K . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 30 μM cisplatin 6 days after germination , i . e . 3 days after transfer from control plates . L . cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs . M . cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . N . cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs .

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

K. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 30 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.L. cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.M. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.N. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.


A . Representative examples of comets of 21 days old wild - type plants , cdkb1 ; 1 cdkb1 ; 2 and cycb1 ; 1 cycb1 ; 3 double mutant seedlings in full spectrum view of the TriTek Comet Score software . Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min ( recovery ) and plants incubated without cisplatin for one hour ( control ) , respectively . B . Box plot of percentage of tail DNA of wild - type cells , cdkb1 ; 1 cdkb1 ; 2 and cycb1 ; 1 cycb1 ; 3 under cisplatin treatment . Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications . The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software . The box represents the interquartile range , the line across the box indicates the median values and whiskers represent 5 - 95 percentile values . Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99 . 99 % calculated with Student ' s t - test .

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A. Representative examples of comets of 21 days old wild-type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for one hour (control), respectively.B. Box plot of percentage of tail DNA of wild-type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values and whiskers represent 5-95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student's t-test.


C . Immunostaining of γ - H2AX foci in wild - type plants and mutant cells after two hours of treatment with 50µM cisplatin . D . Counted number of γ - H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild - type and mutant plants . For each sample the γ - H2AX foci of 100 cells were counted and grouped into six categories : cells with no , 1 - 2 , 3 - 5 , 6 - 10 , 11 - 20 and more than 20 foci per cell .

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C. Immunostaining of γ-H2AX foci in wild-type plants and mutant cells after two hours of treatment with 50µM cisplatin.D. Counted number of γ-H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the γ-H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1-2, 3-5, 6-10, 11-20 and more than 20 foci per cell.


B . Wild - type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors . C . cycb1 ; 1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors .

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B. Wild-type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.C. cycb1;1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.


D . Graphs show numbers of blue sectors per plant grown without drug treatment . E . Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days . F . Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days .

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D. Graphs show numbers of blue sectors per plant grown without drug treatment.E. Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days.F. Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days.


A . The wildtype and CDKA ; 1 - DE mutants were grown on control plates for 10 days . Root lengths were measured 10 days after germination . B . The wildtype and CDKA ; 1 - DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days . The mutant wee1 was used as a positive control for hydroxy urea sensitivity . Root lengths were measured 10 days after germination . C . The wildtype and CDKA ; 1 - DE mutants were grown on plates containing 0 , 6 μg / mL bleomycin for 10 days . The mutant ku70 was used as a positive control for bleomycin sensitivity . Root lengths were measured 10 days after germination . D . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on control plates for 10 days . Root lengths were measured 10 days after germination . E . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity . Root lengths were measured 10 days after germination . F . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on plates containing 0 , 6 μg / mL bleomycin for 10 days . The mutant ku70 was used as a positive control for bleomycin sensitivity . Root lengths were measured 10 days after germination .

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A. The wildtype and CDKA;1-DE mutants were grown on control plates for 10 days. Root lengths were measured 10 days after germination.B. The wildtype and CDKA;1-DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days. The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.C. The wildtype and CDKA;1-DE mutants were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.D. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on control plates for 10 days. Root lengths were measured 10 days after germination.E. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.F. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.


G . The wildtype , CDKA ; 1 - DE ( grey bar ) and the double mutant cdkb1 ; 1cdkb1 ; 2 ( purple bar ) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs . H . Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype , CDKA ; 1 - DE and cdkb1 ; 1cdkb1 ; 2 . I . Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype , CDKA ; 1 - DE and cdkb1 ; 1cdkb1 ; 2 .

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G. The wildtype, CDKA;1-DE (grey bar) and the double mutant cdkb1;1cdkb1;2(purple bar) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.H. Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.I. Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.


J - N . Images show a wild - type plant , CDKA ; 1 - DE and the double mutant cdkb1 ; 1 cdkb1 ; 2 ( from left to right ) on the indicated day after germination and the indicated drug treatment .

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J-N. Images show a wild-type plant, CDKA;1-DE and the double mutant cdkb1;1 cdkb1;2 (from left to right) on the indicated day after germination and the indicated drug treatment.


A . WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin , respectively , three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph . Asterisk indicates significant differences within a 5 % confidence interval ( Student ' s t - test ) . B . Image shows a wild - type plant ( left ) and rad51 mutant ( right ) grown on control plates . Images were taken 6 days after germination . C . Image shows a wild - type plant ( left ) and rad51 mutant ( right ) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination . Images were taken 6 days after germination , i . e . 3 days after transfer to cisplatin .

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A. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin, respectively, three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student's t-test).B. Image shows a wild-type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination.C. Image shows a wild-type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination. Images were taken 6 days after germination, i.e. 3 days after transfer to cisplatin.


D . Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin .

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D. Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin.


E . Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild - type and mutant plants . For each sample the RAD51 foci of 100 cells were counted and grouped into six categories : cells with 0 , 1 , 2 , 3 , 4 or > 5 foci per cell .

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E. Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4 or >5 foci per cell.


F . In vitro kinase assay of purified CDK complexes phosphorylating RAD51 . RAD51 and histone H1 kinase assays were performed with [ γ - 32P ] ATP as a phosphate donor . Proteins were subjected to SDS - PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates ( lower panels ) . Phosphorylated proteins were detected by autoradiograph ( upper panels ) . The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK - cyclin complexes , protein blots indicate the relative amounts of the CDKs in the reaction ( bottom panel ) . Abbreviation : p - RAD51 and p - Histone H1 for [ 32P ] - phosphorylated MBP - RAD51 - His6 and recombinant human histone H1 , respectively , resulting from kinase assays with radio - labeled ATP . Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure . 1 : without kinase , 2 : CDKA ; 1 - CYCA2 ; 3 , 3 : CDKB1 ; 1 - CYCA2 ; 3 , 4 : CDKB1 ; 1 - CYCD2 ; 1 , 5 : CDKA ; 1 - CYCB1 ; 1 , 6 : CDKB1 ; 1 - CYCB1 ; 1 .

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F. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ-32P]ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiograph (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK-cyclin complexes, protein blots indicate the relative amounts of the CDKs in the reaction (bottom panel).Abbreviation: p-RAD51 and p-Histone H1 for [32P]-phosphorylated MBP-RAD51-His6 and recombinant human histone H1, respectively, resulting from kinase assays with radio-labeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1-CYCA2;3, 3: CDKB1;1-CYCA2;3, 4: CDKB1;1-CYCD2;1, 5: CDKA;1-CYCB1;1, 6: CDKB1;1-CYCB1;1.


A . Transgenic Arabidopsis plants harboring the CYCB1 ; 1 promoter and a GFP fused to the N - terminal part of CYCB1 ; 1 including the destruction box grown on control plates and imaged five days after germination . D . Transgenic Arabidopsis plants harboring the CYCB1 ; 1 promoter and a GFP fused to the N - terminal part of CYCB1 ; 1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application .

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A. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box grown on control plates and imaged five days after germination.D. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.


B . Transgenic Arabidopsis plants harboring the CYCB1 ; 2 promoter and a GFP fused to the N - terminal part of CYCB1 ; 2 including the destruction box grown on control plates and imaged five days after germination . E . Transgenic Arabidopsis plants harboring the CYCB1 ; 2 promoter and a GFP fused to the N - terminal part of CYCB1 ; 2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application .

B. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box grown on control plates and imaged five days after germination.E. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.


C . Transgenic Arabidopsis plants harboring CDKB1 ; 1 promoter fused to GUS grown on control plates , were stained and imaged five days after germination . F . Transgenic Arabidopsis plants harboring CDKB1 ; 1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application .

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C. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS grown on control plates, were stained and imaged five days after germination.F. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application.


G . Chromatin immunoprecipitation ( ChIP ) of wild - type plants and PROSOG1 : SOG1 - Myc lines and anti - MYC antibody . The promoter region of CYCB1 ; 1 is enriched in SOG1 - MYC after cisplatin treatment . H . Structure of genes tested by ChIP with an antibody anti - MYC in PROSOG1 : SOG1 - Myc and wild - type plants . Red arrowheads indicate the primer binding sites for PCR . A total of four regions were tested as indicated by arrowheads . Asterisks indicates significant differences within a 5 % confidence interval ( Student ' s t - test ) .

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G. Chromatin immunoprecipitation (ChIP) of wild-type plants and PROSOG1:SOG1-Myc lines and anti-MYC antibody. The promoter region of CYCB1;1 is enriched in SOG1-MYC after cisplatin treatment.H. Structure of genes tested by ChIP with an antibody anti-MYC in PROSOG1:SOG1-Myc and wild-type plants. Red arrowheads indicate the primer binding sites for PCR. A total of four regions were tested as indicated by arrowheads. Asterisks indicates significant differences within a 5% confidence interval (Student's t-test).


A . The wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 were grown on control plates and root lengths were measured 10 days after germination . B . The wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 were grown on plates containing 0 , 6 μg / mL BLM . Root lengths were measured 10 days after germination . C . Graph represents the ratio of the mean growth rate on 0 , 6 µg / mL BLM compared to control experiments on plates lacking BLM for the wildtype , ku70 , cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 .

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A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on control plates and root lengths were measured 10 days after germination.B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70.


D . Image shows the wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 ( from left to right ) grown on control plates 10 days after germination . E . Images show the wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 ( from left to right ) on plates containing 0 , 6 μg / mL BLM .

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D. Image shows the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) grown on control plates 10 days after germination.E. Images show the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) on plates containing 0,6 μg/mL BLM.


B : CL formation was measured in the presence of 60 nmol MLCL ( 18 : 2 ) 3 , 40 nmol PC ( 18 : 2 ) 2 , 4 µg TAZ , and 0 . 2 mg MBP or 0 . 2 mg Alb . C : CL formation was measured as in B with different amounts of MBP or Alb .

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B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb. C: CL formation was measured as in B with different amounts of MBP or Alb.


G : CL formation was measured in the presence of 40 nmol PC ( 18 : 2 ) 2 , 10 nmol MLCL ( 18 : 2 ) 3 , 8 µg TAZ , 1250 nmol Triton X - 100 ( TX ) , and 5 . 6 nmol bacteriorhodopsin ( BR ) . H : PC formation was measured in the presence of 180 nmol CL ( 18 : 2 ) 4 , 20 nmol LPC16 : 0 , 8 µg TAZ , 20 mM Ca2 + , and 5 . 6 nmol BR .

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G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR). H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR.


J : Transacylations were measured as in I in the presence of 0 . 9 g BR / g lipid . The ratio of total linoleoyl transfer over total oleoyl transfer was calculated .

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J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.


A : Inner ( IM ) and outer ( OM ) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM / OM surface area ratio . Multiple micrographs were analyzed per biological replica .

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A: Inner (IM) and outer (OM) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM/OM surface area ratio. Multiple micrographs were analyzed per biological replica.


A : Mitochondrial membranes were traced in electron micrographs to determine the IM / OM surface area ratio . Tracings of the IM are shown in yellow boxes .

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A: Mitochondrial membranes were traced in electron micrographs to determine the IM/OM surface area ratio. Tracings of the IM are shown in yellow boxes.


C : Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence - labeled secondary antibodies . The abundances of IM proteins ( NDUFB6 , UQCR2 , ATP5A1 ) are expressed relative to the abundance of the OM protein TOM70 .

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C: Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence-labeled secondary antibodies. The abundances of IM proteins (NDUFB6, UQCR2, ATP5A1) are expressed relative to the abundance of the OM protein TOM70.


Tissues were analyzed by quantitative electron microscopy . Multiple micrographs were analyzed per biological

Tissues were analyzed by quantitative electron microscopy. Multiple micrographs were analyzed per biological replica


F , G : Tissues were analyzed by label - free quantitative proteomics . C I - V , complex I - V ; CAC , citric acid cycle enzymes ; MICOS , contact site complex ; mt TA , mitochondrial transaminases ; NS , not significant ; PDH pyruvate dehydrogenase ; VDAC , voltage - dependent anion channel

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F, G: Tissues were analyzed by label-free quantitative proteomics. C I-V, complex I-V; CAC, citric acid cycle enzymes; MICOS, contact site complex; mt TA, mitochondrial transaminases; NS, not significant; PDH pyruvate dehydrogenase; VDAC, voltage-dependent anion channel


Glycolytic flux of wildtype ( WT ) and TM6 strains strongly correlates with fructose - 1 , 6 - bisphosphate ( FBP ) concentration . The glycolytic flux is reported here as the flux between the metabolites fructose 6 - phosphate ( F6P ) and FBP . Glycolytic fluxes were obtained on the basis of physiological and metabolome data , and via a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . While on high glucose , the WT strain accomplishes a high glucose uptake rate ( and thus glycolytic flux ) , the mutant strain ( TM6 ) only generates a low glucose uptake ( and thus glycolytic flux ) . On maltose , also the mutant strain achieves a high glycolytic flux , since maltose is transported by a separate transporter ( Chang et al , 1989 ) . To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions , we implemented Pearson ' s correlation analysis assisted by bootstrapping . Specifically , we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions ( combinations of strains and carbon sources ) , biological and technical replicates . We paired each of these FBP measurements with the mean and standard deviation of the model - derived glycolytic flux ( A ) in the corresponding metabolic condition . We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition , and implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth - rate distributions to calculate the correlation statistics .

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

Glycolytic flux of wildtype (WT) and TM6 strains strongly correlates with fructose-1,6-bisphosphate (FBP) concentration. The glycolytic flux is reported here as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were obtained on the basis of physiological and metabolome data, and via a novel method to estimate intracellular fluxes (Niebel et al, 2019). While on high glucose, the WT strain accomplishes a high glucose uptake rate (and thus glycolytic flux), the mutant strain (TM6) only generates a low glucose uptake (and thus glycolytic flux). On maltose, also the mutant strain achieves a high glycolytic flux, since maltose is transported by a separate transporter (Chang et al, 1989). To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the model-derived glycolytic flux (A) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


FBP concentration as a function of cellular growth rate shows weaker correlation . For metabolite levels and growth rates , error bars correspond to the standard deviation between three independent experiments ; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space ( cf . Methods ) . The carbon sources were used at a final concentration of 10 gL - 1 and are indicated : Glucose ( GLU ) ; Galactose ( GAL ) ; Maltose ( MAL ) and Pyruvate ( PYR ) . To assess the linear correlation between the FBP concentration and the growth rate ( B ) across the studied conditions , we implemented Pearson ' s correlation analysis assisted by bootstrapping . Specifically , we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions ( combinations of strains and carbon sources ) , biological and technical replicates . We paired each of these FBP measurements with the mean and standard deviation of the growth rate ( B ) in the corresponding metabolic condition . We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition , and implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth - rate distributions to calculate the correlation statistics .

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

FBP concentration as a function of cellular growth rate shows weaker correlation. For metabolite levels and growth rates, error bars correspond to the standard deviation between three independent experiments; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space (cf. Methods). The carbon sources were used at a final concentration of 10 gL-1 and are indicated: Glucose (GLU); Galactose (GAL); Maltose (MAL) and Pyruvate (PYR). To assess the linear correlation between the FBP concentration and the growth rate (B) across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the growth rate (B) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


CggR intracellular abundance in the wildtype ( WT ) strain on glucose strongly varies with the promoter used . The CggR intracellular levels were quantified by proteomics in steady - state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL - 1 . Error bars represent the standard deviation of at least three replicate experiments .

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CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments.


The relative abundance of CggR ( normalized to the abundance measured on glucose and the same promoter ) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells , but not with PTEFmut2 and PCMV . The CggR intracellular levels were quantified by proteomics in steady - state cultures grown in minimal media with glucose , galactose , maltose or pyruvate as carbon sources at a final concentration of 10 gL - 1 . WT data includes all three promoters , whereas TM6 only includes the PTEFmut2 and PTEFmut7 data . Error bars represent the standard deviation of at least three replicate experiments .

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The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.


Thermal shift assays were used to determine the melting curves of wild - type CggR and mutants . Error bars correspond to the standard deviation of at least five replicates .

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Thermal shift assays were used to determine the melting curves of wild-type CggR and mutants. Error bars correspond to the standard deviation of at least five replicates.


CggR - FBP affinity constants ( KD ' s ) of the wildtype and mutant variants , determined by fitting a simple cooperative binding model to the melting curves data . Error bars indicate the 95 % confidence intervals .

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CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals.


Quantification of CggR binding to DNA . The CggR bound to DNA fraction was calculated by dividing the intensity of the protein - DNA complex band by the total DNA . The background - subtracted total intensities of the CggR - DNA complex and the free - DNA bands were assessed with ImageJ . Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype .

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

Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype.


The four reporter plasmids were transferred to the wildtype strain containing the CggR ( R250A ) under the control of the PTEFmut7 . The strength of the four promoters was assessed by quantifying YFP ( FL1 ) and mCherry ( FL3 ) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l - 1 glucose . The FL1 and FL3 fluorescence shown is the non - background corrected median of 100 000 cell events . The non - FL control is the signal from a wildtype strain grown under the same conditions . The background fluorescence , assessed by the wildtype harboring the YCplac33 plasmid , was substracted from FL1 and FL3 . The final reporter activity is the ratio of the background - corrected YFP and mCherry values . Error bars represent the standard deviation of three independently determined ratios from three replicate experiments .

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The four reporter plasmids were transferred to the wildtype strain containing the CggR (R250A) under the control of the PTEFmut7. The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l-1 glucose. The FL1 and FL3 fluorescence shown is the non-background corrected median of 100 000 cell events. The non-FL control is the signal from a wildtype strain grown under the same conditions. The background fluorescence, assessed by the wildtype harboring the YCplac33 plasmid, was substracted from FL1 and FL3. The final reporter activity is the ratio of the background-corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments.


Reporter activity of the sensor across ( D ) multiple FBP levels Reporter activity is given by the YFP / mCherry ratio , calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry . Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ) . The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR . Error bars represent the standard deviation of at least three replicate experiments .

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Reporter activity of the sensor across (D) multiple FBP levels Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.


Reporter activity of the sensor across glycolytic fluxes . The glycolytic flux is reported as the flux between the metabolites fructose 6 - phosphate ( F6P ) and fructose - 1 , 6 - bisphosphate ( FBP ) . Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . Reporter activity is given by the YFP / mCherry ratio , calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry . Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ) . The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR . Error bars represent the standard deviation of at least three replicate experiments .

Reporter activity of the sensor across glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.


Fraction of CggR bound to FBP across FBP concentrations . The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant . The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound / bound state using the Tm at 0 mM FBP as unbound and at 36 mM ( corresponding to maximum FBP concentration used ) as total bound states . The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one - site specific binding model in GraphPad . The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant . Vertical lines delimit the physiological FBP range .

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Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound/bound state using the Tm at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one-site specific binding model in GraphPad. The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.


Sub - populations of WT and TM6 cells , grown separately and mixed in different fractions as indicated in percentages , can easily be distinguished by flow cytometry , FL1 YFP channel , FL3 mCherry channel . Histogram of single - cell ratios of FL1 / FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry . Here , a sub - population of minimally 5 % can be distinguished .

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Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished.


Tukey boxplots showing the YFP / mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux . At least 35 cells were analyzed in each condition . The glycolytic flux is here reported as the flux between the metabolites fructose 6 - phosphate ( F6P ) and FBP . Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) .

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Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019).


YFP / mCherry ratio measured by microscopy in co - existing dividing ( high - flux ) versus non - dividing ( low - flux ) isogenic TM6 cells on 10 gL - 1 glucose .

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YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose.


Brightfield ( BF ) , YFP and mCherry microscopy images for a co - existing dividing ( high - flux ) and a non - dividing ( low - flux ) TM6 cell expressing the flux sensor in 10g L - 1 glucose minimal medium .

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Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium.


The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor - expressing strain ( F ) , which reflects the cell - cycle dynamics of intracellular FBP concentration and glycolytic flux . In a control strain , lacking CggR , the production rates of YFP and mCherry are coupled ( G ) . The uncoupling was calculated for individual cell - cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale ( see more details in Materials and methods ; Appendix Figure S10 ) . Each curve represents the mean across the indicated number of cell cycles in a replicate experiment . The corresponding shaded areas denote the 95 % confidence intervals of the means ( bootstrapping with 5 000 iterations ) . We smoothed the single - cell - cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression , and used these trajectories to derive the YFP and mCherry production rates , accounting for fluorescent - protein maturation in a first - order kinetics model . To align the cell - cycle trajectories and to calculate the phase , we used the array of three cell cycle events E { cytokinesis ( cyt ) , budding , next cyt } as reference points . The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes , with the mean duration presented in parentheses for each replicate experiment . The cells belonged to the TM6 strain and were cultivated on 20 gL - 1 glucose in the microfluidic device .

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The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.


P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . A - F . Clinical parameters determined in the study include age ( A ) , gender ( B ) , parasitemia ( C ) , hemoglobin ( g / dL blood ) ( D ) , hematocrit ( E ) , and platelet count ( F ) . Boxes represent the 25th to 75th percentiles , whiskers show the range ( minimum to maximum ) , and lines represent the median . Significance was determined by the Kruskal - Wallis test with Dunn ' s multiple comparisons ( A , D - F ) , the Chi - square test ( B ) , and the Mann - Whitney test ( C ) using 30 ( symptomatic ) , 40 ( asymptomatic ) and 31 ( healthy immune controls ) biological replicates . * * * * p < 0 . 0001 .

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P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. A-F. Clinical parameters determined in the study include age (A), gender (B), parasitemia (C), hemoglobin (g/dL blood) (D), hematocrit (E), and platelet count (F). Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median. Significance was determined by the Kruskal-Wallis test with Dunn's multiple comparisons (A, D-F), the Chi-square test (B), and the Mann-Whitney test (C) using 30 (symptomatic), 40 (asymptomatic) and 31 (healthy immune controls) biological replicates. ****p<0.0001.


P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . G . Antibody titers specific for P . falciparum parasite lysate . Bars represent the mean ± SD of using 29 ( symptomatic ) , 35 ( asymptomatic ) and 26 ( healthy immune controls ) biological replicates . The dotted line depicts the average antibody background levels of malaria - naive healthy Melbourne controls . H . Mean antibody titers as determined by ELISA to the P . falciparum recombinant proteins EBA - 175 , EBA - 140 , PfRh2 , PfRh4 , PfRh5 , and PfRipr .

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P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. G. Antibody titers specific for P. falciparum parasite lysate. Bars represent the mean ± SD of using 29 (symptomatic), 35 (asymptomatic) and 26 (healthy immune controls) biological replicates. The dotted line depicts the average antibody background levels of malaria-naive healthy Melbourne controls. H. Mean antibody titers as determined by ELISA to the P. falciparum recombinant proteins EBA-175, EBA-140, PfRh2, PfRh4, PfRh5, and PfRipr.


PBMCs from P . falciparum symptomatic ( n = 16 ) and asymptomatic ( n = 24 ) infected individuals as well as healthy immune controls ( n = 24 ) were stained with a panel of metal - labelled antibodies and analysed by CyTOF . tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub - populations within gated : A . TH1 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 + ) B . TH2 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 - ) C . Circulating memory TFH cells ( CD19 - CD3 + CD4 + CD45RA - CXCR5 + ) D . Classical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 + ) E . Atypical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 - CD21 - ) F . Activated MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 - ) The tSNE plots in the top panel display cell density and represent the pooled data for each group , while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot . Heatmaps show the median marker expression for each FlowSOM cluster .

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PBMCs from P. falciparum symptomatic (n=16) and asymptomatic (n=24) infected individuals as well as healthy immune controls (n=24) were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated: A. TH1-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3+) B. TH2-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3-) C. Circulating memory TFH cells (CD19-CD3+CD4+CD45RA-CXCR5+) D. Classical MBCs (CD3-CD19+CD20+CD10-CD27+CD21+) E. Atypical MBCs (CD3-CD19+CD20+CD10-CD27-CD21-) F. Activated MBCs (CD3-CD19+CD20+CD10-CD27+CD21-) The tSNE plots in the top panel display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster.


Unsupervised identification of differentially abundant high - dimensional cell populations between clinical groups was performed using CITRUS ( FDR < 5 % ) . between P . falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls . A - E . tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers , relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3 + CCR6 - memory CD4 + T cells ( A ) , CXCR3 - CCR6 - memory CD4 + T cells ( B ) , circulating memory TFH cells ( C ) , classical MBCs ( D ) , and atypical MBCs ( E ) . The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays . The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay , whiskers showing the range ( minimum to maximum ) , with lines representing the median of 16 ( symptomatic ) , 24 ( asymptomatic ) and 24 ( healthy immune controls ) biological replicates , while the pink histograms illustrate marker expression in identified differentially abundant populations , relative to background expression , shown in lilac .

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Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac.


PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . A . Estimated proportions of PBMC sub - populations determined from cell - type deconvolution from study participant transcriptional profiles .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. A. Estimated proportions of PBMC sub-populations determined from cell-type deconvolution from study participant transcriptional profiles.


PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . D . Blood transcriptional module ( BTM ) analysis showing significant BTMs differentially enriched for pairwise comparisons ( FDR < 15 % ) .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. D. Blood transcriptional module (BTM) analysis showing significant BTMs differentially enriched for pairwise comparisons (FDR<15%).


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . A . Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes ( DEGs ) between symptomatic P . falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance . B . Mean - difference plot displaying genes differentially expressed between symptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in symptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. A. Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes (DEGs) between symptomatic P. falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance. B. Mean-difference plot displaying genes differentially expressed between symptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in symptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . F . Upstream regulator analysis of the 922 DEGs between symptomatic P . falciparum malaria and healthy immune controls . The red lines represent a significant activation z - score of ± 2 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. F. Upstream regulator analysis of the 922 DEGs between symptomatic P. falciparum malaria and healthy immune controls. The red lines represent a significant activation z-score of ±2.


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . Hierarchical clustering heatmap ( G ) displaying DEGs in symptomatic P . falciparum malaria and healthy immune controls predicted to be controlled by IFN - γ and IL - 13 upstream regulators .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. Hierarchical clustering heatmap (G) displaying DEGs in symptomatic P. falciparum malaria and healthy immune controls predicted to be controlled by IFN-γ and IL-13 upstream regulators.


Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . C . Heat map of the 418 DEGs in P . falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls . E . Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2 - fold - change and - Log10 ( P - value ) differentially expressed between P . falciparum symptomatic and asymptomatic infected individuals . Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. C. Heat map of the 418 DEGs in P. falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls. E. Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2-fold-change and -Log10(P-value) differentially expressed between P. falciparum symptomatic and asymptomatic infected individuals. Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . F . Mean RPKMs + SEM of selected genes in P . falciparum malaria symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) individuals . Dotted red lines depict transcriptional levels of healthy immune controls , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. F. Mean RPKMs + SEM of selected genes in P. falciparum malaria symptomatic (n=6) and asymptomatic (n=5) individuals. Dotted red lines depict transcriptional levels of healthy immune controls, Mann-Whitney test of biological replicates,*p<0.05.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . A . Hierarchical clustering heat map of the 171 differentially expressed genes ( DEGs ) in P . falciparum asymptomatic and healthy immune control participants . C . Mean - difference plot displaying DEGs between asyptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in asymptomatic malaria .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. A. Hierarchical clustering heat map of the 171 differentially expressed genes (DEGs) in P. falciparum asymptomatic and healthy immune control participants. C. Mean-difference plot displaying DEGs between asyptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in asymptomatic malaria.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . E . Mean RPKMs + SEM of selected genes in P . falciparum malaria asymptomatic individuals ( n = 5 ) and healthy immune controls ( n = 6 ) . Dotted red lines depict transcriptional levels of P . falciparum - infected symptomatic individuals , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. E. Mean RPKMs + SEM of selected genes in P. falciparum malaria asymptomatic individuals (n=5) and healthy immune controls (n=6). Dotted red lines depict transcriptional levels of P. falciparum-infected symptomatic individuals, Mann-Whitney test of biological replicates, *p<0.05.


A . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM + activated MBCs . B . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T - bet + CD4 + T cells .

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A. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM+ activated MBCs. B. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T-bet+ CD4+ T cells.


B - G . PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 6 ) infected individuals as well as healthy immune controls ( n = 6 ) were stained with a panel of antibodies and analyzed by flow cytometry . tSNE plots ( B ) display cell density of the pooled data for each group , ( C ) shows a projection of the FlowSOM clusters on a tSNE plot , and ( D ) is a heatmap depicting the celluar phenotype of clusters of CTLA - 4 + CD3 + CD19 - cells . Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups ( E ) , Mann - Whitney test , * p < 0 . 05 . The panel on the left shows the average CTLA - 4 mean fluorescent intensity ( MFI ) or 6 biological replicates in cluster 4 + SEM , Mann - Whitney test , * * p < 0 . 01 , while representative histograms of CTLA - 4 expression in cluster 4 are shown on the right ( F ) . Marker intensity within clusters 1 and 6 ( G ) .

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B-G. PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=6) infected individuals as well as healthy immune controls (n=6) were stained with a panel of antibodies and analyzed by flow cytometry. tSNE plots (B) display cell density of the pooled data for each group, (C) shows a projection of the FlowSOM clusters on a tSNE plot, and (D) is a heatmap depicting the celluar phenotype of clusters of CTLA-4+CD3+CD19- cells. Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups (E), Mann-Whitney test, *p<0.05. The panel on the left shows the average CTLA-4 mean fluorescent intensity (MFI) or 6 biological replicates in cluster 4 + SEM, Mann-Whitney test, **p<0.01, while representative histograms of CTLA-4 expression in cluster 4 are shown on the right (F). Marker intensity within clusters 1 and 6 (G).


B . DCC and netrin - 1 expression ( FACS analysis ) in B - cells lymphocytes isolated from DCC + / + and DCCmut / mut mice spleen and lymph nodes . Results are presented as expression ratios between means expression levels in mutant animals ( n = 3 ) and control ones ( n = 3 ) . No significant difference was observed between wild - type and mutant animals .

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B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.


C . DCCmut / mut mouse with splenomegaly and systemic lymphadenopathy . Left panel : black and white arrows indicate respectively the inguinal and mesenteric lymph nodes . Right panel : enlarged spleen in a lymphoma - bearing mutant mouse ( left ) as compared to control organ ( right ) .

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C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right).


D . Incidence of lymphoid proliferations and lymphoma in DCC + / + ( n = 19 ) and DCCmut / mut ( n = 29 ) mice . Tumors classification was performed blinded to genotype according to anatomopathologists consensus ( 29 ) . * : p = 0 . 036 ; one - tailed Fisher ' s exact test .

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D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test.


E . Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice . Sections from control spleen , low - grade FL and high - grade DLBCL were stained with hematoxylin - eosin - safran or with antibodies to B220 or Ki67 . w : white pulp , r : red pulp , cr : compressed red pulp .

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E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.


A - B . Analysis of DCC ( A ) and netrin - 1 ( B ) expression in a cohort of normal tonsils ( n = 9 ) , Germinal - Center - B - Cell - like - DLBCL ( n = 13 ) , Activated - B - Cell - like - DLBCL ( n = 18 ) and MCL ( n = 36 ) . Quantification of genes expression was performed by Q - RT - PCR , relatively to HPRT housekeeping gene . * * : p < 0 . 005 ; * * * * : p < 0 . 0001 ; two - sided Mann - Whitney U - test .

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A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.


C - D . Analysis of netrin - 1 expression in DLBCL ( C ) and MCL ( D ) by immunohistochemistry . C . 21 DLBCL patients biopsies were used to build a TMA . Normal tissues ( normal tonsils , reactive lymph nodes , muscle , liver ) are included ( the left - most spot , upper panel , corresponds to liver ) . Enlarged representative image of netrin - 1 expression in DLBCL is shown ( lower panel ) . D . Representative images of netrin - 1 expression in paraffin - embedded biopsies of MCL ( n = 9 ) . Five tumors were positive for netrin - 1 ( left panel ) and four were negative ( right panel ) .

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C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).


A . Expression of netrin - 1 and DCC in lymphoma cell lines . Quantification was performed by Q - RT - PCR in 21 lymphoma cell lines . HPRT housekeeping gene was used as a standardization control . Netrin - 1 and DCC levels are indicated as follow : − , not detectable ; + to + + + + , moderate to very high expression . ABC and GC - DLBCL cell lines are highlighted respectively in blue and green , MCL ones are in grey . Expression of netrin - 1 ( in green ) by immunofluorescence using netrin - 1 antibody on OCI - Ly3 and Granta - 519 cell lines is shown on the right panel . Nuclei are counterstained in blue by Hoechst .

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A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.


B . Caspase - 3 activity in Granta - 519 cells after transfection of scrambled siRNA ( si - Ctl ) or netrin - 1 siRNA ( si - netrin - 1 ) , with or without DCC siRNA ( si - DCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 03 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test .

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B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.


D . Effect of net - 1 mAb on Granta - 519 ( left panel ) and OCI - Ly3 ( right panel ) cell density . Results are means + / - std indexed to control of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 03 ; two - sided Mann - Whitney U - test .

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D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.


E . Effect of net - 1 mAb on induction of Granta - 519 cells apoptosis , detected by TUNEL staining . Left panel : representative images are shown . TUNEL positive cells are labelled in red . Nuclei are counterstained in blue by Hoechst staining . Right panel : quantification of one representative experiment out of three performed is exposed . Results are presented as percentage of TUNEL - positive cells per field . * : p = 0 . 04 ; two - sided Mann - Whitney U - test .

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

E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.


F . Caspase - 3 activity in Granta - 519 cells treated with net - 1 mAb antibody or with an unrelated Ig - G1 antibody ( Ctl ) , with or without silencing of DCC via siRNA ( siDCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test .

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

F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.


G - H . Effect of netrin - 1 interfering antibody net - 1 mAb on OCI - Ly3 ( G ) and Granta - 519 ( H ) xenograft tumors growth . Results are presented as means + / - 95 % CIs of tumor growth rates from the beginning of treatment . * : p = 0 . 02 ; * * : p = 0 . 01 ; $ : p = 0 . 007 ; $ $ : p = 0 . 002 ; * * * : p = 0 . 0002 ; * * * * : p < 0 . 0001 ; two - sided Student t - test ( Granta - 519 ) or Welch t - test ( OCI - Ly3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3).


I . Caspase - 3 activity in tumors from control ( n = 4 ) or netrin - 1 - interfering antibody ( n = 4 ) treated mice . Tumors resection was performed at the end of the treatment . Results are means + / - std of caspase - 3 activity . * : p = 0 . 02 ; two - sided Mann - Whitney test .

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

I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.


( B ) Volcano plot showing differentially expressed genes between WT ( WT1 and WT2 ) and patient ( V . 1 ( F1 ) , V . 5 ( F1 ) ) primary dermal fibroblasts . The vertical axis ( y - axis ) shows the - log10 P - value , whereas the horizontal axis ( x - axis ) displays the log2 fold change value . The red dots represent the upregulated transcripts ; the blue dots represent the downregulated transcripts . A total of 172 genes were found significantly dysregulated . TAPT1 , a gene located in the IBD region , appeared among the most significantly downregulated genes in the patients .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot showing differentially expressed genes between WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the -log10 P-value, whereas the horizontal axis (x-axis) displays the log2 fold change value. The red dots represent the upregulated transcripts; the blue dots represent the downregulated transcripts. A total of 172 genes were found significantly dysregulated. TAPT1, a gene located in the IBD region, appeared among the most significantly downregulated genes in the patients.


( B ) qPCR results using specific primers for TAPT1 and TAPT1 - AS1 in 3 WT ( WT1 , WT2 , V . 2 ( F1 ) ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts . TAPT1 mRNA is significantly reduced in all patients compared to WTs , whereas TAPT1 - AS1 transcript levels are unaffected . Fold change relative to V . 2 ( F1 ) is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) .

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(B) qPCR results using specific primers for TAPT1 and TAPT1-AS1 in 3 WT (WT1, WT2 , V.2 (F1)) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts. TAPT1 mRNA is significantly reduced in all patients compared to WTs, whereas TAPT1-AS1 transcript levels are unaffected. Fold change relative to V.2 (F1) is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).


( C ) Western blot analysis of endogenous TAPT1 protein ( ~ 60 kDa ) using whole protein extracts from primary dermal fibroblasts from WT ( WT1 and WT2 ) , heterozygous ( IV . 3 ( F1 ) ) and homozygous ( V . 1 ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) individuals and two different commercial antibodies ( top : Sigma , HPA042567 ; bottom : Sigma , HPA048658 ) . Results show a complete absence of TAPT1 protein in patient samples . GAPDH was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of endogenous TAPT1 protein (~60 kDa) using whole protein extracts from primary dermal fibroblasts from WT (WT1 and WT2), heterozygous (IV.3 (F1)) and homozygous (V.1 (F1), V.5 (F1) and IV.1 (F2)) individuals and two different commercial antibodies (top: Sigma, HPA042567; bottom: Sigma, HPA048658). Results show a complete absence of TAPT1 protein in patient samples. GAPDH was used as a loading control.


( D ) qPCR analysis of TAPT1 expression in 3 WT ( WT1 , WT2 , WT3 ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts treated with cycloheximide ( CHX ) . CHX was used to block nonsense mediated decay ( NMD ) . Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells . For each graph , fold change relative to non - treated condition is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) .

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

(D) qPCR analysis of TAPT1 expression in 3 WT (WT1, WT2, WT3) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts treated with cycloheximide (CHX). CHX was used to block nonsense mediated decay (NMD). Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells. For each graph, fold change relative to non-treated condition is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).


( B ) Western blot analyses for TAPT1 ( ~ 60 kDa ) using cytosolic , Mito / ER / Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs ( WT1 and WT2 ) and two patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . TAPT1 protein is highly enriched in the Mito / ER / Golgi fraction , and to a lower extent in the nuclear fraction . GAPDH , TGN46 and BiP served as a cytosolic , Golgi network and ER markers , respectively . Adenylate Kinase ( AK2 ) was used as a mitochondrial marker . Laminin A / C was used as a nuclear marker .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analyses for TAPT1 (~60 kDa) using cytosolic, Mito/ER/Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs (WT1 and WT2) and two patients (V.5 (F1) and IV.1 (F2)). TAPT1 protein is highly enriched in the Mito/ER/Golgi fraction, and to a lower extent in the nuclear fraction. GAPDH, TGN46 and BiP served as a cytosolic, Golgi network and ER markers, respectively. Adenylate Kinase (AK2) was used as a mitochondrial marker. Laminin A/C was used as a nuclear marker.


( C ) Immunofluorescence staining of mitochondria using anti - TOM20 ( green ) , ER using anti - CANX ( red ) and Golgi using anti - GLG1 ( red ) in primary dermal fibroblasts from WT1 and V . 5 ( F1 ) . Similar staining patterns are observed with the three antibodies in both cell lines . Scale bar represents 10 µm .

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

(C) Immunofluorescence staining of mitochondria using anti-TOM20 (green), ER using anti-CANX (red) and Golgi using anti-GLG1 (red) in primary dermal fibroblasts from WT1 and V.5 (F1). Similar staining patterns are observed with the three antibodies in both cell lines. Scale bar represents 10 µm.


( D ) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients ( - / - ) ( V . I ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) primary dermal fibroblasts compared to WTs ( + / + ) ( WT1 , WT2 and WT3 ) cells . Fold change relative to WT is plotted as mean ± SD . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients (-/-) (V.I (F1), V.5 (F1) and IV.1 (F2)) primary dermal fibroblasts compared to WTs (+/+) (WT1, WT2 and WT3) cells. Fold change relative to WT is plotted as mean ± SD. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).


( E ) CMV cell infection assay on 2 patient ( V . 1 ( F1 ) and V . 5 ( F1 ) ) and 3 WT ( WT1 , WT4 and WT5 ) primary dermal fibroblast cell lines , using β - galactosidase activity as a readout . MRC5 cell line was used as a positive control . All of the cells were infected by the HCMV strain RC256 at a MOI = 0 . 1 . Data are shown as mean ± SD of three technical replicates . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) .

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

(E) CMV cell infection assay on 2 patient (V.1 (F1) and V.5 (F1)) and 3 WT (WT1, WT4 and WT5) primary dermal fibroblast cell lines, using β-galactosidase activity as a readout. MRC5 cell line was used as a positive control. All of the cells were infected by the HCMV strain RC256 at a MOI=0.1. Data are shown as mean ± SD of three technical replicates. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).


( A ) Volcano plot showing differentially expressed genes as determined by RNA - seq in patient primary dermal fibroblasts ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) cells . The y - axis shows the - log10 P - value , whereas the x - axis displays the log2 fold change value . The red dots represent 75 significantly upregulated genes , and the blue dots represent 97 significantly downregulated genes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot showing differentially expressed genes as determined by RNA-seq in patient primary dermal fibroblasts (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) cells. The y-axis shows the -log10 P-value, whereas the x-axis displays the log2 fold change value. The red dots represent 75 significantly upregulated genes, and the blue dots represent 97 significantly downregulated genes.