g , Degradation of long - lived proteins in STX17 knockdown cells . St , starved ; wor , wortmannin . All error bars denote s . d . Scale bars , 1 µm ( a , b ) , 200 nm ( c ) and 5 µm ( d ) .

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

g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 µm (a, b), 200 nm (c) and 5 µm (d).


PS decarboxylase activity in wild - type ( WT , W303 ) and the indicated mutant strains expressed as ( B ) the molecular species composition of 13C315N - labeled PS ( * PS ) and 13C215N - labeled PE ( * PE ) , after 20 min incubation with 13C315N - serine .

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

PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.


( C ) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between * PS and * PE as percentages of the total amount of label incorporated in the molecular species 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

(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ( B ) the molecular species composition of * PS and * PE after 20 min incubation with 13C315N -

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serine


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ; ( C ) the corresponding molecular signatures of PSD activity .

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.


( B ) Acyl chain composition of WT and psd1 ∆ cells overexpressing SCT1 ( pSCT1 ) vs . empty vector control ( pEmpty ) . Data is presented as mean ± SD ( n > 10 ) . * * * * p < 0 . 0001 , multiple t - test .

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

(B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.


( C ) Ratio of the proportions of saturated over unsaturated acyl chains ( SFA / UFA ) of WT , psd1 ∆ , crd1 ∆ and psd2 ∆ overexpressing SCT1 vs . empty vector control . Data is presented as mean ± SD ( n ≥ 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed t - test .

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 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

(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.


( D ) OLE1 transcript levels in WT and psd1 ∆ transformed with pSCT1 vs . pEmpty as determined by RT - qPCR . Data were normalized to endogenous ACT1 mRNA , and to the average of WT pEmpty ( n = 3 ) .

O O O B-MEASURED_VAR 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

(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).


( A ) SFA / UFA ratios of WT and indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . control ( pEmpty ) . Data is presented as mean ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n . s . not significant , unpaired two - tailed t - test of the indicated bar compared to WT pSCT1 .

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

(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.


( B ) Growth of WT and indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . pEmpty . Serial dilutions ( 10 - 1 - 10 - 4 ) were spotted on SGal + 0 . 05 % glucose and incubated for 3 days at 30 ° C .

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

(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.


PS decarboxylase activity in wild - type ( WT , BY4741 ) and the indicated mutant strains transformed with pEmpty , expressed , and ( D ) molecular species composition of 13C315N - labeled PS ( * PS ) and 13C215N - labeled PE ( * PE ) after 20 min incubation with 13C315N - serine . The differences in molecular species profiles of * PS and * PE between the BY4741 WT shown here and those obtained by 2H3 - serine labeling ( Figure 4A ) are attributed to the different culture media , lipid extraction procedures and mass spectrometry techniques used .

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

PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.


( E ) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between * PS and * PE as percentages of the total amount of label incorporated in the molecular species indicated .

(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


SFA / UFA - ratio of WT and indicated mutant strains overexpressing SCT1 cultured without ( data taken from Fig 5A ) and with 1 mM ethanolamine ( etn ) or 1 mM choline ( cho ) as indicated . Data is presented as average ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t - test of the indicated bar compared to the corresponding condition of WT . Underlying data for this figure can be found in Appendix Table S1 .

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

SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.


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 .

O O O O O O O O O O O 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

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 ) , 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 .

O O O O O O O O O O O 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 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 , and pep4prb1Δ cells under nitrogen starvation . All data are means of triplicates . The error bars represent the 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 O B-CONTROLLED_VAR O O O O 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 ) 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 )

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

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 , 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 .

O O O O O O O B-CONTROLLED_VAR 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 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

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 , 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 .

O O O O O O O B-CONTROLLED_VAR 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 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

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 , 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-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 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 .

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

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

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

O 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 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

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 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 .

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 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

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 ) 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 .

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 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 ) 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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.


Immunoprecipitation was performed with GFP - binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings , expressing either YFP - MKKK7 or Lti6B . Seedlings were treated with 1 μM flg22 for the indicated time . YFP - MKKK7 and Lti6B - GFP were detected with an anti - GFP antibody while FLS2 was detected with an FLS - specific antibody . Upper panel shows ( co ) - immunoprecipated proteins , lower panel shows input levels of protein . Arrowheads indicate the position of proteins of interest .

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 B-CONTROLLED_VAR 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 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

Immunoprecipitation was performed with GFP-binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings, expressing either YFP-MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated time. YFP-MKKK7 and Lti6B-GFP were detected with an anti-GFP antibody while FLS2 was detected with an FLS-specific antibody. Upper panel shows (co)-immunoprecipated proteins, lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.


Application of selected reaction monitoring ( SRM ) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22 . Bars represent the mean ratio of endogenous phosphopeptide versus spiked - in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM ( n = 3 . ) . Asterisks indicate significant difference level compared to t = 0 ( student t - test , * > 0 . 05 , * * > 0 . 01 and * * * > 0 . 001 ) . The color of each bar corresponds to the different time points ( 0 minutes = dark blue , 5 minutes = red , 10 minutes = green , 20 minutes = purple , 30 minutes = light blue ) . Above each graph is the protein name and the phosphorylated residue ( in brackets ) is indicated and below the corresponding phosphopeptide is shown with the Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation site indicated by " [ + 80 ] " .

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Application of selected reaction monitoring (SRM) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22. Bars represent the mean ratio of endogenous phosphopeptide versus spiked-in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM (n=3.). Asterisks indicate significant difference level compared to t = 0 (student t-test, *>0.05, **>0.01 and ***>0.001). The color of each bar corresponds to the different time points (0 minutes = dark blue, 5 minutes = red, 10 minutes = green, 20 minutes = purple, 30 minutes = light blue). Above each graph is the protein name and the phosphorylated residue (in brackets) is indicated and below the corresponding phosphopeptide is shown with the Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation site indicated by "[+80]".


A ) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col - 0 and in mkkk7 . Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t = 0 , 10 and 30 min post induction . The p44 / 42 antibody was used to detect phosphorylated MAPKs . Position of the individual phosphorylated MAPKs is indicated at the right . Equal loading of proteins is shown with an α - Actin antibody as a loading control ( bottom panel ) . Three biological replicates were done with identical

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

A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results


B ) MPK6 phosphorylation is specifically enhanced in mkkk7 . Comparison of phosphopeptide abundances from selected MAP kinases in Col - 0 ( blue ) and mkkk7 ( red ) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring ( SRM ) mass spectrometry . Phosphopeptide corresponding to MKKK7 are only detectable in Col - 0 seedlings and are non - detectable ( ND ) in mkkk7 . Bars represent means of measured peptide areas ( sum of all transition areas ) for three biological replicates , with error bars ± SEM ( n = 3 ) . Asterisks indicate significant difference between Col - 0 and mkkk7 at individual time points ( student t - test , * > 0 . 05 , * * > 0 . 01 and * * * > 0 . 001 ) . ND indicates integration of an area without transitions significantly above background . Above each graph the protein name and the phosphorylated residue ( in brackets ) is indicated as well as the corresponding phosphopeptide sequence . Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation is indicated by " [ + 80 ] " .

O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 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

B) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *>0.05, **>0.01 and ***>0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by "[+80]".


A ) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment . Protoplasts were isolated from 4 week old plants and transfected with pWRKY29 : fLUC ( WRKY29 ) or pFRK1 : fLUC ( FRK1 ) constructs together with 35S : rLUC , as indicated in the graph . Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated . The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample , as fold induction . All measurements were normalized to the rLUC activity . Bars represent means ± STDEV ( n = 2 ) . Experiment was repeated 6 times with similar results .

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A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWRKY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results.


B ) WRKY29 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 and mkkk7 were treated with 1 µM flg22 for t = 0 , 1 , 2 , and 4h . WRKY29 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value and error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - test ) .

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B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


C ) FRK1 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 and mkkk7 were treated with 1 µM flg22 fort = 0 , 1 , 2 , and 4h . FRK1 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value and error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - test ) .

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C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


A ) Transient co - expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22 - induced WRKY29 gene expression . Protoplasts were transfected with pWRKY29 : fLUC , 35S : rLUC and indicated overexpression constructs of MKKK7 ( OE - MKKK7 , OE - MKKK7AA or OE - MKKK7DD ) as indicated on the horizontal axis . Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs . All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample , shown as fold induction on the vertical axis . Results shown are means ± STDEV ( n = 2 ) . At least two biological replicates were done with similar

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A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results


A ) Four - week - old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored . Data represents mean values ± SEM ( n = 20 ; * * * , p < 0 . 001 ; paired t - test ) . Three biological experiments were done showing similar results .

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A) Four-week-old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored. Data represents mean values ± SEM (n=20;***, p<0.001; paired t-test). Three biological experiments were done showing similar results.


B ) Quantification of bacterial growth in Arabidopsis lines Col - 0 , mkkk7 and p35S : MKKK7 - GFP in the mkkk7 background . Four to five - week - old plants were pressure - infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re - isolated on selective media . The number of colony forming units ( cfu / cm2 ) was determined at t = 0 , 2 and 3days post inoculation ( dpi ) . Data represents mean values ± SEM ( n = 6 ; * * , p < 0 . 01 ; paired t - test ) . Experiments were done at least twice with similar results .

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B) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p<0.01; paired t-test). Experiments were done at least twice with similar results.


C ) Disease symptom development in Pst - infected lines with estradiol inducible constructs of ind - MKKK7AA L8 , ind - MKKK7AA L10 , ind - MKKK7DD L1 and ind - MKKK7DD L3 . Two independent transgenic lines for each construct were grown under short - day conditions and disease symptoms were scored 3 dpi . Data represents mean values ± SEM ( n = 20 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; paired t - test ) . The vertical axis represents the percentage disease symptoms . Experiments were done at least twice with similar results .

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C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p<0.05; **, p<0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.


A ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col - 0 and mkkk7 .

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A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7.


B ) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col - 0 and two independent inducible MKKK7AA transgenic lines .

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B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.


C ) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col - 0 and two independent inducible MKKK7DD transgenic lines . Graphs represent means with error bars ± SEM ( n = 24 ) . The vertical axis represents the relative increase in ROS production ( photon counts ) after PAMP treatment . At least three biological replicate experiments were done with similar results .

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C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.


( A ) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15 - days old ( upper panel ) and 1 - month old animals ( lower panel ) .

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(A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel).


( B ) Body weight comparison over time of Ndufs3 smKO male mice ( filled blue squares ; n = 14 ) and age matched wild - type ( WTFlx ; n = 15 ) males ( empty blue squares ) . Ndufs3 smKO females ( filled red circles ) showed similar results ( n = 10 for smKO , and n = 12 for WTFlx group ) . P values were calculated by Student ' s t test ;

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(B) Body weight comparison over time of Ndufs3 smKO male mice (filled blue squares; n=14) and age matched wild-type (WTFlx; n=15) males (empty blue squares). Ndufs3 smKO females (filled red circles) showed similar results (n=10 for smKO, and n=12 for WTFlx group). P values were calculated by Student's t test;


( C ) Representative image of a smKO male Ndfu3s3 - smKO mouse and a WTFlx littermate of 6 - month old showing decreased body weight .

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(C) Representative image of a smKO male Ndfu3s3-smKO mouse and a WTFlx littermate of 6-month old showing decreased body weight.


( D ) Survival curve of Ndufs3 smKO female mice ( red line ) and males ( blue line ) . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0010 for males ( n = 19 ) , and p = 0 . 0004 for females ( n = 10 ) .

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(D) Survival curve of Ndufs3 smKO female mice (red line) and males (blue line). P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0010 for males (n=19), and p=0.0004 for females (n=10).


( E ) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild - type littermates from 1 and 2 months old male mice . P values were calculated by Student ' s t test . P values were calculated by Student ' s t test to determine the level of statistical difference . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 0001 .

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(E) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild-type littermates from 1 and 2 months old male mice. P values were calculated by Student's t test. P values were calculated by Student's t test to determine the level of statistical difference. ; *p<0.05, **p<0.01 and ***p<0.0001.


( F ) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice , starting at 2 months ( n = 8 ) . Bars represent means ± standard error ( SE ) . P values were calculated by Student ' s t test to determine the level of statistical difference . n . s . - not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 0001 .

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(F) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice, starting at 2 months (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.


( G ) Rotarod performed by Ndufs3 smKO and wild - type male mice . ( n = 8 ) . Bars represent means ± standard error ( SE ) . P values were calculated by Student ' s t test to determine the level of statistical difference . n . s . - not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 0001 .

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(G) Rotarod performed by Ndufs3 smKO and wild-type male mice. (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.


( A ) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age . Muscle weight from Ndufs3 smKO males ( black bar ) and wild - type mice ( white bar ) from 6 , and 8 months of age ( n = 5 each group ) . Bars represent means ± standard error ( SEM ) .

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(A) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age. Muscle weight from Ndufs3 smKO males (black bar) and wild-type mice (white bar) from 6, and 8 months of age (n=5 each group). Bars represent means ± standard error (SEM).


( B ) Representative H & E , COX , and SDH staining of quadriceps from WTFlx and Ndufs3 - Mlc1f smKO at 8 months old .

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(B) Representative H&E, COX, and SDH staining of quadriceps from WTFlx and Ndufs3-Mlc1f smKO at 8 months old.


( C ) Cross - sectional area ( CSA ) of quadriceps muscle fibers . A minimum of 100 fibers / sample were analyzed ( n = 3 / genotype ) . The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months ( n = 3 mice for WTFlx and n = 4 mice for Ndufs3 smKO ) . P values were calculated by Student ' s t test .

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(C) Cross-sectional area (CSA) of quadriceps muscle fibers. A minimum of 100 fibers/sample were analyzed (n =3/genotype). The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months (n =3 mice for WTFlx and n=4 mice for Ndufs3 smKO). P values were calculated by Student's t test.


( D ) Serum lactate levels of WTFlx and Ndufs3 - smKO animals at different ages . Ndufs3 smKO male mice ( filled black squares ) showed increased levels compared to matched WTFlx males ( empty black squares ) . Error bars represent SEM .

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(D) Serum lactate levels of WTFlx and Ndufs3-smKO animals at different ages. Ndufs3 smKO male mice (filled black squares) showed increased levels compared to matched WTFlx males (empty black squares). Error bars represent SEM.


( E ) BN - PAGE In - Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild - type and Ndufs3 - Mlc1f smKO animals at 2 and 4 months .

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(E) BN-PAGE In-Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild-type and Ndufs3-Mlc1f smKO animals at 2 and 4 months.


( F - G ) Western blots of muscle homogenates ( quadriceps ) of WTFlx and Ndufs3 - Mlc1f smKO animals at different ages ( 2 and 4 months , respectively ) using antibodies against PGC - 1α , COX1 ( complex IV subunit ) , NDUFS3 , VDAC1 , SDHA ( complex II subunit ) , and β - actin . ( H ) Quantification of the western blots in panels F and G . Bars represent means ± SEM of n = 4 for each group . P values were determined by Student ' s t test .

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(F-G) Western blots of muscle homogenates (quadriceps) of WTFlx and Ndufs3-Mlc1f smKO animals at different ages (2 and 4 months, respectively) using antibodies against PGC-1α, COX1 (complex IV subunit), NDUFS3, VDAC1, SDHA (complex II subunit), and β-actin. (H) Quantification of the western blots in panels F and G. Bars represent means ±SEM of n=4 for each group. P values were determined by Student's t test.


( C ) The KO - GFP mouse was visibly smaller and weaker than a KO - NDUFS3 mouse , at 7 months .

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(C) The KO-GFP mouse was visibly smaller and weaker than a KO-NDUFS3 mouse, at 7 months.


( D ) Females body weight injected with rAAV9 - Ndufs3 ( heretofore referred to as KO - NDUFS3 ) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9 - eGFP injected mice ( KO - GFP ) starting at 5 months . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group .

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(D) Females body weight injected with rAAV9-Ndufs3 (heretofore referred to as KO-NDUFS3) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9-eGFP injected mice (KO-GFP) starting at 5 months. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( E ) Males body weight of KO - NDUFS3 mice were similar to WTFlx mice , and significantly different from KO - GFP from 4 months onwards . Statistical analysis was performed by One - way ANOVA followed by a Tukey post - test . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group .

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(E) Males body weight of KO-NDUFS3 mice were similar to WTFlx mice, and significantly different from KO-GFP from 4 months onwards. Statistical analysis was performed by One-way ANOVA followed by a Tukey post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( F ) Treadmill performance was significantly improved in the KO - NDUFS3 mice when compared to the KO - GFP , from 2 months onwards . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group .

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(F) Treadmill performance was significantly improved in the KO-NDUFS3 mice when compared to the KO-GFP, from 2 months onwards. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( G ) Four limb strength was evaluated by the grid hang test . KO - NDUFS3 mice performed significantly better starting at 3 months , when compared to the KO - GFP mice . F ) and G ) Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group .

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(G) Four limb strength was evaluated by the grid hang test. KO-NDUFS3 mice performed significantly better starting at 3 months, when compared to the KO-GFP mice. F) and G) Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( H ) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO - NDUFS3 mice . Quadriceps weight was significantly increased in KO - NDUFS3 mice when compared to KO - GFP injected . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . ns - not significant . P values were calculated using Log - rank ( Mantel - Cox ) Test ; p = 0 . 0016 ( KO - NDUFS3 vs KO - GFP ) ; p = 0 . 0014 ( KO - GFP vs WTFlx ) ; n - indicates the number of animals used per group .

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(H) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO-NDUFS3 mice. Quadriceps weight was significantly increased in KO-NDUFS3 mice when compared to KO-GFP injected. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. ns- not significant. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.


( I ) Survival curve of Ndufs3 smKO mice injected with rAVV9 - Ndufs3 ( blue line ) and with rAVV9 - eGFP ( green line ) . WTFlx mice are depicted in black . rAAV9 - Ndufs3 injections extended the survival of the smKO mice .

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(I) Survival curve of Ndufs3 smKO mice injected with rAVV9-Ndufs3 (blue line) and with rAVV9-eGFP (green line). WTFlx mice are depicted in black. rAAV9-Ndufs3 injections extended the survival of the smKO mice.


( A ) Western blots of 6 months old quadriceps of WTFlx , KO and KO - NDUFS3 injected mice at 15 - 18 days old . GFP expression was observed only in the KO - GFP mice . Protein loading was obtained with stain - free gel technology . ( B ) Quantification of NDUFS3 , COXI and SDHA expression levels in female mice showed in A .

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(A) Western blots of 6 months old quadriceps of WTFlx, KO and KO-NDUFS3 injected mice at 15-18 days old. GFP expression was observed only in the KO-GFP mice. Protein loading was obtained with stain-free gel technology. (B) Quantification of NDUFS3, COXI and SDHA expression levels in female mice showed in A.


( C ) Western blot of BN - PAGE of quadriceps of 6 - month - old mice . We analyzed : CI ( NDUFA9 , NDUFB8 ) , CIV ( COX1 ) , CII ( SDHA ) . ( D ) Quantification of the BN - PAGE western blots from female mice shown in panel C .

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(C) Western blot of BN-PAGE of quadriceps of 6-month-old mice. We analyzed: CI (NDUFA9, NDUFB8), CIV (COX1), CII (SDHA). (D) Quantification of the BN-PAGE western blots from female mice shown in panel C.


( E ) BN - PAGE in - gel activity of CI and CIV in quadriceps of 6 - month - old mice . ( F ) Quantification of BN - PAGE in - gel activities in females .

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(E) BN-PAGE in-gel activity of CI and CIV in quadriceps of 6-month-old mice. (F) Quantification of BN-PAGE in-gel activities in females.


( G ) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1 / 18S . Inset shows the appearance of the quadriceps , reflecting normalization of mitochondrial levels after replacement of NDUFS3 .

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(G) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1/18S. Inset shows the appearance of the quadriceps, reflecting normalization of mitochondrial levels after replacement of NDUFS3.


( H ) Spectrophotometric CI / citrate synthase activity ratio was measured in quadriceps homogenates of 6 - month - old males and females .

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(H) Spectrophotometric CI/citrate synthase activity ratio was measured in quadriceps homogenates of 6-month-old males and females.


( I ) Plasma lactate levels were determined as described in methods . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test .

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(I) Plasma lactate levels were determined as described in methods. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test.


( B ) The smKO mice group that received retro - orbital injections at 2 months was tested prior to the rAAV9 - Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill . The KO - NDUFS3 gradually recovered over time . Individual KO - NDUFS3 mice are color coded . Green triangles represent the animals injected with GFP whereas white triangles represent non - injected mice . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test .

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(B) The smKO mice group that received retro-orbital injections at 2 months was tested prior to the rAAV9-Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill. The KO-NDUFS3 gradually recovered over time. Individual KO-NDUFS3 mice are color coded. Green triangles represent the animals injected with GFP whereas white triangles represent non-injected mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.


( C ) The hang test was performed in symptomatic mice which had a significant decrease in four - limb muscle strength and coordination , at 2 months . The KO - NDUFS3 mice showed a significant recovery 3 . 5 months after injection . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test .

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(C) The hang test was performed in symptomatic mice which had a significant decrease in four-limb muscle strength and coordination, at 2 months. The KO-NDUFS3 mice showed a significant recovery 3.5 months after injection. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.


( D ) KO - NDUFS3 muscle weight was similar to WTFlx mice , and it was significantly increased in gastrocnemius when compared to KO - GFP mice . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test .

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(D) KO-NDUFS3 muscle weight was similar to WTFlx mice, and it was significantly increased in gastrocnemius when compared to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test.


( E ) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6 - month - old animals . The expression of NDUFB8 ( representative of assembled CI ) was recovered in the KO - NDUFS3 mice and COX1 expression was similar to WTFlx animals .

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(E) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6-month-old animals. The expression of NDUFB8 (representative of assembled CI) was recovered in the KO-NDUFS3 mice and COX1 expression was similar to WTFlx animals.


( F ) COX and COX / SDH activity staining showed that KO - NDUFS3 quadriceps were reverted to levels found in wild - type , at 6 months . n - indicates the number of animals used per group , scale : 100 µm .

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(F) COX and COX/SDH activity staining showed that KO-NDUFS3 quadriceps were reverted to levels found in wild-type, at 6 months. n-indicates the number of animals used per group, scale: 100 µm.


( A ) Western blots showed NDUFS3 expression in KO - NDUFS3 and GFP expression in the KO - GFP quadriceps samples . PGC - 1α expression was normalized in the KO - NDUFS3 mice which was abnormally elevated in the KO - GFP . The same was observed for CIV ( COX1 ) , CIII ( Core 2 ) and CII ( SDHA ) . ( B ) Quantification of western blots showed in A . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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(A) Western blots showed NDUFS3 expression in KO-NDUFS3 and GFP expression in the KO-GFP quadriceps samples. PGC-1α expression was normalized in the KO-NDUFS3 mice which was abnormally elevated in the KO-GFP. The same was observed for CIV (COX1), CIII (Core 2) and CII (SDHA). (B) Quantification of western blots showed in A. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( C ) BN - PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO - NDUFS3 injected mice . CIV and CII were similar to wild - type in the KO - NDUFS3 samples , at 6 months .

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(C) BN-PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO-NDUFS3 injected mice. CIV and CII were similar to wild-type in the KO-NDUFS3 samples, at 6 months.


( D ) Quantification of BN - PAGE western blots Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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(D) Quantification of BN-PAGE western blots Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( E ) BN - PAGE In - gel CI and IV activities of KO - NDUFS3 mice were also reverted to levels similar to the WTFlx samples ( F ) Quantification of in - gel activities . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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(E) BN-PAGE In-gel CI and IV activities of KO-NDUFS3 mice were also reverted to levels similar to the WTFlx samples (F) Quantification of in-gel activities. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( G ) Complex I / Citrate synthase activity of KO - NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO - GFP mice . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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(G) Complex I/Citrate synthase activity of KO-NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( H ) Total mtDNA levels were determined by qPCR . MtDNA levels in KO - NDUFS3 mice were decreased when compared to KO - GFP mice and not different from the levels in WTFlx mice . Statistical analysis was performed by One - way ANOVA followed by Bonferroni post - test . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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(H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


( I ) The serum lactate levels were similar to WTFlx in KO - NDUFS3 samples and significantly decreased when compared to KO - GFP levels . The individual KO - NDUFS3 animals are color coded . Green triangles represent the animals injected with rAAV9 - eGFP whereas black triangles represent animals which did not receive an rAAV9 - eGFP injection . Statistical analysis was performed by One - way ANOVA followed by Tukey post - test . Data represent means ± SEM .

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(I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.


Population doublings ( PDs ) determined in BJ cells cultured with and without folate for 28 days .

Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days.


Example of a single combed DNA molecule labeled with IdU ( green ) and CldU ( red ) , showing replication from three adjacent origins . Horizontal white arrows indicate fork orientation .

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Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation.


Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate - free medium .

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Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium.


Fork rate ( Kb / min ) distribution . Light blue bars : BJ cells ( n = 126 ) ; black bars : BJ cells that were cultured for 7 days in a folate - free medium ( n = 131 ) ; blue bars : BJ cells cultured for 7 days in a folate - free medium and supplemented with A , G , C , and T nucleosides for the last 48 h of the experiment ( n = 138 ) . Box plot summarizing the fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 360 ) ; − folate ( n = 372 ) ; − folate + AGCT ( n = 361 ) .

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Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361).


Fork distance ( Kb ) distribution . The color code is as in ( D ) . Control ( n = 72 ) ; − folate ( n = 71 ) ; − folate + AGCT ( n = 75 ) . Box plot summarizing the fork distance distribution ( Kb ) of three independent experiments . Control ( n = 212 ) ; − folate ( n = 215 ) ; − folate + AGCT ( n = 209 ) .

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Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75).Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209).


A Population doublings ( PDs ) determined in BJ cells cultured at the indicated folate concentrations for 48 days .

A Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days.


B , C The average replication rate ± SEM ( B ) and the average fork distance ± SEM ( C ) in the indicated folate concentrations at 14 and 21 days . At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate . At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance .

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B, C The average replication rate ± SEM (B) and the average fork distance ± SEM (C) in the indicated folate concentrations at 14 and 21 days. At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate. At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance.


D - G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation . ( D ) Fork rate ( Kb / min ) distribution . Light blue bars : BJ cells ( n = 115 ) ; gray bars : BJ cells that were cultured for 14 days in 100 nM folate ( n = 117 ) ; blue bars : BJ cells cultured for 14 days in 100 nM folate and supplemented with A , G , C , and T nucleosides for the last 48 h of the experiment ( n = 117 ) . ( E ) Box plot summarizing the fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 352 ) ; 100 nM folate ( n = 364 ) ; 100 nM folate + AGCT ( n = 355 ) . Main box represents the values from the lower to upper quartile ( 25th to 75th percentile ) . The middle line represents the median . ( F ) Fork distance ( Kb ) distribution . The color code is as in ( D ) . Control ( n = 69 ) ; 100 nM folate ( n = 74 ) ; 100 nM folate + AGCT ( n = 72 ) . ( G ) Box plot summarizing the fork distance distribution ( Kb ) of three independent experiments . Control ( n = 201 ) ; 100 nM folate ( n = 220 ) ; 100 nM folate + AGCT ( n = 228 ) . Main box represents the values from the lower to upper quartile ( 25th to 75th percentile ) . The middle line represents the median . * * P < 0 . 001 .

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D-G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation. (D) Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 115); gray bars: BJ cells that were cultured for 14 days in 100 nM folate (n = 117); blue bars: BJ cells cultured for 14 days in 100 nM folate and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 117). (E) Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 352); 100 nM folate (n = 364); 100 nM folate + AGCT (n = 355). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. (F) Fork distance (Kb) distribution. The color code is as in (D). Control (n = 69); 100 nM folate (n = 74); 100 nM folate + AGCT (n = 72). (G) Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 201); 100 nM folate (n = 220); 100 nM folate + AGCT (n = 228). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.001.


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Fork rate ( Kb / min ) distribution . White bars : BJ cells expressing an empty vector ( n = 145 ) ; dark gray bars : BJ cells expressing the cyclin E oncogene ( n = 147 ) ; light gray bars : BJ cells cultured for 7 days in a folate - free medium ( n = 135 ) ; black bars : BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate - free medium ( n = 138 ) .

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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138).


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Fork distance distribution ( Kb ) . The color code is as in ( A ) . Empty vector ( n = 78 ) ; CycE ( n = 79 ) ; empty vector − folate ( n = 71 ) ; CycE − folate ( n = 80 ) .

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 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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80).


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Percent of origins with the indicated progression ratio between sister forks . Empty vector ( n = 158 ) ; CycE ( n = 155 ) ; empty vector − folate ( n = 160 ) ; CycE − folate ( n = 154 ) . * P < 0 . 05 .

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 B-CONTROLLED_VAR 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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05.


Cyclin E - expressing BJ cells were grown for 7 days with and without folate . Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression ( CycE ) ( n = 65 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( empty vector − folate ) ( n = 67 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 70 ) . Red : γH2AX ; green : 53BP1 ; blue : DAPI staining . Percent of nuclei with the indicated number of γH2AX - 53BP1 co - localized foci . * * P < 0 . 01 .

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 B-MEASURED_VAR O O B-CONTROLLED_VAR I-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 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 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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining.Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01.


Cyclin E - expressing BJ cells grown for 7 days with and without folate . Immunoblotting with anti - phosphorylated ATM and anti - phosphorylated CHK1 antibodies . Anti - β - catenin and anti - actin antibodies were used as loading controls . Protein level quantification .

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

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification.


Cyclin E - expressing BJ cells grown for 7 days with and without folate . Examples of nuclei with RAD51 foci following cyclin E expression ( CycE ) ( n = 67 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( − folate ) ( n = 71 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 75 ) . Green : RAD51 , blue : DAPI staining . Percent of nuclei with the indicated number of RAD51 foci ( n = 66 ) . * * P < 0 . 01 .

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

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.


A , B Cyclin E - and Ras ( H - RasV12 ) - expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium . Control cells were grown in a normal medium for the whole period . ( A ) Examples of anchorage - independent growth in soft agar of 3T3 cells . ( B ) Average number of colonies per soft agar plate of 3T3 cells . The number of colonies per plate is expressed as the average ± SEM from three independent experiments .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O 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 Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments.


C - E Ras ( H - RasV12 ) - expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium . Control cells were grown in a normal medium for the whole period . ( C ) Examples of anchorage - independent growth in soft agar of MCF10A cells . ( D ) Average number of colonies per soft agar plate of MCF10A cells . The number of colonies per plate is expressed as the average ± SEM from three independent experiments . ( E ) Percentage of tumor - free flanks at the indicated time points after cell injection . Ten mice were injected in both sides in each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.


C ) Time course analysis of triubiquitin ( 2 µM ) hydrolysis using 250 nM SARS2 PLpro , resolved on a Coomassie stained SDS - PAGE gel . Linkage specific cleavage of Lys48 - linked triubiquitin to di - and monoubiquitin resembles SARS PLpro activity ( Békés et al , 2015 ; 2016 ) . See Source Data for uncropped gels , and Appendix Fig S1B - D for gel - based cleavage quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time course analysis of triubiquitin (2 µM) hydrolysis using 250 nM SARS2 PLpro, resolved on a Coomassie stained SDS-PAGE gel. Linkage specific cleavage of Lys48-linked triubiquitin to di- and monoubiquitin resembles SARS PLpro activity (Békés et al, 2015; 2016). See Source Data for uncropped gels, and Appendix Fig S1B-D for gel-based cleavage quantification.


B ) Fluorescence polarisation assays against ubiquitin - TAMRA and ISG15 - TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n = 2 for each mutant , and compared to wild - type PLpro as shown in Appendix Fig S1A . Catalytic efficiencies were calculated as described in Methods .

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B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods.


C ) Gel based analysis of PLpro variant activity against Lys48 - triubiquitin and proISG15 . Experiments were performed in duplicate , see Fig EV3 and Source Data for uncropped gels .

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

C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.


C ) Calculated enzymatic efficiencies for K48 - diUb - TAMRA cleavage , followed by fluorescence polarisation with PLpro wild - type ( reproduced from Appendix Fig S1A ) , and PLpro F69S . ( Left ) Using the same concentration range of SARS2 PLpro , the F69S mutant activity could not be fitted ; ( middle ) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin - TAMRA ( 45 vs . 86 M - 1s - 1 , compare Fig 1D , Appendix Fig S1A ) . ( Right ) A ~ 3 - fold lower efficiency for PLpro F69S cleaving ISG15 - TAMRA yields values similar to cleavage of ISG15CTD - TAMRA ( Appendix Fig S1A ) , suggesting that the S2 site contributes the difference in binding for the N - terminal Ubl - fold . Experiments for F69S were performed in technical triplicate and biological duplicate ; wild - type data is reproduced from experiments shown in Fig 1D , Appendix Fig S1A .

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

C) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A.


D - F ) Gel based analysis showing hydrolysis time course of triubiquitin ( D ) , proISG15CTD ( E ) and proISG15 ( F ) using wild - type PLpro ( left , wild - type gels reproduced from Appendix Fig S1B - D to enable direct comparison ) or PLpro F69S ( right ) . PLpro F69S has a strong effect on triubiquitin hydrolysis ( D ) , no marked effect on hydrolysis of proISG15CTD ( E ) and reduces proISG15 cleavage to the same levels as proISG15CTD ( compare E , F ) , Experiments were performed in duplicate , see Source Data for uncropped gels .

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

D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.


B ) Hit compounds and compound rac5c ( see Fig 5 ) , were further assessed in 10 - point IC50 titrations using the Ub - Rhodamine assay , using a starting concentration of 100 µM serially diluted in 1 : 3 steps . Degree of inhibition is shown as a titration heat - map from dark ( full inhibition ) to light blue ( low / no inhibition ) . The catalytic domain of human USP21 ( Ye et al , 2011 ) was used as a counterscreen . Each PLpro hit compound showed either no activity in the titration analysis , or an identical inhibition profile against PLpro and USP21 , suggesting assay interference . Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM . IC50 assays were performed in technical triplicate in two 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 O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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

B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.


C ) In vitro inhibition ( IC50 ) for rac5c inhibiting SARS2 PLpro . Experiments were performed using the HTS assay ( Fig 4 ) , in technical triplicate in three independent experiments . A geometric mean was used to determine IC50 .

O O O O O O O O O O O O O O O O O O O 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) In vitro inhibition (IC50) for rac5c inhibiting SARS2 PLpro. Experiments were performed using the HTS assay (Fig 4), in technical triplicate in three independent experiments. A geometric mean was used to determine IC50.


D ) Full - length nsp3 was expressed from a C - terminally GFP - tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h . GFP is released from the C - terminus , presumably by nsp3 protease activity . Nsp3 can be detected by a SARS / SARS2 PLpro antibody ( see Fig EV5E for antibody validation ) . Lysates were blotted for Lys48 - linked polyubiquitin with a linkage specific antibody ( K48 ) . Experiments were performed in duplicate with similar results . Also see Fig EV5F , G and Source Data for uncropped blots .

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

D) Full-length nsp3 was expressed from a C-terminally GFP-tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h. GFP is released from the C-terminus, presumably by nsp3 protease activity. Nsp3 can be detected by a SARS/SARS2 PLpro antibody (see Fig EV5E for antibody validation). Lysates were blotted for Lys48-linked polyubiquitin with a linkage specific antibody (K48). Experiments were performed in duplicate with similar results. Also see Fig EV5F, G and Source Data for uncropped blots.


B ) Reduction in SARS - CoV - 2 induced cytopathic effect with rac5c , Remdesivir ( RDV ) and hydroxychloroquine ( HCQ ) treatment . DMSO 0 . 3 % ( v / v ) was required to keep 33 µM rac5c in solution ( see Fig EV6A , B ) . Mean ( black line ) is provided for 18 samples in each group , representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c . HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats . P values were calculated using a one - way ANOVA , with regular Dunnet ' s post - hoc test for multiple comparisons between treatment arms and infected / vehicle treated control using a single pooled variance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reduction in SARS-CoV-2 induced cytopathic effect with rac5c, Remdesivir (RDV) and hydroxychloroquine (HCQ) treatment. DMSO 0.3% (v/v) was required to keep 33 µM rac5c in solution (see Fig EV6A, B). Mean (black line) is provided for 18 samples in each group, representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c. HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats. P values were calculated using a one-way ANOVA, with regular Dunnet's post-hoc test for multiple comparisons between treatment arms and infected/vehicle treated control using a single pooled variance.


C ) TCID50 data , mean and SD , for one representative experiment from B with 6 technical replicates .

C) TCID50 data, mean and SD, for one representative experiment from B with 6 technical replicates.


( A - F ) TNF - α , Cxcl1 and Cxcl2 ( A , C , and E respectively ) were measured at 24 h after stimulation of bone - marrow - derived neutrophils ( 5 x 105 cells ) with increasing concentrations of bacterial nucleic acids ( 1 , 5 , 10 and 20 µg of DNA + RNA in equal proportions ) , HK - bacteria ( 1 , 5 , 10 or 20 μg / ml ) or live bacteria ( MOIs of 2 , 5 , 10 and 20 ) . LPS ( 1 , 10 , 100 and 1000 ng / ml ) was used as a positive control stimulus . ( B , D and F ) , Effect of FPR antagonist Boc - 2 on GBS - induced TNF - α , Cxcl1 and Cxcl2 production , respectively . Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 50 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

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

(A-F) TNF-α, Cxcl1 and Cxcl2 (A, C, and E respectively) were measured at 24 h after stimulation of bone-marrow-derived neutrophils (5 x 105 cells) with increasing concentrations of bacterial nucleic acids (1, 5, 10 and 20 µg of DNA + RNA in equal proportions), HK-bacteria (1, 5, 10 or 20 μg/ml) or live bacteria (MOIs of 2, 5, 10 and 20). LPS (1, 10, 100 and 1000 ng/ml) was used as a positive control stimulus. (B, D and F), Effect of FPR antagonist Boc-2 on GBS-induced TNF-α, Cxcl1 and Cxcl2 production, respectively. Bone-marrow-derived neutrophils were exposed to Boc-2 (50 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 10 µM ) or WRW4 ( 1 , 5 and 10 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

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

Bone-marrow-derived neutrophils were exposed to Boc-2 (10 µM) or WRW4 (1, 5 and 10 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.