ventral anterior (VA) sub-compartments or the (F) dorsal anterior (DA) and dorsal posterior (DP) sub-compartments of apGalts>PtcRNAi wing discs. Shaded regions indicate SD; dots represent the mean per timepoint; solid lines illustrate a fit of the mean data.(I) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition.(J) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings. Shaded regions indicate SD; dots represent the average per timepoint; solid lines illustrate a fit of the mean data.


Figure 4 ( A ) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants . To highlight the variability in this experiment , we show example discs from two separate experiments . ( B , D ) Mean FRET efficiency measured over time in non - organizers compared to ( B ) Anterior - Posterior boundary ( APB ) or ( D ) all organizer regions . Shaded regions indicate SD ; dots are the means per each timepoint , and the solid lines indicate the fit to the mean .

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

Figure 4(A) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants. To highlight the variability in this experiment, we show example discs from two separate experiments.(B, D) Mean FRET efficiency measured over time in non-organizers compared to (B) Anterior-Posterior boundary (APB) or (D) all organizer regions. Shaded regions indicate SD; dots are the means per each timepoint, and the solid lines indicate the fit to the mean.


Figure 1 ( A - H ) Six to eight weeks old female outbred CD - 1 mice were infected via tail vein injection with 5x107 CFU of WT S . aureus Newman or isogenic mutant strains grown in TSB medium to an OD600 of 0 . 5 . Organs were harvested after ( A - D ) four days or ( E - H ) eight days post - infection , and bacterial burdens in the ( A and E ) liver , ( B and F ) heart , ( C and G ) spleen and ( D and H ) kidney were determined . Data represent mean ± SEM ( n = 7 biological replicates ) . . * Denotes p < 0 . 05 , * * denotes p < 0 . 005 and * * * denotes p < 0 . 0007 , using the Mann - Whitney test .

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

Figure 1(A-H) Six to eight weeks old female outbred CD-1 mice were infected via tail vein injection with 5x107 CFU of WT S. aureus Newman or isogenic mutant strains grown in TSB medium to an OD600 of 0.5. Organs were harvested after (A-D) four days or (E-H) eight days post-infection, and bacterial burdens in the (A and E) liver, (B and F) heart, (C and G) spleen and (D and H) kidney were determined. Data represent mean ± SEM (n=7 biological replicates).. * Denotes p < 0.05, ** denotes p < 0.005 and *** denotes p < 0.0007, using the Mann-Whitney test.


Figure 2Cells were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( A ) Bacterial growth was measured by optical density at 600 nm . Data are representative of five independent experiments . Cells were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( B ) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth . Data are expressed as average ± SEM of three independent experiments . * * * Denotes p < 0 . 001 compared to ndhC expression in WT strain grown in MHB - ca medium via two - way ANOVA with Tukey ' s posttest .

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

Figure 2Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (A) Bacterial growth was measured by optical density at 600 nm. Data are representative of five independent experiments. Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (B) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth. Data are expressed as average ± SEM of three independent experiments. *** Denotes p < 0.001 compared to ndhC expression in WT strain grown in MHB-ca medium via two-way ANOVA with Tukey's posttest.


Figure 3 ( A ) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode . Cells were grown in MHB - ca medium until exponential phase ( OD600 = 0 . 6 ) or until late stationary phase ( 24 h ) . Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol . Data are expressed as average ± SD of four independent experiments . * * * denotes p < 0 . 001 compared to WT via two - way ANOVA with Tukey ' s posttest . ( B ) NADH / NAD + ratios were measured in cells grown aerobically in MHB - ca medium for 20 h were measured . 100 % corresponds to a NADH / NAD + ratio of 0 . 003 . Data are expressed as average ± SD of four independent experiments . * Denotes p < 0 . 05 , and * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest . ( C ) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * * Denotes p < 0 . 005 and * * * denotes p < 0 . 001 compared to WT via two - way ANOVA with Tukey ' s posttest . ( D ) MKH2 / MK ratios were measured in cells grown aerobically for 20 h in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * Denotes p < 0 . 05 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

Figure 3(A) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode. Cells were grown in MHB-ca medium until exponential phase (OD600 = 0.6) or until late stationary phase (24 h). Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol. Data are expressed as average ± SD of four independent experiments. *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.(B) NADH/NAD+ ratios were measured in cells grown aerobically in MHB-ca medium for 20 h were measured. 100% corresponds to a NADH/NAD+ ratio of 0.003. Data are expressed as average ± SD of four independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.(C) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.(D) MKH2/MK ratios were measured in cells grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.


Figure 4 ( A ) The carotenoid pigment of different S . aureus strains was extracted by methanol and quantified by the optical density at 465 nm , and the values were normalized to WT Newman strain , which was set at 100 . Data are expressed as the average ± SD of five independent experiments . * * Denotes p < 0 . 005 and * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest . ( B ) Hydrogen peroxide sensitivity was measured by incubating WT and isogenic strains , previously grown for 20 h in MHB - ca , with different concentrations of hydrogen peroxide . Serial dilutions were plated in TSB medium for measuring CFU . Data are expressed as the average ± SD of three independent experiments .

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

Figure 4(A) The carotenoid pigment of different S. aureus strains was extracted by methanol and quantified by the optical density at 465 nm, and the values were normalized to WT Newman strain, which was set at 100. Data are expressed as the average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.(B) Hydrogen peroxide sensitivity was measured by incubating WT and isogenic strains, previously grown for 20 h in MHB-ca, with different concentrations of hydrogen peroxide. Serial dilutions were plated in TSB medium for measuring CFU. Data are expressed as the average ± SD of three independent experiments.


Figure 5 ( A ) PndhC and PndhF YFP reporter constructs were used to follow gene expression under the biofilm growth conditions . Data collected after 24 h of static incubation was normalized by optical density at 600 nm . Data are expressed as average ± SEM of three independent experiments . No significant differences were found when ndhC expression in WT strain was compared via one - way ANOVA with Dunnett ' s posttest . ( B ) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 ° C for 20 h . The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose and 2 % NaCl . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest . ( C ) MKH2 / MK ratios were measured in cells grown under the same conditions as for biofilm formation . Data are expressed as average ± SD of three independent experiments . * Denotes p < 0 . 05 compared to WT via one - way ANOVA with Dunnett ' s posttest . ( D ) NADH / NAD + ratios were measured in cells grown under the same conditions as for biofilm formation . 100 % corresponds to a NADH / NAD + ratio of 0 . 09 . Data are expressed as average ± SD of four independent experiments performed by duplicates . * Denotes p < 0 . 05 , and * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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

Figure 5(A) PndhC and PndhF YFP reporter constructs were used to follow gene expression under the biofilm growth conditions. Data collected after 24 h of static incubation was normalized by optical density at 600 nm. Data are expressed as average ± SEM of three independent experiments. No significant differences were found when ndhC expression in WT strain was compared via one-way ANOVA with Dunnett's posttest.(B) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.(C) MKH2/MK ratios were measured in cells grown under the same conditions as for biofilm formation. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.(D) NADH/NAD+ ratios were measured in cells grown under the same conditions as for biofilm formation. 100% corresponds to a NADH/NAD+ ratio of 0.09. Data are expressed as average ± SD of four independent experiments performed by duplicates. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


Figure 6Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB - ca medium without and with 25 mM glucose , 40 mM sodium pyruvate or 60 mM sodium acetate . Lysis was monitored by the release of hemoglobin . Data are expressed as average ± SD of five independent experiments . * Denotes p < 0 . 05 , and * * * denotes p < 0 . 001 , ns denotes no significant compared to WT grown in MHB - ca and WT grown in MHB - ca + glucose via two - way ANOVA with Tukey ' s posttest .

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

Figure 6Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium without and with 25 mM glucose, 40 mM sodium pyruvate or 60 mM sodium acetate. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of five independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001, ns denotes no significant compared to WT grown in MHB-ca and WT grown in MHB-ca + glucose via two-way ANOVA with Tukey's posttest.


Figure 7 ( A ) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB - ca medium . Lysis was monitored by the release of hemoglobin . Data are expressed as average ± SD of four independent experiments . * * * Denotes p < 0 . 001 , ns denotes no significant compared to WT via one - way ANOVA with Dunnett ' s posttest . ( B ) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 ° C for 20 h . The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose and 2 % NaCl . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * Denotes p < 0 . 005 , ns denotes no significant compared to WT via one - way ANOVA with Dunnett ' s posttest . ( C ) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB - ca medium , in the absence or presence of 10 mg / ml FA - deficient BSA and 0 . 01 % oleic acid . Data are expressed as average ± SD of five independent experiments . * * Denotes p < 0 . 005 and * * * denotes p < 0 . 001 compared to each strain without any BSA or oleic acid addition via two - way ANOVA with Tukey ' s posttest . ( D ) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose and 2 % NaCl , in the absence or presence of 10 mg / ml FA - deficient BSA and 0 . 01 % oleic acid . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to each strain without any BSA or oleic acid addition via two - way ANOVA with Tukey ' s posttest . ( E ) Hemolytic activity of Newman ∆ ndhC strain overexpressing the N . gonorrhea acyl - ACP synthetase and grown aerobically for 20 h in MHB - ca medium . Data are expressed as average ± SD of four independent experiments . * * Denotes p < 0 . 005 compared to ∆ ndhC strain with the empty plasmid via Student ' s t - test . ( F ) Biofilm formation of Newman ∆ ndhC strain overexpressing the N . gonorrhea acyl - ACP synthetase and grown under biofilm producing conditions . Data are expressed as average ± SD of three independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to ∆ ndhC strain with the empty plasmid via Student ' s t - test .

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

Figure 7(A) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of four independent experiments. *** Denotes p < 0.001, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.(B) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. ** Denotes p < 0.005, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.(C) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB-ca medium, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.(D) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.(E) Hemolytic activity of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of four independent experiments. ** Denotes p < 0.005 compared to ∆ndhC strain with the empty plasmid via Student's t-test.(F) Biofilm formation of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown under biofilm producing conditions. Data are expressed as average ± SD of three independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to ∆ndhC strain with the empty plasmid via Student's t-test.


Figure 1Primary wild type MEF were treated with DMSO or Aurora kinase inhibitor ZM447439 ( ZM , 2μM ) for 72h , followed by RNA isolation and mRNA - sequencing . ( A ) Volcano plot representation of differentially expressed genes ( DEGs ) between ZM and DMSO treatment . Adjusted p value ≤ 0 . 01 and log2 FC ≥ 1 or log2 FC ≤ - 1 .

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

Figure 1Primary wild type MEF were treated with DMSO or Aurora kinase inhibitor ZM447439 (ZM, 2μM) for 72h, followed by RNA isolation and mRNA-sequencing. (A) Volcano plot representation of differentially expressed genes (DEGs) between ZM and DMSO treatment. Adjusted p value ≤ 0.01 and log2 FC ≥ 1 or log2 FC ≤ -1.


Figure 2 ( A ) Time - lapse analysis of NF - κB - driven EGFP reporter activity in A549kB cells where cytokinesis failure was induced by treatment with ZM ( 2μM ) or DHCB ( 4μM ) . Single cells were imaged every 20 min using IncuCyte life cell imaging technology . Left , representative images of A549kB cells 48h after starting the treatment . Scale bars : 10 μm . Right , EGFP integrated fluorescence intensity of individually tracked cells was measured over time . Time zero was determined as the moment of cytokinesis failure ( CF ) of each individual cell analyzed . Dashed lines indicate the integrated EGFP intensity of the cells at time zero that was used to calculate the fold - change over time . Solid lines indicate the mean EGFP integrated fluorescence intensity of the cells 24 h after failing cytokinesis . An n ≥ 10 cells per condition were followed individually in a single experiment , data points represent means ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( B ) Top , experimental design . Left , densitometric analysis of IKBα protein levels in synchronized A549 cells detected over time after release . Bars represent ZM values normalized with respect to DMSO . n = 3 independent experiments , data represent mean values ± SEM . Right , time course immunoblot analysis of synchronized A549 cells , released in DMSO or ZM ( 2μM ) . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( D , E ) ( D ) Western blot analysis confirming lack of PIDD1 expression in A549 cells and ( E ) MDM2 processing after induction of cytokinesis failure using ZM ( 2µM ) for 48h . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( F ) Live - cell imaging analysis of NF - κB EGFP - reporter activity in CRISPR - edited polyclonal A549κB cells . NF - κB activity of ZM - treated cells is represented as fold - change with respect their untreated counterparts ( DMSO - treated cells , grey lines ) . n = 5 independent experiments . Error bars represent ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( H ) Live - cell imaging analysis of NF - κB EGFP - reporter activity in CRISPR - edited polyclonal RPE1kB cells analyzed as in as in F . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

Figure 2(A) Time-lapse analysis of NF-κB-driven EGFP reporter activity in A549kB cells where cytokinesis failure was induced by treatment with ZM (2μM ) or DHCB (4μM). Single cells were imaged every 20 min using IncuCyte life cell imaging technology. Left, representative images of A549kB cells 48h after starting the treatment. Scale bars: 10 μm. Right, EGFP integrated fluorescence intensity of individually tracked cells was measured over time. Time zero was determined as the moment of cytokinesis failure (CF) of each individual cell analyzed. Dashed lines indicate the integrated EGFP intensity of the cells at time zero that was used to calculate the fold-change over time. Solid lines indicate the mean EGFP integrated fluorescence intensity of the cells 24 h after failing cytokinesis. An n ≥ 10 cells per condition were followed individually in a single experiment, data points represent means ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(B) Top, experimental design. Left, densitometric analysis of IKBα protein levels in synchronized A549 cells detected over time after release. Bars represent ZM values normalized with respect to DMSO. n=3 independent experiments, data represent mean values ± SEM. Right, time course immunoblot analysis of synchronized A549 cells, released in DMSO or ZM (2μM). Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(D, E) (D) Western blot analysis confirming lack of PIDD1 expression in A549 cells and (E) MDM2 processing after induction of cytokinesis failure using ZM (2µM) for 48h. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(F) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal A549κB cells. NF-κB activity of ZM-treated cells is represented as fold-change with respect their untreated counterparts (DMSO-treated cells, grey lines). n=5 independent experiments. Error bars represent ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).(H) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal RPE1kB cells analyzed as in as in F. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).


Figure 3 ( A ) Representative immunofluorescence images of γ - tubulin staining to mark and enumerate centrosomes in A549κB reporter cells . Cells were pre - treated for 24 h with centrinone ( 125nM ) or solvent control ( DMSO ) and cultured further in medium either containing DMSO or ZM to promote cytokinesis failure . Size bar : 10µM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( B ) Cells treated as indicated in A were analyzed for NF - κB - driven EGFP - reporter activation by IncuCyte live - cell imaging . Left , representative pictures of live - cell imaging NF - κB activation after treatment with DMSO , ZM ( 2μM , 48 h ) or TNF ( 20 ng / mL , 24h ) . Size bar : 20µm . Right , EGFP MFI quantification of ZM - treated cells was plotted as fold - change expression relative to DMSO - treated controls . Data points represent means of n = 4 biological replicates ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( C ) Left , representative immunofluorescence images of supernumerary centrosomes originated from Dox - inducible PLK4 overexpression in CRISPR - edited polyclonal U2OSPLK4 cells . Size scale bar : 10µm . Right , representative cell cycle profiles of U2OSPLK4 control and PIDD1 - deficient cells . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( E ) Live - cell imaging analysis of NF - κB EGFP - reporter activity in CRISPR - edited polyclonal PIDD1 - competent or - deficient U2OSκB - PLK4 and A549κB - PLK4 cells . NF - κB activity of ZM , Dox or TNF - treated cells is represented as fold - change with respect to their untreated counterparts . Bars represent means of n = 5 independent experiments ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) . ( F ) Representative immunofluorescence images of A549 cells co - stained with the indicated antibodies and documenting PIDD1 localization at centrosomes . Scale bars : 10 μm . ( G ) Luciferase reporter analysis of NF - κB activity in CRISPR - edited SCLT1 - competent or - deficient A549 cells . Luciferase activity was normalized respect renilla control . n = 3 independent experiments ; represented mean values ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

Figure 3(A) Representative immunofluorescence images of γ-tubulin staining to mark and enumerate centrosomes in A549κB reporter cells. Cells were pre-treated for 24 h with centrinone (125nM) or solvent control (DMSO) and cultured further in medium either containing DMSO or ZM to promote cytokinesis failure. Size bar: 10µM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(B) Cells treated as indicated in A were analyzed for NF-κB-driven EGFP-reporter activation by IncuCyte live-cell imaging. Left, representative pictures of live-cell imaging NF-κB activation after treatment with DMSO, ZM (2μM, 48 h) or TNF (20 ng/mL, 24h). Size bar: 20µm. Right, EGFP MFI quantification of ZM-treated cells was plotted as fold-change expression relative to DMSO-treated controls. Data points represent means of n=4 biological replicates ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(C) Left, representative immunofluorescence images of supernumerary centrosomes originated from Dox-inducible PLK4 overexpression in CRISPR-edited polyclonal U2OSPLK4 cells. Size scale bar: 10µm. Right, representative cell cycle profiles of U2OSPLK4 control and PIDD1-deficient cells. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(E) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal PIDD1-competent or -deficient U2OSκB-PLK4 and A549κB-PLK4 cells. NF-κB activity of ZM, Dox or TNF-treated cells is represented as fold-change with respect to their untreated counterparts. Bars represent means of n=5 independent experiments ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).(F) Representative immunofluorescence images of A549 cells co-stained with the indicated antibodies and documenting PIDD1 localization at centrosomes. Scale bars: 10 μm. (G) Luciferase reporter analysis of NF-κB activity in CRISPR-edited SCLT1- competent or -deficient A549 cells. Luciferase activity was normalized respect renilla control. n=3 independent experiments; represented mean values ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).


Figure 4 ( B ) Left , immunoblot of CRISPR - edited polyclonal RIPK1 - or NEMO - deficient A549κB derivatives . Right , luciferase assay of A549 cells indicated genotypes , treated for 48h with either ZM ( 2 μM ) or DHCB ( 4 μM ) . n = 4 independent experiments . Data are represented as the mean values ± SEM . The statistical significance was determined using two - way ANOVA ; p < 0 . 01 ( * * ) ; p < 0 . 001 ( * * * ) ; p < 0 . 0001 ( * * * * ) . ( C ) Representative confocal fluorescence images documenting centrosome accumulation , as identified by CEP63 staining in 293T - Trex - Flip - in cells . Time course analysis was performed in cells synchronized in G1 / S after a 24h single thymidine block and release in either DMSO or ZM ( 2 μM ) . Cells were fixed at the indicated times for IF analyses . Scale bars : 10 μm . ( D ) Different time point immunoprecipitations ( IP ) after release in ZM ( 2 μM ) of endogenous RAIDD , RIPK1 and NEMO proteins in PIDD1 - deficient 293T - Trex - Flip - in cells re - expressing a FLAG - tagged PIDD1 - FL protein . One representative experiment out of three is shown . ( E ) Analysis of NEMO - PIDDosome formation in PIDD1 - deficient 293Trex - Flip - In cells re - expressing different PIDD1 mutants depicted in G . Cells were treated as in C and processed for FLAG - IP 11h after release into DMSO or ZM ( 2 μM ) . One out of two independent experiments is shown . ( F ) 293T cells were transiently transfected with constructs allowing expression of GFP , a full length ( FL ) single ( PIDD1 - FL _ FLAG ) or double - tagged ( HA _ PIDD1 - FL _ FLAG ) version of PIDD1 , or an HA - tagged N - terminal fragment of PIDD1 ( HA _ PIDD1 - N ) . Co - immunoprecipitation was performed to probe for interaction with endogenous RIPK1 after either anti - FLAG or anti - HA pull - down . One out of two independent experiments is shown .

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

Figure 4(B) Left, immunoblot of CRISPR-edited polyclonal RIPK1- or NEMO-deficient A549κB derivatives. Right, luciferase assay of A549 cells indicated genotypes, treated for 48h with either ZM (2 μM) or DHCB (4 μM). n=4 independent experiments. Data are represented as the mean values ± SEM. The statistical significance was determined using two-way ANOVA; p< 0.01 (**);p< 0.001 (***); p< 0.0001 (****).(C) Representative confocal fluorescence images documenting centrosome accumulation, as identified by CEP63 staining in 293T-Trex-Flip-in cells. Time course analysis was performed in cells synchronized in G1/S after a 24h single thymidine block and release in either DMSO or ZM (2 μM). Cells were fixed at the indicated times for IF analyses. Scale bars: 10 μm.(D) Different time point immunoprecipitations (IP) after release in ZM (2 μM) of endogenous RAIDD, RIPK1 and NEMO proteins in PIDD1-deficient 293T-Trex-Flip-in cells re-expressing a FLAG-tagged PIDD1-FL protein. One representative experiment out of three is shown.(E) Analysis of NEMO-PIDDosome formation in PIDD1-deficient 293Trex-Flip-In cells re-expressing different PIDD1 mutants depicted in G. Cells were treated as in C and processed for FLAG-IP 11h after release into DMSO or ZM (2 μM). One out of two independent experiments is shown.(F) 293T cells were transiently transfected with constructs allowing expression of GFP, a full length (FL) single (PIDD1-FL_FLAG) or double-tagged (HA_PIDD1-FL_FLAG) version of PIDD1, or an HA-tagged N-terminal fragment of PIDD1 (HA_PIDD1-N). Co-immunoprecipitation was performed to probe for interaction with endogenous RIPK1 after either anti-FLAG or anti-HA pull-down. One out of two independent experiments is shown.


Figure 5Primary MEF from Pidd1 - / - mice were treated for 72h with ZM or DMSO and subjected to mRNA - sequencing analysis . ( B ) qRT - PCR analysis confirm expression of different pro - inflammatory genes in Pidd1 + / + but not Pidd1 - / - MEF . Hprt1 , β - actin and Gapdh were used as housekeeping gene expression controls . n = 3 independent samples ; data represent mean values ± SEM . The statistical significance was determined using two - way ANOVA test ; p ≤ 0 . 01 ( * * ) , p ≤ 0 . 001 ( * * * ) . Primary MEF from Pidd1 - / - mice were treated for 72h with ZM or DMSO and subjected to mRNA - sequencing analysis . ( D ) qRT - qPCR analysis of macrophage activation markers indicative of a pro - inflammatory state . Relative expression values are plotted as the fold - change expression relative to BMDM stimulated with conditioned medium from untreated MEFPLK4 . β - actin was used as housekeeping gene for normalization . Bars represent mean ± SEM ( n = 9 ) biological replicates . The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 01 ( * * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

Figure 5Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (B) qRT-PCR analysis confirm expression of different pro-inflammatory genes in Pidd1+/+ but not Pidd1-/- MEF. Hprt1, β-actin and Gapdh were used as housekeeping gene expression controls. n=3 independent samples; data represent mean values ± SEM. The statistical significance was determined using two-way ANOVA test; p ≤ 0.01 (**), p ≤ 0.001 (***).Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (D) qRT-qPCR analysis of macrophage activation markers indicative of a pro-inflammatory state. Relative expression values are plotted as the fold-change expression relative to BMDM stimulated with conditioned medium from untreated MEFPLK4. β-actin was used as housekeeping gene for normalization. Bars represent mean ± SEM (n=9) biological replicates. The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).


Figure 6A549EGFP cells were treated with DMSO or ZM ( 2μM ) for 72h , followed by co - culture with NK - 92 cells and live - cell imaging analysis . ( A ) Representative images of target A549EGFP cells pre - treated for 72 h with ZM ( 2µM ) and co - cultured subsequently with NK - 92 cells in a 1 : 10 target to effector ratio . Interactions were recorded by live - cell imaging for 18 h at a 1 min interval . White arrow indicate polyploid cell undergoing cell death . EGFP release coincides with loss of cell membrane integrity . All images were acquired at the same exposure time and light intensity . Scale bar : 10 µm . Data information : The statistical significance was determined using two - tailed t - test ; P < 0 . 001 ( * * ) ; P < 0 . 001 ( * * * ) . A549EGFP cells were treated with DMSO or ZM ( 2μM ) for 72h , followed by co - culture with NK - 92 cells and live - cell imaging analysis . ( D ) Confluence of ZM pre - treated A549EGFP cells expressing or lacking PIDD1 was measured over time in presence of NK cells and normalized with respect to their DMSO treated counterparts . Middle , NK cell - mediated killing interpolated from cell confluence graphs at 50 h . n ≥ 4 independent experiments performed in duplicates , mean ± SEM . ( E ) Confluence measurement over time of A549EGFP derivatives impaired in either NEMO - PIDDosome ( RIPK1 KO ) or Caspase - 2 - PIDDosome formation ( RAIDD KO ) . Middle , NK cell - mediated killing interpolated from cell confluence graphs at 50 h . n ≥ 3 independent experiments ; mean ± SEM ; unpaired t - test . ( F ) Confluence analysis of Dox pre - treated A549EGFP cells overexpressing PLK4 was performed over time in the presence of NK cells and normalized with respect to their DMSO treated controls . n = 5 independent experiments ; mean ± SEM . ( G ) Confluence analysis of centriole - depleted and ZM pre - treated A549EGFP cells cultured in presence of NK cells . Values are normalized with respect to their DMSO ± Centrinone ( 125nM ) treated controls . n = 4 independent experiments ; mean ± SEM . Data information : The statistical significance was determined using two - tailed t - test ; P < 0 . 001 ( * * ) ; P < 0 . 001 ( * * * ) .

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

Figure 6A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (A) Representative images of target A549EGFP cells pre-treated for 72 h with ZM (2µM) and co-cultured subsequently with NK-92 cells in a 1:10 target to effector ratio. Interactions were recorded by live-cell imaging for 18 h at a 1 min interval. White arrow indicate polyploid cell undergoing cell death. EGFP release coincides with loss of cell membrane integrity. All images were acquired at the same exposure time and light intensity. Scale bar: 10 µm. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (D) Confluence of ZM pre-treated A549EGFP cells expressing or lacking PIDD1 was measured over time in presence of NK cells and normalized with respect to their DMSO treated counterparts. Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥4 independent experiments performed in duplicates, mean ± SEM. (E) Confluence measurement over time of A549EGFP derivatives impaired in either NEMO-PIDDosome (RIPK1 KO) or Caspase-2-PIDDosome formation (RAIDD KO). Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥3 independent experiments; mean ± SEM; unpaired t-test. (F) Confluence analysis of Dox pre-treated A549EGFP cells overexpressing PLK4 was performed over time in the presence of NK cells and normalized with respect to their DMSO treated controls. n=5 independent experiments; mean ± SEM. (G) Confluence analysis of centriole-depleted and ZM pre-treated A549EGFP cells cultured in presence of NK cells. Values are normalized with respect to their DMSO ± Centrinone (125nM) treated controls. n=4 independent experiments; mean ± SEM. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).


Figure 1Phenotypes of wild type and nine microtubule - associated gene mutants . Comparison of the filament and stigma phenotypes of wild type and nine microtubule - associated gene mutants .

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Figure 1Phenotypes of wild type and nine microtubule-associated gene mutants. Comparison of the filament and stigma phenotypes of wild type and nine microtubule-associated gene mutants.


Figure 2Microtubule organization in wild type stamen filament cells . ( D - F ) Microtubule organization in the stamen filament cells of lue1 and ktn80 . 1234 . Arrows indicate microtubule crossovers . Dotted lines indicate abnormal microtubules .

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Figure 2Microtubule organization in wild type stamen filament cells. (D-F) Microtubule organization in the stamen filament cells of lue1 and ktn80.1234. Arrows indicate microtubule crossovers. Dotted lines indicate abnormal microtubules.