( B ) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues esophagus mucosa from GTEx database . Sample sizes for each genotype are shown in x - axis . y - axis shows normalized expression of SPINK6 ( Norm . expression ) in indicated trusses . Median interquartile range are represented by a box respectively . Data distribution is colored with blue ; a wider section of violin plot indicates data on section have a higher frequency . Linear regression was used for data .

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 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

(B) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database. Sample sizes for each genotype are shown in the x-axis. The y-axis shows the normalized expression of SPINK6 (Norm. expression) in the indicated trusses. Median and interquartile range are represented by a white line and dark box respectively. Data distribution is colored with light blue; a wider section of the violin plot indicates the data on the section have a higher frequency. Linear regression was used for data analysis.


A Electron microscopy analyses to see autophagic bodies in vacuoles under both growth nitrogen starvation conditions . Autophagic bodies typically contain ribosomes . pep4 ∆ prb1 ∆ pep4 ∆ prb1 ∆ atg2 ∆ were grown in SD to mid ‐ log phase ( OD600 = 1 ) transferred to SD ( ‐ N ) for 5 h . were examined by transmission electron microscopy as described in . Scale bar , 500 nm .

O O O O O O O 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

A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm.


B - D Time ‐ dependent changes in nucleoside contents under nitrogen starvation ( B ) , rapamycin treatment ( C ) , carbon starvation ( D ) conditions . Wild ‐ type atg2Δ were grown in SD transferred to SD ( ‐ N ) , SD medium with 0 . 2 μM rapamycin , or SD ( ‐ C ) at time 0 . At indicated time points , nucleosides were analyzed by LC / MS as described in . Results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . data are means of triplicate samples . error bars represent standard deviation .

O O O O O O O O O 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 O O O O 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-D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.


A Time ‐ dependent changes in nucleoside contents under nitrogen starvation . Nucleosides in wild ‐ type , atg2Δ , atg7Δ , atg11Δ , atg17Δ , atg19Δ , atg32Δ , nvj1Δ , pep4prb1Δ under nitrogen starvation . data are means of triplicates . error bars represent standard deviation .

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

A Time‐dependent changes in nucleoside contents under nitrogen starvation. Nucleosides in wild‐type, atg2Δ, atg7Δ, atg11Δ, atg17Δ, atg19Δ, atg32Δ, nvj1Δ, and pep4prb1Δ cells under nitrogen starvation. All data are means of triplicates. The error bars represent the standard deviation.


B Alkaline phosphatase ( ALP ) ( Pho8 ∆ 60 ) . Wild ‐ type , atg2 ∆ , atg11Δ , atg17Δ , atg19Δ expressing Pho8 ∆ 60 were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , lysates were prepared subjected to ALP . bars represent standard deviation of three independent experiments . * P 0 . 05 ; * * P 0 . 005 ; * * * P 0 . 0005 ( paired t ‐ , two ‐ )

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

B Alkaline phosphatase (ALP) assay (Pho8∆60). Wild‐type, atg2∆, atg11Δ, atg17Δ, and atg19Δ cells expressing Pho8∆60 were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, lysates were prepared and subjected to the ALP assay. The bars represent the standard deviation of three independent experiments. *P 0.05; **P 0.005; ***P 0.0005 (paired t‐test, two‐tailed)


A - C Wild ‐ type , atg2Δ , pho8Δ , rny1Δ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , nucleosides ( A ) were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . data are means of triplicates . error bars represent standard deviation .

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

A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, nucleosides (A) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


A - C Wild ‐ type , atg2Δ , pho8Δ , rny1Δ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , 5 ′ ‐ NMPs ( B ) were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . data are means of triplicates . error bars represent standard deviation .

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

A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 5′‐NMPs (B) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


A - C Wild ‐ type , atg2Δ , pho8Δ , rny1Δ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , 3 ′ ‐ NMPs ( C ) were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . Note that 3 ′ ‐ NMPs in pho8Δ are presented in a different scale . data are means of triplicates . error bars represent standard deviation .

A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 3′‐NMPs (C) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.


A , B Expression localization of Rny1 Pho8 . Wild ‐ type atg2Δ expressing Rny1 ‐ GFP or Pho8 ‐ GFP were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) . After 2 h of starvation , GFP ‐ tagged were observed by microscopy ( A ) . Scale bar , 5 μm .

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

A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2Δ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were observed by fluorescence microscopy (A). Scale bar, 5 μm.


A , B Expression localization of Rny1 Pho8 . Wild ‐ type atg2 ∆ expressing Rny1 ‐ GFP or Pho8 ‐ GFP were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) . After 2 h of starvation , GFP ‐ tagged were analyzed by Western blot with anti ‐ GFP antibody ( B ) .

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

A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2∆ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were analyzed by Western blot with anti‐GFP antibody (B).


C Detection of free . Wild ‐ type , atg2 ∆ , rny1 ∆ , rny1 ∆ atg2 ∆ grown in SD ( left ) or SD ( ‐ N ) for 2 h ( right ) were stained with FM4 ‐ 64 GR Green observed by microscopy . Scale bar , 5 μm .

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

C Detection of free RNA. Wild‐type, atg2∆, rny1∆, and rny1∆atg2∆ cells grown in SD (left) or SD(‐N) for 2 h (right) were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.


A Time ‐ dependent change in nucleoside after nitrogen starvation . Wild ‐ type , atg2 ∆ , ubp3 ∆ , bre5 ∆ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . Metabolites were analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . data are means of triplicates . error bars represent standard deviation .

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

A Time‐dependent change in nucleoside levels after nitrogen starvation. Wild‐type, atg2∆, ubp3∆, and bre5∆ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. Metabolites were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.


B Detection of free within . rny1 ∆ , rny1 ∆ atg2 ∆ , rny1 ∆ ubp3 ∆ , rny1 ∆ bre5 ∆ grown in SD ( ‐ N ) for 2 h were stained with FM4 ‐ 64 GR Green observed by microscopy . Scale bar , 5 μm .

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 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 Detection of free RNA within cells. rny1∆, rny1∆atg2∆, rny1∆ubp3∆, and rny1∆bre5∆ cells grown in SD(‐N) for 2 h were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.


B , C Nucleosides ( B ) nucleobases ( C ) within . Wild ‐ type atg2 ∆ were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , were collected ; their metabolites were extracted , nucleosides nucleobases were analyzed by LC / MS . level of each nucleoside base is presented as its absolute , as described in Supplementary Methods . data are means of duplicates except wild ‐ type at 1 h .

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 O O O O O O O O O O

B, C Nucleosides (B) and nucleobases (C) within the cells. Wild‐type and atg2∆ were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; their metabolites were extracted, and the nucleosides and nucleobases were analyzed by LC/MS. The level of each nucleoside and base is presented as its absolute concentration, as described in and Supplementary Methods. All data are means of duplicates except the wild‐type strain at 1 h.


D , E Nucleosides ( D ) nucleobases ( E ) in medium . culture medium of above cultures was filtered , nucleosides nucleobases were directly analyzed by LC / MS as described in Supplementary Methods . data are means of duplicates .

O O O O O O O O O O O O O O O O O O O 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, E Nucleosides (D) and nucleobases (E) in the medium. The culture medium of the above cultures was filtered, and nucleosides and nucleobases were directly analyzed by LC/MS as described in and Supplementary Methods. All data are means of duplicates.


F , G Destinations of nucleosides bases . on data from ( B - E ) , level of each nucleoside base in ( open circle ) in medium ( closed circle ) of wild ‐ type were estimated . See Supplementary Methods in detail ( flow chart of calculation procedure ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G Destinations of nucleosides and bases. Based on the data from (B-E), the level of each nucleoside and base in cells (open circle) and in cells and medium (closed circle) of wild‐type strain were estimated. See Supplementary Methods in detail (flow chart of the calculation procedure).


Wild ‐ type atg1Δ of S . pombe were grown in SD to mid ‐ log phase transferred to SD ( ‐ N ) at time 0 . At indicated time points , were collected ; metabolites in these were extracted analyzed by LC / MS as described in . results are presented as normalized intensities on basis of peak height of each metabolite in wild ‐ type . data are means of triplicates . error bars represent standard deviation .

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

Wild‐type and atg1Δ of S. pombe were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; the metabolites in these cells were extracted and analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.


B , C GOT2 can be acetylated . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T treated with NAM ( 5 mM ) / or TSA ( 0 . 5 mM ) for indicated time period . Acetylation of Flag ‐ bead ‐ purified GOT2 were determined by Western blot using a pan ‐ anti ‐ acetyllysine antibody ( α ‐ Ac ) . IB IP denote immunoblotting immunoprecipitation , respectively . Relative GOT2 acetylation ratios were calculated after normalizing against Flag .

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

B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.


D GOT2 acetylation enhances interaction between ectopically expressed GOT2 MDH2 . Flag ‐ GOT2 Myc ‐ MDH2 were co ‐ overexpressed in HEK293T treated without or with NAM ( 5 mM ) for indicated time period . acetylation level of Flag ‐ bead ‐ purified GOT2 association between ectopic of GOT2 MDH2 were determined by Western blot .

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

D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.


E Acetylation enhances interaction between ectopically expressed GOT2 endogenous MDH2 . Flag ‐ GOT2 was co ‐ overexpressed in HEK293T treated without or with NAM ( 5 mM ) for indicated time period . acetylation level of Flag ‐ GOT2 its association with endogenous MDH2 were determined by Western blot .

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

E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.


F Acetylation enhances interaction between ectopically expressed MDH2 endogenous GOT2 . Flag ‐ MDH2 was co ‐ overexpressed in HEK293T treated without or with NAM ( 5 mM ) for indicated time period . association between Flag ‐ MDH2 endogenous GOT2 was determined by Western blot .

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

F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis.


H Mapping major lysine residue ( s ) of acetylation in GOT2 whose acetylation can affect interaction between GOT2 MDH2 . Putative acetylated residues were divided into six groups according to their position in structure of GOT2 . Each group of putative acetylated lysine ( K ) sites was mutated to arginine ( R ) , deacetylated mimic K ‐ to ‐ R mutants were examined for their association with ectopically expressed Myc ‐ MDH2 by Western blot .

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

H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis.


I GOT2 coupled with MDH2 shows a high level of K159 acetylation . Flag ‐ GOT2 was overexpressed in HEK293T without or with co ‐ overexpression of Myc ‐ MDH2 . These transfected were treated with NAM ( 5 mM ) for 5 h . Double immunoprecipitation was performed to obtain Flag ‐ GOT2 from Myc ‐ MDH2 immunoprecipitates ( Ppt , supposed to be GOT2 bound with MDH2 ) . remaining supernatants ( Sup , supposed to be GOT2 not bound with MDH2 ) were also harvested precipitated by Flag antibody to K159 acetylation level of Flag ‐ GOT2 by Western blot .

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

I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis.


J 3KQ / R mutations do not change GOT2 enzyme activity . Wild ‐ type 3K mutant GOT2 were overexpressed purified from E . coli , enzyme activity of GOT2 was determined as described in " " . Shown are average values with standard deviation ( SD ) of triplicated experiments . n . s . = not significant for indicated comparison .

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

J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild‐type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in "". Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.


A , B Glucose glutamine increase GOT2 K159 acetylation GOT2 - MDH2 association in HEK293T . Flag ‐ GOT2 Myc ‐ MDH2 were overexpressed in treated with increased concentrations of glucose ( A ) or glutamine ( B ) for 6 h . GOT2 were purified by Flag beads , K159 acetylation level of GOT2 its association with Myc ‐ MDH2 were determined by Western blot .

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

A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.


C , D Glucose glutamine increase GOT2 K159 acetylation in Panc ‐ 1 . Flag ‐ GOT2 was overexpressed in treated with different concentrations of glucose ( C ) glutamine ( D ) for 4 h . GOT2proteins were purified by Flag beads , K159 acetylation level of GOT2 was determined by Western blot . Relative GOT2 K159 acetylation were normalized against Flagprotein .

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

C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels.


E , F of of K159 ‐ acetylated endogenous GOT2 in Panc ‐ 1 . Recombinant fully K159 ‐ acetylated GOT2 was loaded onto same gel , together with endogenous GOT2 from Panc ‐ 1 treated without or with glucose ( 12 mM ) ( E ) or glutamine ( 2 mM ) ( F ) for 4 h . GOT2 K159 acetylation were detected by Western blot . percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 .

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

E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.


G , H 3K mutant GOT2 displays negligible response in changing association with MDH2 after glucose or glutamine treatment . Panc ‐ 1 with GOT2 knockdown re ‐ expression of wild ‐ type or 3K mutant GOT2 were treated with glucose ( G ) or glutamine ( H ) at indicated concentrations for 4 h . association between Flag ‐ tagged wild ‐ type or 3K mutant GOT2 endogenous MDH2 was determined by Western blot .

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G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.


A GOT2 interacts with SIRT3 , but not SIRT4 SIRT5 . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T together with HA ‐ tagged SIRT as indicated . were purified by IP with Flag beads , following Western blot to detect SIRTs with an HA antibody .

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A GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA‐tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.


B Endogenous of GOT2 SIRT3 interact with each other . SIRT3 in HEK293T was purified by IP with an anti ‐ SIRT3 antibody , following Western blot to detect GOT2 with an anti ‐ GOT2 antibody .

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B Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti‐SIRT3 antibody, following Western blot to detect GOT2 with an anti‐GOT2 antibody.


C GOT2 deacetylation is dependent on SIRT3 catalytic activity . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T together with HA ‐ tagged wild ‐ type SIRT3 a catalytically inactive mutant , SIRT3 H248Y . GOT2 were purified by Flag beads , following Western blot to detect GOT2 K159 acetylation .

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C GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with HA‐tagged wild‐type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.


D SIRT3 impairs interaction between GOT2 MDH2 . HEK293T were transfected with plasmids as indicated . GOT2 were purified by Flag beads , their interaction with Myc ‐ MDH2 was determined by Western blot .

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D SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc‐MDH2 was determined by Western blot.


E Knockdown of SIRT3 increases GOT2 K159 acetylation enhances GOT2 - MDH2 association . HEK293T with or without transient SIRT3 knockdown were transfected with plasmids as indicated . GOT2 were purified by Flag beads , K159 acetylation level of GOT2 its association with endogenous MDH2 were determined by Western blot .

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E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2-MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.


F , G Knockdown of SIRT3 diminishes effect of glucose or glutamine on changing GOT2 K159 acetylation . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T without or with transient SIRT3 knockdown . These were treated with different concentrations of glucose ( F ) glutamine ( G ) as indicated . GOT2 were purified by Flag beads , K159 acetylation level of GOT2 was determined by Western blot .

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F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.


H , I Up ‐ regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting . C57BL / 6 mice ( , 20 ; n = 2 per group ) were fed a normal chow diet or fasted overnight . Upon sacrifice , mouse liver adipose tissues were harvested , of indicated were determined by Western blot .

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H, I Up‐regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.


A malate - aspartate shuttle , such as LDH , also plays a key role in regulating NADH in Panc ‐ 1 . were treated with AOA ( 1 . 6 mM , 20 min ) , oxamate ( 5 mM , 20 min ) , EGCG ( 0 . 5 mM , 20 min ) . NADH level was measured as described in .

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A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in .


B , C GOT2 3K acetylation promotes net transfer of NADH into mitochondria . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , NADH level in cytosol ( B ) mitochondrion ( C ) was determined in extracts as described in .

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B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in .


D GOT2 3K acetylation regulates mitochondrial NADH / NAD + redox in . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , of NADH / NAD + in mitochondria of was measured as described in .

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D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in .


E GOT2 3K acetylation promotes ATP production . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , ATP production was determined in extracts as described in .

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E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in .


F GOT2 3K acetylation promotes NADPH production . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , of NADPH / NADP + was determined in extracts as described in .

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F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in .


G GOT2 3K acetylation promotes GSH production . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , GSH / GSSG was determined in extracts as described in .

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G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in .


H GOT2 3K acetylation suppresses ROS . In Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated , ROS was determined as described in in under non ‐ stressed condition or exposed to menadione ( 50 μM for 30 min ) .

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H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min).


I GOT2 3K acetylation protects from oxidative damage . Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated were treated with increasing concentrations of menadione for 3 h as indicated , viability was determined by counting remaining adherent .

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I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.


J , K Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated were seeded in a 6 ‐ well plate , were maintained under condition of low glucose ( 0 . 5 mM , J ) or high glucose ( 12 mM , K ) . Culture medium was refreshed every day , numbers were counted every 1 - 2 days over a period of 7 days .

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J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days.


L , M Panc ‐ 1 stable in ( J , K ) were grown under low glucose ( 0 . 5 mM ) or high glucose ( 12 mM ) for 7 days . level of histone H3 phosphorylation at Ser10 was determined by Western blot ( L ) . percentages of living apoptotic were determined by flow cytometry as described in ( M ) .

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L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).


A - C GOT2 3K acetylation promotes xenograft tumorgrowth . Panc ‐ 1 stable with GOT2 knockdown re ‐ expressing indicated ( 2 × 106cells ) were injected subcutaneously into flanks of nude mice . volume was carefully monitored over indicated time period ( A ) . At 8 after injection , tumors from six mice were extracted , photographed , weighted ( B , C ) .

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A-C GOT2 3K acetylation promotes xenograft tumorgrowth. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins (2 × 106cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C).


D GOT2 3K acetylation promotes cellproliferation . sections from xenografts were prepared for , PCNA ‐ stained sections were analyzed by quantifying PCNA ‐ positive .

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D GOT2 3K acetylation promotes tumor cellproliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA‐stained tumor sections were analyzed by quantifying the PCNA‐positive area.


E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) adjacent normal tissue ( shown as N ) were lysed . acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot . F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) adjacent normal tissue ( shown as N ) were lysed . SIRT3protein were compared against tubulin by Western blot . G , H of GOT2 K159 acetylation SIRT3protein expression in clinical cases as described in ( E ) ( F ) .

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E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot.F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3protein levels were compared against tubulin by Western blot.G, H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in (E) and (F).


( a ) Subcellular compartments from U937 extracts were separated by sedimentation velocity on discontinuous sucrose gradient probed for indicated by immunoblotting . Numbers represent fractions from gradient ( 1 , top ; 5 , bottom ) .

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(a) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom).


( b ) Immunoblot of compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit .

(b) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit.


( c ) Immunoblot of KDEL IRGM distribution in different fractions after purification of mitochondria as in b .

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(c) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b.


d ) Intracellular localization of IRGM analysed by confocal microscopy . were stained with antibodies against indicated or vitally with MitoTracker Red ( MTR ) where indicated . on right are merged from on left . A - L ; endogenous IRGM localization in HeLa relative to calnexin , MTR , cytochrome c COX IV . M - O ; IRGM localization in human macrophages relative to mitochondria visualized with cytochrome c antibody . Scale bars , 5 μm .

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d) Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A-L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M-O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm.


( e ) of IRGM ( green ) COX IV ( red ) as a function of along shown in inset . Image in inset corresponds to panels J - L in d . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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(e) Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J-L in d. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Purified mitochondria were left untreated , or were treated with Proteinase K ( Pr . K ) following osmotic shock ( OS ) , or solubilization of total using Triton X - 100 ( TX - 100 ) , as indicated . Samples were analysed by immunoblotting with antibodies against indicated .

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(a) Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins.


( b ) Mitochondrial preparations were subjected to disruption by freeze - thaw cycles as indicated accessibility of indicated to Proteinase K was examined by immunoblotting .

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(b) Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting.


( c ) Immunoblot of membranous organelles from U937 separated by isopycnic sucrose density - gradient centrifugation . Numbers represent fractions from gradient ( 1 , top ; 12 , bottom ) . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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(c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) were transfected with control siRNA or with siRNA specific to IRGM ( top ) DRP1 ( bottom ) for 48 h samples from these were then analysed by immunoblotting with anti - IRGM anti - DRP1 antibodies . Actin was used as a loading control .

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(a) Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control.


( b ) HeLa were treated with siRNA specific to either IRGM or DRP1 as indicated were then labelled with MTR analysed by live - confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm.


( c ) were treated with either control siRNA or with DRP1 , IRGM , ATG7 or BECN1 ( Beclin 1 ) siRNAs , labelled with MTR of with mitochondrial morphologies ranging from normal , punctiform ( dots ) elongated were quantified . Definition of mitochondrial morphologies criteria are given in Supplementary Information , Figure S2 .

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(c) Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2.


( d ) were transfected with either control siRNA or with siRNA specific to MFN1 / MFN2 , IRGM or IRGM MFN1 / MFN2 , then labelled with MTR analysed for mitochondrial morphologies . Data are means ± s . e . m . ( c , n = 3 ; d , n = 4 ) . Asterisk indicates P 0 . 05 , double asterisks indicate P 0 . 01 dagger indicates P > 0 . 05 ( t - ) .

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(d) Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial morphologies. Data are means ± s.e.m. (c, n = 3; d, n = 4). Asterisk indicates P 0.05, double asterisks indicate P 0.01 and dagger indicates P > 0.05 (t-test).


( a ) U937 were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by treatment with hIFN - γ . GFP - LC3 in were imaged by confocal microscopy . Scale bars , 5 μm . ( b ) LC3 per were quantified from treated as in a .

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(a) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by treatment with hIFN-γ. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. (b) LC3 puncta per cell were quantified from cells treated as in a.


( c ) U937 were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by starvation . GFP - LC3 in were imaged by confocal microscopy . Scale bars , 5 μm .

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(c) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by starvation. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm.


( d ) U937 were transfected with either control siRNA or siRNA specific to MFN1 / MFN2 , endogenous LC3 per were quantified .

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(d) U937 cells were transfected with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified.


( e ) U937 were transfected with indicated siRNA infected with BCG . Autophagy was induced by starvation phagosome maturation was analysed using LysoTracker probe ( LTR ) as a reporter of acidification .

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(e) U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification.


( f ) U937 were treated with indicated siRNA infected with virulent M . tuberculosis H37Rv . Autophagy was induced by starvation , or were kept in full medium , as indicated . Colony forming units were counted , expressed as a of number of plated colonies from untreated , to assess survival . Data are means ± s . e . m . ( n = 6 ; two independent transfections , 6 independent infections ) . Asterisk indicates P 0 . 05 , dagger indicates P > 0 . 05 ( t - ) for indicated data .

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(f) U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data.


( b ) HeLa were transfected with plasmids encoding - tagged IRGMb , IRGMd WT ( wild - type IRGMd ) or IRGMS47N mutant ( control ; vector expressing GFP only ) , after 48 h were labelled with MTR imaged by live - confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm.


( c ) of green red channels along a drawn through two adjacent , one GFP - IRGMd positive one GFP - IRGMd negative , as indicated in inset .

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(c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative, as indicated in the inset.


( d ) of GFP + MTR + from an experiment performed as in b .

(d) Quantification of GFP+ MTR+ cells from an experiment performed as in b.


( e ) Nitrocellulose filters spotted with indicated lipids were incubated with GST or GST - IRGM . Binding was detected by immunoblotting .

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(e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting.


( f ) Agarose beads ( control beads ) cardiolipin - bound agarose beads ( CL beads ) were incubated with GST or GST - IRGMd . were eluted from beads immunoblotted . Two lanes on left indicate immunoblotting of samples used to incubate beads .

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(f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads.


( g ) Nitrocellulose filters spotted with indicated lipids ( defined in e ) were incubated with wild - type IRGMd , IRGMdS47N , IRGM isoform IRGMb at indicated concentrations . GST control is shown in Supplementary Information , Figure S1d . Membranes in e - g were probed with anti - GST antibody .

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(g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.


( a ) HeLa transfected with plasmids encoding GFP or GFP - IRGMd were stained with MTR imaged by live - confocal microscopy . Scale bars , 5 μm . ( b - d ) of mitochondrial morphologies in transfected with IRGMd , IRGMdS47N or IRGMb GFP fusions ( control express GFP only ) .

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(a) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b-d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only).


( e ) HeLa were transfected with indicated siRNAs vector encoding GFP - IRGMd , labelled with MTR . of with mitochondrial morphologies ranging from normal , punctiform ( dots ) elongated were quantified .

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(e)HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified.


( f ) HeLa were transfected with indicated siRNAs vector encoding GFP - IRGMd ( or vector encoding GFP only , as a control ) labelled with MTR . Percentages of GFP + with MTR at 48 h were quantified .

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(f) HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified.


( g ) Confocal microscopy of HeLa transfected with vector encoding GFP - DRP1 ( A ) or GFP - IRGMd ( B - G ) labelled with MTR ( A - D ) or antibodies against COX - IV ( F ) . A , steady - state DRP1 distribution ; B - D , translocation of IRGMd from cytosol to mitochondria at indicated times post - transfection ; E - G , co - localization of IRGMd COX - IV ( G is merged from E F ) . Scale bars , 5 μm .

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(g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP-DRP1 (A) or GFP-IRGMd (B-G) and labelled with MTR (A-D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B-D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E-G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm.


( h ) HeLa , co - transfected with GFP - IRGMd , control , BECN1 or ATG7 siRNA , were labelled with MTR 48 h post - transfection , imaged by live - microscopy , percentages of GFP + that were also MTR + were quantified .

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(h) HeLa cells, co-transfected with GFP-IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.


( i ) from Atg7 wild - type ( WT ) or Atg7 − / − MEFs transfected with vectors encoding GFP - IRGMd for 48 h were stained with MTR , imaged , of GFP + that were also MTR + was quantified .

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(i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP-IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified.


( j ) Wild - type W2 ( Bax / Bak + / + ) or mutant D3 ( Bax / Bak − / − ) BMK were transfected with vectors encoding GFP - IRGMd after 48 h were labelled with MTR . were imaged by live - microscopy , percentages of GFP + that were also MTR + were quantified .

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(j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP-IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.


( k ) HeLa were transfected with vectors encoding GFP - IRGMd , treated with z - VAD as indicated , after 48 h stained with MTR . were imaged by live - microscopy , percentages of GFP + that were also MTR + were quantified . Data are means ± s . e . m . ( n = 3 ) . Dagger indicates P ≥ 0 . 05 , asterisk indicates P 0 . 05 double asterisks indicate P 0 . 01 ( t - ) for indicated data .

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(k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.


( a ) of HeLa transfected with vectors encoding GFP or GFP - IRGMd that were rounded 48 h post - transfection .

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(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP-IRGMd that were rounded 48 h post-transfection.


( b ) HeLa transfected with plasmids encoding GFP or GFP - IRGMd were stained with 7 - AAD after 48 h percentages of GFP + that were also 7 - AAD + were quantified .

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(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.


( c ) HeLa transfected with vectors encoding GFP - IRGMd versus GFP - IRGMdS47N YFP - IRGMb ( vector encoding GFP only was transfected as a control ) were stained with 7 - AAD after 48 h , percentages of GFP + ( for IRGMd IRGMS47N ) or YFP + ( for IRGMb ) that were also 7 - AAD + were quantified .

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(c) HeLa cells transfected with vectors encoding GFP-IRGMd versus GFP-IRGMdS47N and YFP-IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified.


( d ) HeLa were transfected with plamsids encoding GFP - IRGMd , GFP - IRGMDS47N or YFP - IRGMb ( vector encoding GFP only was transfected as a control ) , after 48 h were stained with propidium iodide ( PI ) . Percentages of GFP + or YFP + that were also PI + were quantified .

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(d) HeLa cells were transfected with plamsids encoding GFP-IRGMd, GFP-IRGMDS47N or YFP-IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified.


( e ) of HeLa transfected with vectors encoding GFP - IRGMd ( bottom ) were stained for active caspases 3 7 using FLICA dye . Staurosporine ( STS ; middle ) was used as a positive control . were transfected with vectors encoding GFP only as a control ( top ) .

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(e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top).


( f ) HeLa were transfected with plasmids encoding GFP - IRGMd were stained with antibodies against HMGB1 ( absence of HMGB1 stain is a marker of HMGB1 release ) or DAPI . Top : indicate GFP - IRGMd ( left ) , HMGB1 ( middle ) DAPI ( right ) localization . Bottom : phase - contrast microscopy image of ( left ) image merged from other panels ( right ) . Scale bars , 5 μm .

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(f) HeLa cells were transfected with plasmids encoding GFP-IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP-IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm.


( g - i ) were transfected with plasmids encoding GFP or GFP - IRGMd , control or DRP1 siRNA , as indicated . After 48 h , ( g ) were stained with 7 - AAD percentages of GFP + that were also 7 - AAD + were quantified , ( h ) rounded were quantified , or ( i ) were stained with HMGB1 HMGB1 + nuclei were quantified . Data are means ± s . e . m . ( n = 3 ) . Asterisk indicates P 0 . 05 double asterisk indicate P 0 . 01 ( t - ) .

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(g-i) Cells were transfected with plasmids encoding GFP or GFP-IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).


( a , b ) HeLa were transfected with plasmids encoding GFP , CFP - IRGMa , GFP - IRGMc or GFP - IRGMd , after 24 h ( a ) or 48 h ( b ) , stained with MTR . Percentages of positively transfected that were also MTR + were then quantified . ( c , d ) were transfected as in a , b , after 24 h ( c ) or 48 h ( d ) percentages of positively transfected that were also rounded by were quantified .

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(a, b) HeLa cells were transfected with plasmids encoding GFP, CFP-IRGMa, GFP-IRGMc or GFP-IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified.


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Number of EMPs per equivalent ( e . e . ) of wild type Sl / Sl E9 . 5 E11 . 5 YS , determined by flow cytometry ( panels in B , D ) . E9 . 5 Kit + CD41 + CD16 / 32 + EMP numbers are mean ± SD from 4 wild type 3 Sl / Sl biological replicates , with each replicate consisting of single or 2 pooled YS of same genotype . Total number of analyzed : 7 + / + ( 14 - 23sp ) , 5 Sl / Sl ( 17 - 25sp ) . E11 . 5 Kit + CD41 + EMP numbers are mean ± SD of 5 + / + 6 Sl / Sl YS analyzed individually over 2 independent experiments . Total live per YS : 1 . 7 ± 0 . 3x105 ( wt ) , 1 . 7 ± 0 . 2x105 ( Sl / Sl ) at E9 . 5 ; 4 . 2 ± 1 . 4x105 ( wt ) , 4 . 0 ± 2 . 2x105 ( Sl / Sl ) at E11 . 5 .

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Number of EMPs per embryo equivalent (e.e.) of wild type and Sl/Sl E9.5 and E11.5 YS, determined by flow cytometry (panels in B,D). E9.5 Kit+ CD41+ CD16/32+ EMP numbers are the mean±SD from 4 wild type and 3 Sl/Sl biological replicates, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of embryos analyzed: 7 +/+ (14-23sp), 5 Sl/Sl (17-25sp). E11.5 Kit+ CD41+ EMP numbers are the mean±SD of 5 +/+ and 6 Sl/Sl YS analyzed individually over 2 independent experiments. Total live cells per YS: 1.7±0.3x105 (wt), 1.7±0.2x105 (Sl/Sl) at E9.5; 4.2±1.4x105 (wt), 4.0±2.2x105 (Sl/Sl) at E11.5.


Example of flow cytometry of E9 . 5 YS EMPs quantified in panel ( A ) . Analyses were performed on Ter119 - live .

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Example of flow cytometry analysis of E9.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.


Number of CFU - C in wild type Sl / Sl E9 . 5 E11 . 5 YS . E9 . 5 data ( mean ± SD ) are from 3 wild type 2 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of single or 2 pooled YS of same genotype . Total number of analyzed : 5 + / + ( 17 - 23sp ) , 3 Sl / Sl ( 17 - 25sp ) over 2 independent experiments . For E11 . 5 , data are from 4 wild type 6 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of single or 2 pooled YSs of same genotype . Total number of analyzed : 6 + / + , 7 Sl / Sl over 4 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid .

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Number of CFU-C in wild type and Sl/Sl E9.5 and E11.5 YS. E9.5 data (mean±SD) are from 3 wild type and 2 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of analyzed embryos: 5 +/+ (17-23sp), 3 Sl/Sl (17-25sp) over 2 independent experiments. For E11.5, data are from 4 wild type and 6 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YSs of the same genotype. Total number of embryos analyzed: 6 +/+, 7 Sl/Sl over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.


Example of flow cytometry of E11 . 5 YS EMPs quantified in panel ( A ) . Analyses were performed on Ter119 - live .

Example of flow cytometry analysis of E11.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.


Confocal WM - IF of CD31 + pHH3 + Kit + proliferating EMPs ( arrowheads ) in E9 . 5 wild type Sl / Sl YS . A single 2 . 5μm - thick Z section is shown . For each YS , CD31 + Kit + pHH3 + were counted in 3 to 5 fields using a 25x objective of pHH3 + proliferating EMPs was calculated . Bar graphs represent mean ± SD from 3 wild type ( 21 - 24sp ) 3 Sl / Sl ( 22 - 23sp ) YS . Scale bars : 50μm .

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Confocal WM-IF analysis of CD31+ pHH3+ Kit+ proliferating EMPs (arrowheads) in E9.5 wild type and Sl/Sl YS. A single 2.5μm-thick Z section is shown. For each YS, CD31+ Kit+ pHH3+ cells were counted in 3 to 5 fields using a 25x objective and the percentage of pHH3+ proliferating EMPs was calculated. Bar graphs represent the mean±SD from 3 wild type (21-24sp) and 3 Sl/Sl (22-23sp) YS. Scale bars: 50μm.


cycle of wild type Sl / Sl E11 . 5 YS EMPs . of EMPs ( Ter119 - Kit + CD41 + ) in G0 / G1 or S / G2 / M was determined by flow cytometry on basis of BrdU 7 - AAD incorporation . a : apoptotic ( not shown ) . were analyzed individually . N = 6 ( + / + ) , N = 5 ( Sl / Sl ) over 3 independent experiments . E11 . 5 were staged according to tail somite as described in [ 76 ] were : 12 - 17 ( + / + ) ; 12 - 17 ( Sl / Sl ) . Bar graphs show mean ± SD , FACS plots representative results .

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Cell cycle analysis of wild type and Sl/Sl E11.5 YS EMPs. The percentage of EMPs (Ter119- Kit+ CD41+) in G0/G1 or S/G2/M was determined by flow cytometry on the basis of BrdU and 7-AAD incorporation. a: apoptotic (percentage not shown). Embryos were analyzed individually. N=6 (+/+), N=5 (Sl/Sl) over 3 independent experiments. E11.5 embryos were staged according to tail somite counts as described in [76] and were: 12-17 (+/+); 12-17 (Sl/Sl). Bar graphs show mean±SD, and FACS plots representative results.


Flow cytometry of EMPs ( Ter119 - Kit + CD41 + CD16 / 32 + ) early erythroid progenitors ( Early E : Ter119 - Kit + CD41 - CD71 + CD44 + ) in wild type Sl / Sl E11 . 5 FL . EMP early E data are mean ( ± SD ) of 6 wild type 4 Sl / Sl biological replicates , with a total of 10 wild type 6 Sl / Sl analyzed in pools of 1 - 3 FL per genotype . Bar graphs on right show corresponding absolute numbers ( nr ) per FL . Total FL are mean ( ± SD ) of 12 wild type 13 Sl / Sl . Tail somite range : 10 - 16 ( wild type ) , 12 - 15 ( Sl / Sl ) . Note that although EMP frequency in Sl / Sl FL is increased , their absolute number is severely decreased .

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Flow cytometry analysis of EMPs (Ter119- Kit+ CD41+ CD16/32+) and early erythroid progenitors (Early E: Ter119- Kit+ CD41- CD71+ CD44+) in wild type and Sl/Sl E11.5 FL. EMP and early E data are the mean (±SD) of 6 wild type and 4 Sl/Sl biological replicates, with a total of 10 wild type and 6 Sl/Sl embryos analyzed in pools of 1-3 FL per genotype. Bar graphs on the right show corresponding absolute cell numbers (nr) per FL. Total FL cell counts are the mean (±SD) of 12 wild type and 13 Sl/Sl embryos. Tail somite range: 10-16 (wild type), 12-15 (Sl/Sl). Note that although EMP frequency in Sl/Sl FL is increased, their absolute number is severely decreased.


CFU - C numbers in wild type Sl / Sl E11 . 5 FL . Data are mean ( ± SD ) of 10 wild type 10 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of from 1 - 2 FLs . A total of 12 wild type 11 Sl / Sl FL were plated over 6 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid .

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CFU-C numbers in wild type and Sl/Sl E11.5 FL. Data are the mean (±SD) of 10 wild type and 10 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of cell from 1-2 FLs. A total of 12 wild type and 11 Sl/Sl FL were plated over 6 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.


Total numbers ( nr ) of E12 . 5 wild type ( n = 7 ) Sl / Sl ( n = 8 ) FL . Right : CFU - E numbers per wild type Sl / Sl E12 . 5 FL . Data are mean ( ± SD ) of 3 biological replicates analyzed over 2 independent experiments . Each biological replicate consisted of 1 - 2 FL samples pooled according to genotype was plated in duplicate into a methylcellulose medium containing EPO ; colonies were scored after 2 days . Total number of FLs analyzed : 4 wild type 6 Sl / Sl .

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Total cell numbers (nr) of E12.5 wild type (n=7) and Sl/Sl (n=8) FL. Right: CFU-E numbers per wild type and Sl/Sl E12.5 FL. Data are the mean (±SD) of 3 biological replicates analyzed over 2 independent experiments. Each biological replicate consisted of 1-2 FL samples pooled according to genotype and was plated in duplicate into a methylcellulose based medium containing EPO; colonies were scored after 2 days. Total number of FLs analyzed: 4 wild type and 6 Sl/Sl.


of erythroid lineage in E12 . 5 wild type Sl / Sl FL . Absolute ( top ) percentages ( bottom ) of proerythroblasts ( ProE ) , basophilic erythroblasts ( BasoE ) , polychromatic erythroblasts ( PolyE ) orthochromatic erythroblasts ( OrthoE ) were determined by imaging flow cytometry . number ( nr ) of per FL was on total cellularity their relative proportions . Data are mean ( ± SD ) of 3 biological replicates for each genotype , with replicates consisting of single or 2 pooled FL samples . A total of 4 wild type , 5 Sl / + 6 Sl / Sl FLs were analyzed .

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Quantification of erythroid lineage cells in E12.5 wild type and Sl/Sl FL. Absolute cell counts (top) and percentages (bottom) of proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE) and orthochromatic erythroblasts (OrthoE) were determined by imaging flow cytometry analysis. The number (nr) of cells per FL was based on total cellularity and their relative proportions. Data are the mean (±SD) of 3 biological replicates for each genotype, with replicates consisting of single or 2 pooled FL samples. A total of 4 wild type, 5 Sl/+ and 6 Sl/Sl FLs were analyzed.


Representative examples of maturing erythroblast series in wild type Sl / Sl E12 . 5 FL , as identified by imaging flow cytometry . Scale bars : 10μm .

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Representative examples of maturing erythroblast series in wild type and Sl/Sl E12.5 FL, as identified by imaging flow cytometry. Scale bars: 10μm.


Number of EMPs in peripheral blood ( PB ) of wild type Sl / Sl midgestation , calculated from of PB EMPs ( Ter119 - Kit + CD41 + CD16 / 32 + ) determined by flow cytometry ( Figure EV1E , F ) total ( 4 . 2 ± 1 . 6x105 ( wt ) , 3 . 1 ± 1 . 1x105 ( Sl / Sl ) at E9 . 75 ; 2 . 6 ± 0 . 8x106 ( wt ) , 2 . 3 ± 1 . 4x106 ( Sl / Sl ) at E11 . 5 ) . For E9 . 75 - E10 , PB from 2 wild type ( 28 - 30sp ) 5 Sl / Sl ( 27 - 31 ) was analyzed . For E11 . 5 , PB from 1 - 3 was pooled 6 wild type 4 Sl / Sl biological replicates were analyzed , with 10 wild type 6 Sl / Sl analyzed in total . Graphs show mean ( ± SD ) of PB EMPs per equivalent ( e . e . ) .

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

Number of EMPs in peripheral blood (PB) of wild type and Sl/Sl midgestation embryos, calculated from the percentage of PB EMPs (Ter119- Kit+ CD41+ CD16/32+) determined by flow cytometry (Figure EV1E,F) and the total cell count (4.2±1.6x105 (wt), 3.1±1.1x105 (Sl/Sl) at E9.75; 2.6±0.8x106 (wt), 2.3±1.4x106 (Sl/Sl) at E11.5). For E9.75-E10, PB from 2 wild type (28-30sp) and 5 Sl/Sl (27-31) individual embryos was analyzed. For E11.5, PB from 1-3 embryos was pooled and 6 wild type and 4 Sl/Sl biological replicates were analyzed, with 10 wild type and 6 Sl/Sl embryos analyzed in total. Graphs show the mean (±SD) of PB EMPs per embryo equivalent (e.e.).


Representative confocal whole - mount immunofluorescence of wild type Sl / Sl FL at onset of hematopoietic colonization . Kit + EMPs are shown in , CD31 + vasculature in green . Maximum 3D projections from 350μm - thick Z stacks are shown . Scale bars : 100μm . Number of analyzed : 3 wild type 3 Sl / Sl for E9 . 75 ( 27 - 29sp ) , 7 wild type 8 ( Sl / Sl ) for E10 . 5 ( 34 - 36sp ) .

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Representative confocal whole-mount immunofluorescence images of wild type and Sl/Sl FL at the onset of hematopoietic colonization. Kit+ EMPs are shown in white, CD31+ vasculature in green. Maximum intensity 3D projections from 350μm-thick Z stacks are shown. Scale bars: 100μm. Number of embryos analyzed: 3 wild type and 3 Sl/Sl for E9.75 (27-29sp), 7 wild type and 8 (Sl/Sl) for E10.5 (34-36sp).


Confocal whole - mount immunofluorescence of proliferating hematopoietic progenitors in wild type Sl / Sl E10 . 5 ( 34 - 36sp ) FL . Very few CD31 + pHH3 + Kit + proliferating hematopoietic ( arrowheads ) were detected in Sl / Sl FL . A single 2 . 5μm - thick slice is shown . Scale bars : 20μm . A total of 7 wild type 8 Sl / Sl were analyzed .

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Confocal whole-mount immunofluorescence analysis of proliferating hematopoietic progenitors in wild type and Sl/Sl E10.5 (34-36sp) FL. Very few CD31+ pHH3+ Kit+ proliferating hematopoietic cells (arrowheads) were detected in the Sl/Sl FL. A single 2.5μm-thick slice is shown. Scale bars: 20μm. A total of 7 wild type and 8 Sl/Sl embryos were analyzed.


of cycling Ter119 - Kit + CD41 + CD16 / 32 + EMPs Ter119 - Kit + CD41 - CD71 + CD44 + early erythroid ( Early E ) in E11 . 5 FL , determined by flow cytometric of BrdU 7 - AAD incorporation . Data are mean ( ± SD ) of 6 wild type 5 Sl / Sl FLs analyzed individually over 3 independent experiments . Tail somite range : 12 - 17 ( + / + ) ; 12 - 17 ( Sl / Sl ) .

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

Percentage of cycling Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid (Early E) cells in the E11.5 FL, determined by flow cytometric analysis of BrdU and 7-AAD incorporation. Data are the mean (±SD) of 6 wild type and 5 Sl/Sl FLs analyzed individually over 3 independent experiments. Tail somite range: 12-17 (+/+); 12-17 (Sl/Sl).


of early apoptotic EMPs Early E in wild type Sl / Sl E11 . 5 FL , determined by flow cytometric of AnnexinV among 7 - AAD - Ter119 - CD41 + Kit + CD16 / 32 + EMPs 7 - AAD - Ter119 - Kit + CD41 - CD71 + CD44 + Early E . Data are mean ( ± SD ) of 9 wild type 7 Sl / Sl biological replicates analyzed over 3 independent experiments , with each replicate consisting of 1 - 3 FLs of identical genotypes . A total number of 13 wild type 9 Sl / Sl were analyzed . Tail somite range : 11 - 17 for both genotypes .

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Percentage of early apoptotic EMPs and Early E in wild type and Sl/Sl E11.5 FL, determined by flow cytometric analysis of AnnexinV staining among 7-AAD- Ter119- CD41+ Kit+ CD16/32+ EMPs and 7-AAD- Ter119- Kit+ CD41- CD71+ CD44+ Early E. Data are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments, with each replicate consisting of 1-3 FLs of identical genotypes. A total number of 13 wild type and 9 Sl/Sl embryos were analyzed. Tail somite range: 11-17 for both genotypes.


Percentages of macrophage populations in wild type Sl / Sl E14 . 5 brain , skin ( dissected from back ) , lungs limb buds , as determined by flow cytometry . were gated as Ter119 - CD45 + further gated into F4 / 80hi CD11blo or F4 / 80lo CD11bhi populations . Representative dot plots of skin are shown in Appendix Figure S1A . Data are mean ( ± SD ) of 4 biological replicates for either genotype , with each replicate consisting of or pooled samples ( from up to 3 ) . A total of 8 wild type 5 Sl / Sl were analyzed over 2 independent experiments .

O O O O O O O O O O O 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 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

Percentages of macrophage populations in wild type and Sl/Sl E14.5 brain, skin (dissected from the back), lungs and limb buds, as determined by flow cytometry. Cells were gated as Ter119- CD45+ and further gated into F4/80hi CD11blo or F4/80lo CD11bhi populations. Representative dot plots of skin analysis are shown in Appendix Figure S1A. Data are the mean (±SD) of 4 biological replicates for either genotype, with each replicate consisting of individual or pooled samples (from up to 3 embryos). A total of 8 wild type and 5 Sl/Sl embryos were analyzed over 2 independent experiments.


absolute numbers of tissue macrophages per E14 . 5 FL . Data are mean ( ± SD ) . Number of analyzed : as in ( A ) .

Percentage and absolute numbers of tissue macrophages per E14.5 FL. Data are mean (±SD). Number of embryos analyzed: as in (A).


Representative flow cytometry of macrophages in wild type Sl / Sl E10 . 5 AGM + VU . were gated as shown , with percentages in gates as indicated . Data are mean ( ± SD ) of 4 wild type ( 32 - 35 sp ) or 3 Sl / Sl ( 34 - 35 sp ) biological replicates , analyzed over 2 independent experiments . were analyzed individually . of flow cytometry in ( C ) , showing of F4 / 80 + CD11b + macrophages within live . E11 . 5 data are mean ( ± SD ) of 4 wild type ( 10 - 13 tail somites ) or 3 Sl / Sl ( 10 - 13 tail somites ) biological replicates , analyzed over 3 independent experiments . were analyzed individually .

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Representative flow cytometry analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. Cells were gated as shown, with percentages in individual gates as indicated. Data are the mean (±SD) of 4 wild type (32-35 sp) or 3 Sl/Sl (34-35 sp) biological replicates, analyzed over 2 independent experiments. Embryos were analyzed individually. Quantification of flow cytometry analysis in (C), showing percentage of F4/80+CD11b+ macrophages within live cells. E11.5 data are the mean (±SD) of 4 wild type (10-13 tail somites) or 3 Sl/Sl (10-13 tail somites) biological replicates, analyzed over 3 independent experiments. Embryos were analyzed individually.


Confocal whole - mount immunofluorescence of macrophages in wild type Sl / Sl E10 . 5 AGM + VU . 3D panels show maximum projections from 400μm - thick Z stacks . Slice panels show single 2 . 5μm - thick Z slices of clusters in dorsal aorta ( da ) . A total of 4 wild type ( 31 - 34 sp ) 3 Sl / Sl ( 31 - 33 sp ) were analyzed . Scale bars : 50μm ( 3D ) , 10μm ( slice ) . of whole - mount immunofluorescence in ( E ) , showing absolute numbers ( mean ± SD ) of F4 / 80 + macrophages within hematopoietic clusters in dorsal aorta .

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Confocal whole-mount immunofluorescence analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. 3D panels show maximum intensity projections from 400μm-thick Z stacks. Slice panels show single 2.5μm-thick Z slices of clusters in the dorsal aorta (da). A total of 4 wild type (31-34 sp) and 3 Sl/Sl (31-33 sp) embryos were analyzed. Scale bars: 50μm (3D), 10μm (slice). Quantification of whole-mount immunofluorescence in (E), showing absolute numbers (mean ± SD) of F4/80+ macrophages within hematopoietic clusters in the dorsal aorta.


Confocal whole - mount immunofluorescence ( WM - IF ) of Kit + in wild type Sl / Sl E10 . 5 AGM ( 32 - 36sp ) . are 3D projections from 400μm - thick Z stacks , are representative of 14 wild type 11 Sl / Sl analyzed . Total numbers of Kit + ( ) in dorsal aorta were quantified showed a significant reduction in Sl / Sl compared to wild type ( p = 0 . 008 ) . CD31 + vasculature is shown in green . Arrowheads : hematopoietic clusters . Scale bars : 100μm . of WM - IF in ( A ) , showing absolute numbers ( ± SD ) of clusters of indicated separately shown relative to location . A total of 7 wild type 7 Sl / Sl were counted .

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 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 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

Confocal whole-mount immunofluorescence analysis (WM-IF) of Kit+ cells in wild type and Sl/Sl E10.5 AGM (32-36sp). Images are 3D projections from 400μm-thick Z stacks, and are representative of 14 wild type and 11 Sl/Sl embryos analyzed. Total numbers of Kit+ cells (white) in the dorsal aorta were quantified and showed a significant reduction in Sl/Sl compared to wild type (p=0.008). CD31+ vasculature is shown in green. Arrowheads: hematopoietic cell clusters. Scale bars: 100μm. Quantification of WM-IF in (A), showing absolute numbers (±SD) of clusters of indicated size and separately shown relative to location. A total of 7 wild type and 7 Sl/Sl embryos were counted.


Flow cytometry of HSC lineage in wild type Sl / Sl E11 . 5 AGM + VU . were Ter119 - VE - Cadherin + Kit + gated percentages of within pre - HSC type I gate ( red ) pre - HSC type II / HSC gate ( green ) are shown . Percentages are mean ( ± SD ) of 10 wild type 9 Sl / Sl biological replicates , with each replicate consisting of 1 - 3 pooled AGM + VU . A total of 15 wild type 11 Sl / Sl were analyzed over 5 independent experiments .

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Flow cytometry analysis of the HSC lineage in wild type and Sl/Sl E11.5 AGM+VU. Cells were Ter119- VE-Cadherin+ Kit+ gated and percentages of cells within the pre-HSC type I gate (red) and the pre-HSC type II/HSC gate (green) are shown. Percentages are the mean (±SD) of 10 wild type and 9 Sl/Sl biological replicates, with each replicate consisting of 1-3 pooled AGM+VU. A total of 15 wild type and 11 Sl/Sl embryos were analyzed over 5 independent experiments.


Absolute numbers ( ± SD ) of E10 . 5 E11 . 5 pre - HSC type I ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 - ) pre - HSC type II / HSC ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 + ) , as determined by flow cytometry in ( C ) . Number of E11 . 5 analyzed : as in ( A ) . For E10 . 5 , 5 biological replicates were analyzed for either genotype , with each replicate consisting of 1 - 4 pooled AGM + VU . A total of 12 wild type ( 31 - 35sp ) 9 Sl / Sl ( 30 - 34sp ) were analyzed over 4 independent experiments . Total live per AGM + VU : 2 . 8 ± 1 . 0x105 ( wt ) , 2 . 3 ± 0 . 6x105 ( Sl / Sl ) at E10 . 5 ; 3 . 9 ± 1 . 1x105 ( wt ) , 3 . 8 ± 1 . 4x105 ( Sl / Sl ) at E11 . 5 .

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Absolute numbers (±SD) of E10.5 and E11.5 pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-) and pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+), as determined by flow cytometry in (C). Number of E11.5 embryos analyzed: as in (A). For E10.5, 5 biological replicates were analyzed for either genotype, with each replicate consisting of 1-4 pooled AGM+VU. A total of 12 wild type (31-35sp) and 9 Sl/Sl (30-34sp) embryos were analyzed over 4 independent experiments. Total live cells per AGM+VU: 2.8±1.0x105 (wt), 2.3±0.6x105 (Sl/Sl) at E10.5; 3.9±1.1x105 (wt), 3.8±1.4x105 (Sl/Sl) at E11.5.


CFU - C numbers per wild type Sl / Sl E11 . 5 AGM + VU . are mean ( ± SD ) of 4 wild type 6 Sl / Sl biological replicates plated in duplicate . Biological replicates consisted of from or 2 pooled AGM + VU . A total of 6 wild type 7 Sl / Sl AGM + VU were analyzed over 4 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid .

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CFU-C numbers per wild type and Sl/Sl E11.5 AGM+VU. Counts are the mean (±SD) of 4 wild type and 6 Sl/Sl biological replicates plated in duplicate. Biological replicates consisted of cells from individual or 2 pooled AGM+VU. A total of 6 wild type and 7 Sl/Sl AGM+VU embryos were analyzed over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.


of long - term multi - lineage HSC potential in E11 . 5 Sl / Sl AGM + VU . Irradiated CD45 . 1 syngeneic mice were transplanted with 1 e . e . of wild type , Sl / + or Sl / Sl CD45 . 2 + AGM + VU . PB chimerism is represented as of donor CD45 . 2 + among total CD45 + , 16 after transplant . A total of 14 recipients were transplanted with wild type or Sl / + 8 with Sl / Sl , over 4 independent experiments . Wild type is represented with a circle , Sl / + a triangle Sl / Sl with a square . Tail somite range : 9 - 17 ( + / + , Sl / + ) ; 10 - 17 ( Sl / Sl ) . * : p = 0 . 027 , one - Mann - Whitney ' s U ; p = 0 . 055 , two - Mann - Whitney ' s U .

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

Analysis of long-term multi-lineage HSC potential in E11.5 Sl/Sl AGM+VU. Irradiated CD45.1 syngeneic mice were transplanted with 1 e.e. of wild type, Sl/+ or Sl/Sl CD45.2+ AGM+VU cells. PB chimerism is represented as the percentage of donor CD45.2+ cells among total CD45+ cells, 16 weeks after transplant. A total of 14 recipients were transplanted with wild type or Sl/+ cells and 8 with Sl/Sl cells, over 4 independent experiments. Wild type is represented with a circle, Sl/+ a triangle and Sl/Sl with a square. Tail somite range: 9-17 (+/+, Sl/+); 10-17 (Sl/Sl). *:p=0.027, one-tailed Mann-Whitney's U test; p=0.055, two-tailed Mann-Whitney's U test.


Confocal WM - IF of pHH3 expression in Kit + of wild type Sl / Sl E10 . 5 dorsal aorta ( 33 - 36sp ) . are 2 . 5μm - thick single longitudinal Z slices ; dorsal up . Arrowheads indicate examples of proliferating CD31 + pHH3 + Kit + hematopoietic ( magnified in insets ) ; total number of these per dorsal aorta was quantified showed reduced proliferation in mutant ( p = 0 . 01 ) . A total of 4 per genotype were analyzed . Scale bars : 50μm .

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Confocal WM-IF analysis of pHH3 expression in Kit+ cells of wild type and Sl/Sl E10.5 dorsal aorta (33-36sp). Images are 2.5μm-thick single longitudinal Z slices; dorsal up. Arrowheads indicate examples of proliferating CD31+ pHH3+ Kit+ hematopoietic cells (magnified in insets); the total number of these cells per dorsal aorta was quantified and showed reduced proliferation in the mutant (p=0.01). A total of 4 embryos per genotype were analyzed. Scale bars: 50μm.