( C ) Gsp2 is not involved in H2Bub1 regulation . The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type ( WT ) and gsp2Δ strains by western blotting using anti - total H2B and anti - H2Bub1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Gsp2 is not involved in H2Bub1 regulation. The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type (WT) and gsp2Δ strains by western blotting using anti-total H2B and anti-H2Bub1 antibodies.


B . and C . Molecular models based on the crystal structures previously published [ 44 ] represent configurations of two dimers on DNA : Sox2 - Oct4 heterodimer ( B ) and Oct6 - Oct6 homodimer ( C ) . Oct4 POU domain , green ; Oct4 / Oct6 linker region , magenta ; Sox2 HMG , cyan ; Oct6 POU domain , orange . Individual helixes of POU domains are numbered . In both models , the DNA is represented as a light grey surface . Unless indicated otherwise , the representation is kept throughout the manuscript .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. and C. Molecular models based on the crystal structures previously published [44] represent configurations of two dimers on DNA: Sox2-Oct4 heterodimer (B) and Oct6-Oct6 homodimer (C). Oct4 POU domain, green; Oct4/Oct6 linker region, magenta; Sox2 HMG, cyan; Oct6 POU domain, orange. Individual helixes of POU domains are numbered. In both models, the DNA is represented as a light grey surface. Unless indicated otherwise, the representation is kept throughout the manuscript.


D . EMSAs with a 2 - fold dilutions series starting at 2 μM of the Oct4 POU ( left ) or the Oct6 POU ( right ) in the presence of cy5 - labeled MORE DNA . The free DNA and DNA bound as monomer or dimer are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. EMSAs with a 2-fold dilutions series starting at 2 μM of the Oct4 POU (left) or the Oct6 POU (right) in the presence of cy5-labeled MORE DNA. The free DNA and DNA bound as monomer or dimer are indicated.


E . and F . Bar plots of EMSA - derived cooperativity factors for Oct - Oct homodimers on the MORE ( E ) or Sox2 - Oct heterodimers on the SoxOct element ( F ) are shown for a panel of six POU proteins . The mean is shown with standard deviation as error bars ( n ≥ 3 ) and Tukey multiple comparison of means was used for assessment of statistical significances ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) . N . A . : not assessed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. and F. Bar plots of EMSA-derived cooperativity factors for Oct-Oct homodimers on the MORE (E) or Sox2-Oct heterodimers on the SoxOct element (F) are shown for a panel of six POU proteins. The mean is shown with standard deviation as error bars (n≥3) and Tukey multiple comparison of means was used for assessment of statistical significances (*** p < 0.001, ** p < 0.01, * p < 0.05). N.A.: not assessed.


G . Properties of Oct4 and Oct6 homodimers from molecular dynamics ( MD ) simulations . The structural snapshots illustrate Oct6 ( first monomer in orange , second in dark blue ) and the 3 parameters used : drmsd = the root mean square deviation of interatomic distances measured for the backbone of the structured domains ( excluding the linker ) ; θ = the angle describing the kink in helix α4 ( data for both monomers 1 and 2 are shown ) ; d = the distance between the centers of mass of the 2 helices α5 ; w = the DNA minor groove width in the region between the 2 monomers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Properties of Oct4 and Oct6 homodimers from molecular dynamics (MD) simulations. The structural snapshots illustrate Oct6 (first monomer in orange, second in dark blue) and the 3 parameters used: drmsd = the root mean square deviation of interatomic distances measured for the backbone of the structured domains (excluding the linker); θ = the angle describing the kink in helix α4 (data for both monomers 1 and 2 are shown); d = the distance between the centers of mass of the 2 helices α5; w = the DNA minor groove width in the region between the 2 monomers.


A . Zoom - in view of the Oct6 - Oct6 homodimer interface in the molecular models based on crystal structures of two POU homodimers on MORE DNA . Oct6 POUS , orange ; POUHD of the other monomer , blue . Important residues are labelled and Met ( Oct6 ) in position 151 which was identified to guide the DNA - binding preference is marked with a star . Individual helixes of POU domains are numbered . Colour representation as in Fig . 1B , 1C . For clarity , the second monomer of Oct4 / Oct6 is shown in dark blue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Zoom-in view of the Oct6-Oct6 homodimer interface in the molecular models based on crystal structures of two POU homodimers on MORE DNA. Oct6 POUS, orange; POUHD of the other monomer, blue. Important residues are labelled and Met (Oct6) in position 151 which was identified to guide the DNA-binding preference is marked with a star. Individual helixes of POU domains are numbered. Colour representation as in Fig. 1B, 1C. For clarity, the second monomer of Oct4/Oct6 is shown in dark blue.


B . and C . Network of hydrophobic interactions in Oct6 ( B ) and Oct4 ( C ) . The numbers show the minimal interatomic distances ( excluding hydrogens ) between the residues involved calculated from the simulations . In blue are those distances that change significantly between Oct4 and Oct6 and in red those unchanged . The shown standard deviations represent a measure for the variability during the simulations and not for the uncertainty in the calculations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. and C. Network of hydrophobic interactions in Oct6 (B) and Oct4 (C). The numbers show the minimal interatomic distances (excluding hydrogens) between the residues involved calculated from the simulations. In blue are those distances that change significantly between Oct4 and Oct6 and in red those unchanged. The shown standard deviations represent a measure for the variability during the simulations and not for the uncertainty in the calculations.


D . EMSA showing a differential binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains on MORE ( OctOct ) element . The free DNA and DNA bound by monomer or dimer are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. EMSA showing a differential binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains on MORE (OctOct) element. The free DNA and DNA bound by monomer or dimer are indicated.


E . EMSA showing binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains to SoxOct element in the absence or presence of the Sox2 HMG . The free DNA and DNA bound by monomers or dimer are indicated . Quantifications of cooperativity measurements are shown in Fig . EV3C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. EMSA showing binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains to SoxOct element in the absence or presence of the Sox2 HMG. The free DNA and DNA bound by monomers or dimer are indicated. Quantifications of cooperativity measurements are shown in Fig. EV3C.


F . Bar plot based on single tube EMSAs using cy5 - SoxOct and FAM - MORE DNA elements showing the difference in DNA - binding of WT Oct4 , WT Oct6 , Oct4151M and Oct6151S POU domains . The mean log2 ratio of cy5 ( heterodimer ) and FAM ( homodimer ) band intensities are depicted with standard deviation ( n = 9 ) . Individual data points are shown as gray jitter plots . Tukey multiple comparisons of means was performed to assess significance ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , p > 0 . 05 n . s . ) . Compare to Fig . EV1D and EV3D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Bar plot based on single tube EMSAs using cy5-SoxOct and FAM-MORE DNA elements showing the difference in DNA-binding of WT Oct4, WT Oct6, Oct4151M and Oct6151S POU domains. The mean log2 ratio of cy5 (heterodimer) and FAM (homodimer) band intensities are depicted with standard deviation (n=9). Individual data points are shown as gray jitter plots. Tukey multiple comparisons of means was performed to assess significance (*** p < 0.001, ** p < 0.01, * p < 0.05, p > 0.05 n.s.). Compare to Fig. EV1D and EV3D.


A and B . Molecular models show the position of mutated residues in the structures of two dimers on DNA : Sox2 - Oct4 heterodimer ( A ) and Oct4 - Oct4 homodimer ( B ) . Ser in position 151 ( asterisk ) was mutated to Met in order to shift the preference of Oct4 for homodimerization . The individual helixes of the POU domains are numbered .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A and B. Molecular models show the position of mutated residues in the structures of two dimers on DNA: Sox2-Oct4 heterodimer (A) and Oct4-Oct4 homodimer (B). Ser in position 151 (asterisk) was mutated to Met in order to shift the preference of Oct4 for homodimerization. The individual helixes of the POU domains are numbered.


D . GFP - positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2 , Klf4 , and c - Myc . Colonies were imaged 16 days after second viral infection , using a fluorescence microscope . Scale bars : 250 μm ; objective 10x .

D. GFP-positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2, Klf4, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.


B . Oct4 - GFP - positive colonies of two O6SKM iPSC lines , expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule ( Oct67K , 22T , LinkO4 , 151S ) . Scale bars : 250 μm .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Oct4-GFP-positive colonies of two O6SKM iPSC lines, expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule (Oct67K,22T,LinkO4,151S). Scale bars: 250 μm.


C . Genotyping of two O6SKM iPSC lines . Two stable lines of iPSCs generated with the Oct6 , Sox2 , Klf4 , and c - Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene . OSKM iPSCs and OG2 - MEFs were used as PCR controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Genotyping of two O6SKM iPSC lines. Two stable lines of iPSCs generated with the Oct6, Sox2, Klf4, and c-Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene. OSKM iPSCs and OG2-MEFs were used as PCR controls.


D . Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT - PCR using specific primers . OG2 - MEFs were harvested 5 days after infection and used for comparison as a positive control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT-PCR using specific primers. OG2-MEFs were harvested 5 days after infection and used for comparison as a positive control.


E . Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog . Oct4 expression was confirmed by the Oct4 - GFP transgene . DNA stained by DAPI . Scale bars : 100 μm .

O 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-MEASURED_VAR O O O O O O O O O O

E. Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog. Oct4 expression was confirmed by the Oct4-GFP transgene. DNA stained by DAPI. Scale bars: 100 μm.


F . Bisulfite sequencing of genomic Oct4 , Nanog , and Col1a1 promoter regions of O6SKM iPSCs , OSKM iPSCs , and OG2 - MEFs . White and black circles represent unmethylated and methylated CpG sites , respectively .

F. Bisulfite sequencing of genomic Oct4, Nanog, and Col1a1 promoter regions of O6SKM iPSCs, OSKM iPSCs, and OG2-MEFs. White and black circles represent unmethylated and methylated CpG sites, respectively.


G . Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs ( left ) and O4SK iPSCs ( right ) . Black lines represent a two - fold change in gene expression levels between the paired cell lines . On the right side of the plots , the color bar indicates scattering density . Red and green dots represent up - and downregulated genes , respectively . Positions of selected pluripotency - related genes are highlighted as orange points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs (left) and O4SK iPSCs (right). Black lines represent a two-fold change in gene expression levels between the paired cell lines. On the right side of the plots, the color bar indicates scattering density. Red and green dots represent up- and downregulated genes, respectively. Positions of selected pluripotency-related genes are highlighted as orange points.


H . Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs , illustrating the close relationship between their global gene expression profiles .

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H. Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs, illustrating the close relationship between their global gene expression profiles.


A . In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry : endoderm ( α - fetoprotein , AFP ) , mesoderm ( α - smooth muscle actin , SMA ) , and ectoderm ( α - tubulin , TUJ1 ) . Nuclei ( DNA ) were stained by Hoechst ( blue ) . Scale bars : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O

A. In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry: endoderm (α-fetoprotein, AFP), mesoderm (α-smooth muscle actin, SMA), and ectoderm (α-tubulin, TUJ1). Nuclei (DNA) were stained by Hoechst (blue). Scale bars: 200 μm.


B . F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice .

B. F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice.


C . Pictures demonstrating germline transmission ; endogenous Oct4 - GFP signal was detected in the gonads of F1 pups . Scale bars : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Pictures demonstrating germline transmission; endogenous Oct4-GFP signal was detected in the gonads of F1 pups. Scale bars: 200 μm.


( a ) HEK293 cells were starved of glucose ( 4 h ) as indicated , endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed . Proteins were resolved by SDS - PAGE and visualized with autoradiography ( top ) or western blotting ( WB ; bottom ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS-PAGE and visualized with autoradiography (top) or western blotting (WB; bottom).


( b ) Cells were incubated in glucose - free medium ( 4 h ) as indicated and lysed . Lysates were incubated with lambda phosphatase ( λ PPase ) as indicated . Endogenous Ulk1 mobility was examined by western blotting .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting.


( c ) HA - Ulk1 was transfected into HEK293 cells together with wild - type ( WT ) AMPKα1 or a kinase - dead ( DN ) mutant . Cells were starved of glucose ( 4 h ; Glu ) or amino acids ( - A . A ) and treated with compound C ( 20 μM , C . C ) as indicated . Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting .

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

(c) HA-Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (-A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting.


( d ) HA - Ulk1 proteins were immunopurified from transfected HEK293 cells , which had undergone glucose starvation ( 4 h ) as indicated . The HA - Ulk1 proteins were treated with λ PPase , and in vitro kinase assays were performed in the presence of GST - ATG13 . Proteins were resolved by SDS - PAGE ; phosphorylated proteins were visualized with autoradiography , HA - Ulk1 by western blotting and GST - Atg13 by Coomassie staining .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) HA-Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA-Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography , HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.


( e ) HA - Ulk1 was immunopurified from transfected HEK293 cells under glucose - rich media and treated with AMPK in the presence of cold ATP for 15 min , followed by kinase assays as described in d . In vitro kinase assays were performed in the presence of GST - ATG13 . Proteins were resolved by SDS - PAGE ; phosphorylated proteins were visualized with autoradiography , HA - Ulk1 by western blotting and GST - Atg13 by Coomassie staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) HA-Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. In vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography, HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.


( f ) AMPK wild - type ( WT ) and α1 / α2 double knockout ( DKO ) MEFs were incubated with or without glucose ( 4 h ) . Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured ( mean ± s . d . , n = 3 ) . Autophosphorylation activity was normalized to Ulk1 protein level ; relative activity is calculated by normalization to Ulk1 activity from AMPK wild - type MEFs in glucose - rich conditions .

O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O

(f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions.


( g ) HA - Ulk1 was transfected into HEK293 cells together with vector ( Vec ) or an AMPKα1 kinase - dead mutant ( DN ) . The cells were starved of glucose ( - Glu ) or amino acids ( - A . A ) , or treated with 50 nM rapamycin ( Rapa ) for 3 h before lysis . Left : autophosphorylation activity was assessed and normalized as in f ( mean ± s . d . , n = 3 ) . Right : fold induction in Ulk1 autophosphorylation , compared with Ulk1 autophosphorylation from cells under nutrient - rich conditions . Uncropped images of blots are shown in Supplementary Fig . S5 .

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

(g) HA-Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (-Glu) or amino acids (-A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.


( 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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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.


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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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(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.


A 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 .

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A 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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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.


B 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 .

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B 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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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.


A 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 .

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A 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 .

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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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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.


A 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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 .

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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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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.


A 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 ) .

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A 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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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).


B 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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B 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).


( C ) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A ( ant . A ) .

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(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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(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.


( 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 ) .

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

(A) 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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(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.


( 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 .

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(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 .

(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 .

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(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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(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.


( 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 .

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(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 .

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(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.


( 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 .

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(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.


Cells 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 .

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Cells 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 .

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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.


( 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 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 .

O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.


( 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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 O B-CONTROLLED_VAR I-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR O 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) 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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.


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

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 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.


( 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 .

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) 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.


Primary 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 O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Primary 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.


( 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 ( * * * * ) .

O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 (****).