quantification of macrophages ( E ) in the germband or ( F ) on the yolk next to the germband from control embryos and those expressing a dominant negative c - ring of ATP synthase ( CV - DN ) in macrophages . from Stage 12 embryos For ( E ) control ( n = 24 embryos ) vs CV - DN ( n = 20 ) p = 0 . 0032 . For ( F ) control ( n = 21 embryos ) vs CV - DN ( n = 17 ) p = 0 . 003 . Data information : show Stage 12 embryos . Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Unpaired t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 macrophages (E) in the germband or (F) on the yolk next to the germband from control embryos and those expressing a dominant negative c-ring of ATP synthase (CV-DN) in macrophages. from Stage 12 embryos For (E) control (n=24 embryos) vs CV-DN (n=20) p=0.0032. For (F) control (n=21 embryos) vs CV-DN (n=17) p=0.003. Data information: show Stage 12 embryos. Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests


Confocal images of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III ( or an RNAi against Complex V . Data information : show Stage 12 embryos . Complex III RNAis target Cyt - c1 , UQCR - cp1 - 2 , for Complex V , ATP synthase F1F0 . Scale bars :

O O O O O O O O O O O O O O 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 I-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

Confocal images of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III (or an RNAi against Complex V. Data information: show Stage 12 embryos. Complex III RNAis target Cyt-c1, UQCR-cp1-2, for Complex V, ATP synthase F1F0. Scale bars: 50µm


quantification of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III ( or an RNAi against Complex V . For ( H ) control ( n = 34 embryos ) vs Complex III RNAi 1 ( n = 20 ) p = 0 . 0001 ; vs . Complex III RNAi 2 ( n = 18 ) p = 0 . 027 ; vs . Complex III RNAi 3 ( n = 16 ) p < 0 . 0001 ; vs . Complex V RNAi ( n = 14 ) p < 0 . 0001 . Data information : show Stage 12 embryos . Complex III RNAis target Cyt - c1 , UQCR - cp1 - 2 , for Complex V , ATP synthase F1F0 . Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . one - way ANOVA with Tukey ' s ( H ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-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

quantification of germband macrophages in control embryos and those expressing different RNAis against mitochondrial OxPhos Complex III (or an RNAi against Complex V. For (H) control (n=34 embryos) vs Complex III RNAi 1 (n=20) p=0.0001; vs. Complex III RNAi 2 (n=18) p=0.027; vs. Complex III RNAi 3 (n=16) p<0.0001; vs. Complex V RNAi (n=14) p<0.0001. Data information: show Stage 12 embryos. Complex III RNAis target Cyt-c1, UQCR-cp1-2, for Complex V, ATP synthase F1F0. Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. one-way ANOVA with Tukey's (H).


I Single plane confocal microscope image during germband entry from control or atosPBG embryos , or those expressing pths - RNAi or CV - DN in macrophages . Antibodies against S293 - phosphorylated inactivated Pyruvate Dehydrogenase ( pPDH , green ) or total PDH ( magenta ) in macrophages ( red ) . Higher pPDH levels are usually found when ATP / ADP levels are high and input into the TCA cycle is being downregulated Data information : show Stage 12 embryos . Scale bars :

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

I Single plane confocal microscope image during germband entry from control or atosPBG embryos, or those expressing pths-RNAi or CV-DN in macrophages. Antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH levels are usually found when ATP/ADP levels are high and input into the TCA cycle is being downregulated Data information: show Stage 12 embryos. Scale bars: 10µm


J Quantification of normalized pPDH / PDH levels calculated from fluorescence intensities in macrophages from the genotypes in ( I ) during initial germband invasion . The pPDH / PDH ratio is significantly reduced , arguing that decreased function of CV , Atos or Pths in macrophages results in lower cellular ATP / ADP ratios compared to the control . N = 3 independent experiments . Ctrl 1 ( n = 10 embryos ) vs CV - DN ( n = 9 ) and vs . atos mutant ( n = 13 ) both p = 0 . 0002 ; Ctrl 2 ( n = 7 embryos ) vs pths RNAi ( n = 8 ) p = 0 . 0001 . * * * shown above columns in J are for comparison to relevant control . Data information : Mean ± SEM , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Unpaired t -

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

J Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (I) during initial germband invasion. The pPDH/PDH ratio is significantly reduced, arguing that decreased function of CV, Atos or Pths in macrophages results in lower cellular ATP/ADP ratios compared to the control. N=3 independent experiments. Ctrl 1 (n=10 embryos) vs CV-DN (n=9) and vs. atos mutant (n=13) both p=0.0002; Ctrl 2 (n=7 embryos) vs pths RNAi (n=8) p=0.0001.*** shown above columns in J are for comparison to relevant control. Data information: Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests


A Single plane confocal microscope image during germband entry from control or atosPBG embryos , or those expressing pths , GR / HPR , and LKR / SDH in macrophages ( mac > ) . Embryos were stained for antibodies against S293 - phosphorylated inactivated Pyruvate Dehydrogenase ( pPDH , green ) or total PDH ( magenta ) in macrophages ( red ) . Higher pPDH / PDH ratios are consistent with higher ATP / ADP levels . Data information : Scale bars :

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

A Single plane confocal microscope image during germband entry from control or atosPBG embryos, or those expressing pths, GR/HPR, and LKR/SDH in macrophages (mac>). Embryos were stained for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH/PDH ratios are consistent with higher ATP/ADP levels. Data information: Scale bars: 10µm


B Quantification of normalized pPDH / PDH levels calculated from fluorescence intensities in macrophages from the genotypes in ( A ) during initial germband invasion . The pPDH / PDH ratio is significantly increased in atosPBG embryos expressing either pths , GR / HPR , or LKR / SDH in macrophages compared to the atosPBG embryos . This argues that the decreased Atos function in atosPBG macrophages , resulting in lower cellular ATP / ADP ratios , was restored by expressing either its targets or murine orthologs . N = 3 independent experiments . Control ( n = 9 embryos ) vs . atos mutant ( n = 10 ) p < 0 . 0001 . Control vs . atos mutant rescued with mac > pths ( n = 10 ) p = 0 . 77 ; rescued with mac > GRHPR ( n = 9 ) p = 0 . 48 ; rescued with mac > LKRSDH ( n = 10 ) p = 0 . 012 . For atos mutant vs . atos rescued with mac > pths or mac > GRHPR p < 0 . 0001 . vs . atos rescued with mac > LKRSDH p = 0 . 0014 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . One - way ANOVA with

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

B Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (A) during initial germband invasion. The pPDH/PDH ratio is significantly increased in atosPBG embryos expressing either pths, GR/HPR, or LKR/SDH in macrophages compared to the atosPBG embryos. This argues that the decreased Atos function in atosPBG macrophages, resulting in lower cellular ATP/ADP ratios, was restored by expressing either its targets or murine orthologs. N=3 independent experiments. Control (n=9 embryos) vs. atos mutant (n=10) p<0.0001. Control vs. atos mutant rescued with mac>pths (n=10) p=0.77; rescued with mac>GRHPR (n=9) p=0.48; rescued with mac>LKRSDH (n=10) p=0.012. For atos mutant vs. atos rescued with mac>pths or mac>GRHPR p<0.0001. vs. atos rescued with mac>LKRSDH p=0.0014. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey


C - D ( C ) Confocal images or ( D ) quantification of the macrophages in germband in Stage 12 embryos from the control , atosPBG , and atosPBG expressing atos itself or GR / HPR , or LKR / SDH in macrophages . Germband edge : dotted white line . For ( D ) control ( n = 29 embryos ) vs . atos mutant ( n = 19 ) p < 0 . 0001 . Control vs . atos mutant rescued with mac > atos ( n = 27 embryos ) p = 0 . 99 ; rescued with mac > GRHPR ( n = 28 ) p = 0 . 29 ; rescued with mac > LKRSDH ( n = 20 ) p = 0 . 036 . atos mutant vs . atos rescued with mac > atos or mac > GRHPR p < 0 . 0001 ; rescued with mac > LKRSDH p = 0 . 0134 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . One - way ANOVA with Tukey Scale bars :

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

C-D (C) Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing atos itself or GR/HPR, or LKR/SDH in macrophages. Germband edge: dotted white line. For (D) control (n=29 embryos) vs. atos mutant (n=19) p<0.0001. Control vs. atos mutant rescued with mac>atos (n=27 embryos) p=0.99; rescued with mac>GRHPR (n=28) p=0.29; rescued with mac>LKRSDH (n=20) p=0.036. atos mutant vs. atos rescued with mac>atos or mac>GRHPR p<0.0001; rescued with mac>LKRSDH p=0.0134. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey Scale bars: 50µm


E Single plane confocal microscope images during germband entry from control or atosPBG embryos , or those expressing mammalian orthologs mFAM214A or B ( FAMA - B ) in macrophages ( mac > ) . Embryos were stained for antibodies against S293 - phosphorylated inactivated Pyruvate Dehydrogenase ( pPDH , green ) or total PDH ( magenta ) in macrophages ( red ) . Data information : Scale bars :

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-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 B-MEASURED_VAR I-MEASURED_VAR 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

E Single plane confocal microscope images during germband entry from control or atosPBG embryos, or those expressing mammalian orthologs mFAM214A or B (FAMA-B) in macrophages (mac>). Embryos were stained for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Data information: Scale bars: 10µm


F Quantification of normalized pPDH / PDH levels measured from fluorescence intensities in macrophages from the genotypes in ( E ) during initial germband invasion . The pPDH / PDH ratio is significantly increased in atosPBG embryos expressing either mammalian ortholog mFAM214A or B in macrophages compared to the atosPBG embryos . Results from three independent experiments . Control ( n = 10 embryos ) vs . atos mutant ( n = 11 ) p < 0 . 0001 . Control vs . atos mutant rescued with mac - mFAMA ( n = 14 embryos ) p = 0 . 06 ; rescued with mac - mFAMB ( n = 12 ) p = 0 . 13 . atos mutant vs . atos mutant rescued with mac - mFAMA p = 0 . 0025 or mac - mFAMB p = 0 . 0019 . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . One - way ANOVA with

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

F Quantification of normalized pPDH/PDH levels measured from fluorescence intensities in macrophages from the genotypes in (E) during initial germband invasion. The pPDH/PDH ratio is significantly increased in atosPBG embryos expressing either mammalian ortholog mFAM214A or B in macrophages compared to the atosPBG embryos. Results from three independent experiments. Control (n=10 embryos) vs. atos mutant (n=11) p<0.0001. Control vs. atos mutant rescued with mac-mFAMA (n=14 embryos) p=0.06; rescued with mac-mFAMB (n=12) p=0.13. atos mutant vs. atos mutant rescued with mac-mFAMA p=0.0025 or mac-mFAMB p=0.0019. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . ( B ) Normalized ATP / ADP ratio values are decreased in atosPBG compared to control embryos ( p = 0 . 028 ) . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . unpaired t -

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

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (B) Normalized ATP/ADP ratio values are decreased in atosPBG compared to control embryos (p=0.028). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . Heatmap of non - targeted metabolites in atosPBG compared to wild - type embryos shown with average log2fold change ( FC ) in the non - targeted LC - MS / MS analysis reveals ( C ) an increase in atosPBG in substrates of the dGR / HPR enzyme , including 4 - hydroxy α - ketoglutarate and hydroxyproline ( HLP ) and a smaller decrease in its products , glycolate and glycerate , ( D ) a significant increase in some dipeptides including those containing hydroxyproline . Data information : Values in heat maps are obtained from untargeted metabolomic analysis , unpaired t -

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

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis reveals (C) an increase in atosPBG in substrates of the dGR/HPR enzyme, including 4-hydroxy α-ketoglutarate and hydroxyproline (HLP) and a smaller decrease in its products, glycolate and glycerate, (D) a significant increase in some dipeptides including those containing hydroxyproline. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . ( E ) Quantification shows an increase in atosPBG in the pyruvate / glucose ratio ( p = 0 . 035 ) , but none for the lactate / glucose ratio ( p = 0 . 65 ) . Data information : Mean ± SEM , ns = p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . unpaired t -

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

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (E) Quantification shows an increase in atosPBG in the pyruvate/glucose ratio (p=0.035), but none for the lactate/glucose ratio (p=0.65). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test


Cellular metabolites were measured by LC - MS - based untargeted metabolomics from extracts of Stage 11 embryos ; biological replicates for control n = 5 , for atosPBG n = 7 . Heatmap of non - targeted metabolites in atosPBG compared to wild - type embryos shown with average log2fold change ( FC ) in the non - targeted LC - MS / MS analysis ( F ) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β - oxidation ( FAO ) , including different carnitine - conjugated lipids , and ( F - G ) a significant increase in ketone body substituents compared to the control . Data information : Values in heat maps are obtained from untargeted metabolomic analysis , unpaired t -

O O O O 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-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 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 O O O O O O O O O O O O O O O O O O O O O O O

Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis (F) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β-oxidation (FAO), including different carnitine-conjugated lipids, and (F-G) a significant increase in ketone body substituents compared to the control. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test


C . Kaplan - Meier Survival plot showing low survival of chd7 mutants after 12 dpf ( N = 5 ) .

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

C. Kaplan-Meier Survival plot showing low survival of chd7 mutants after 12 dpf (N=5).


E . Acetylated tubulin staining in 28 hpf controls ( left ) and mutants ( right ) showing severely affected outbranching of the trigeminal nerve ( Vth cranial nerve ) . Notably , chd7 - / - display reduced branching of the Vth cranial nerve ( arrows ) and axonal arborisation in the tectal area . Graphs showing quantitative analyses of percentage ( n = 5 ) and mean total length of peripheral projections ( n = 6 ) per zebrafish in controls and mutants ( * * * p < 0 . 001 ; * * p < 0 . 005 , Student ' s t - test ) .

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 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

E. Acetylated tubulin staining in 28 hpf controls (left) and mutants (right) showing severely affected outbranching of the trigeminal nerve (Vth cranial nerve). Notably, chd7-/- display reduced branching of the Vth cranial nerve (arrows) and axonal arborisation in the tectal area. Graphs showing quantitative analyses of percentage (n=5) and mean total length of peripheral projections (n=6) per zebrafish in controls and mutants (***p<0.001; **p<0.005, Student's t-test).


C - H Total number of GABAergic neurons ( GFP + cells ) in ( C , D ) the optic tectum ( OT ) and cerebellum ( CB ) regions of 5 dpf wild - type and chd7 mutant fish ( n = 16 ; * * * * p < 0 . 0001 ; Student ' s unpaired t - test ) , ( E , F ) the hypothalamus ( hyp ) region ( n = 10 ; * p = 0 . 0182 ; Student ' s t - test ) and ( G , H ) the telencephalon ( tel ) ( n = 7 ; * p = 0 . 0347 ; Student ' s t - test ) .

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

C-H Total number of GABAergic neurons (GFP+ cells) in (C, D) the optic tectum (OT) and cerebellum (CB) regions of 5 dpf wild-type and chd7 mutant fish (n=16; ****p<0.0001; Student's unpaired t-test), (E, F) the hypothalamus (hyp) region (n=10; *p=0.0182; Student's t-test) and (G, H) the telencephalon (tel) (n=7; *p=0.0347; Student's t-test).


F - G . Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron ( n = 10 - 15 ; * * p < 0 . 05 ; * * * * p < 0 . 0001 , one - way ANOVA ) .

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F-G. Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron (n=10-15; **p<0.05; ****p<0.0001, one-way ANOVA).


A . qPCR analysis of PAQR3 expression in a CHD7 - mutation positive patient compared to parental controls set ( N = 4 ; * * * * p < 0 . 0001 ; Student ' s t - test ) . Fold change was calculated according to the 2 ( − ΔΔCt ) method , using HPRT1 and RPS1 as housekeeping genes for normalization . All data were expressed as mean fold change ± SD across replicates , relative to control parents set to 1 ( dotted line ) , N is the number of experimental repeats .

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A. qPCR analysis of PAQR3 expression in a CHD7-mutation positive patient compared to parental controls set (N=4; ****p<0.0001; Student's t-test). Fold change was calculated according to the 2(−ΔΔCt) method, using HPRT1 and RPS1 as housekeeping genes for normalization. All data were expressed as mean fold change ± SD across replicates, relative to control parents set to 1 (dotted line), N is the number of experimental repeats.


C . ChIP - qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7 - mutation positive patient ( N = 3 ) compared to parental controls ( N = 6 ) ; * p < 0 . 05 ; Student ' s t - test , All data were expressed as mean fold change ± SD , N is the number of experimental repeats .

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C. ChIP-qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7-mutation positive patient (N=3) compared to parental controls (N=6); *p< 0.05; Student's t-test, All data were expressed as mean fold change ± SD, N is the number of experimental repeats.


A - B . GABAergic neuron defects in wild - type fish treated with ER / Golgi traffic inhibitor BFA ( n = 9 ; * * * * p < 0 . 0001 ; ns , not significant ; One - way ANOVA ) .

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A-B. GABAergic neuron defects in wild-type fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA).


C - D . Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish ( n = 7 for chd7 - / - and n = 11 for chd7 - / - + paqr3b mRNA ; * * * * p < 0 . 0001 ; * * p < 0 . 05 ; One - way ANOVA ) . Of note , the rescue experiment was performed at 3 dpf given the transient nature of mRNA .

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C-D. Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish (n=7 for chd7-/- and n=11 for chd7-/- + paqr3b mRNA; ****p<0.0001; **p<0.05; One-way ANOVA). Of note, the rescue experiment was performed at 3 dpf given the transient nature of mRNA.


B . Lifespan analyses of chd - 7 ( gk290 ) mutants ( n = 339 ; red ) compared to WT ( n = 444 ; black ) . Log - rank test was performed for statistical analyses . * * * p < 0 . 001 .

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B. Lifespan analyses of chd-7(gk290) mutants (n=339; red) compared to WT (n=444; black). Log-rank test was performed for statistical analyses. ***p < 0.001.


D - F . Example pictures of the GABAergic nervous system cell bodies , commissures , axonal gaps and axonal breaks larger than 50 µm per worm in chd - 7 ( gk290 ) mutants expressing unc - 47p : : mCherry .

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D-F. Example pictures of the GABAergic nervous system cell bodies, commissures, axonal gaps and axonal breaks larger than 50 µm per worm in chd-7(gk290) mutants expressing unc-47p::mCherry.


O . Representative images of GABAergic neurons and total number of GABAergic neurons ( GFP + cells ) in the optic tectum ( OT ) and cerebellum ( CB ) regions of wild - type , chd7 mutant and ephedrine - treated fish ( n = 9 ; * * * * p < 0 . 0001 ; One - way ANOVA ) . Treatment with ephedrine ameliorated the number of GABAergic neurons .

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O. Representative images of GABAergic neurons and total number of GABAergic neurons (GFP+ cells) in the optic tectum (OT) and cerebellum (CB) regions of wild-type, chd7 mutant and ephedrine-treated fish (n=9; ****p<0.0001; One-way ANOVA). Treatment with ephedrine ameliorated the number of GABAergic neurons.


ChEP analysis using EGFP ( control ) - or Piwi - KD OSCs . Signals with an adjusted p - value ( Bonferroni test ) of less than 0 . 05 are highlighted in red . The signals for Lam , LamC , and H1 are labelled within the plots .

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ChEP analysis using EGFP (control)- or Piwi-KD OSCs. Signals with an adjusted p-value (Bonferroni test) of less than 0.05 are highlighted in red. The signals for Lam, LamC, and H1 are labelled within the plots.


Lam - or LamC - KD followed by qRT - PCR of RNA levels for piRNA target ( mdg1 and gypsy ) or non - target ( roo ) TEs . Error bars indicate SD ( n = 3 ) . * = p - value < 0 . 05 ( t - test ) . Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells .

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Lam- or LamC-KD followed by qRT-PCR of RNA levels for piRNA target (mdg1 and gypsy) or non-target (roo) TEs. Error bars indicate SD (n=3). * = p-value<0.05 (t-test). Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells.


Boxplots showing fold changes in the expression of piRNA target or non - target TEs based on RNA - seq upon Piwi - KD ( left ) , and the frequency of piRNAs , which target piRNA target TEs or non - piRNA target TEs ( right ) . Definition of piRNA target and non - target TEs is in Fig EV1E . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test .

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Boxplots showing fold changes in the expression of piRNA target or non-target TEs based on RNA-seq upon Piwi-KD (left), and the frequency of piRNAs, which target piRNA target TEs or non-piRNA target TEs (right). Definition of piRNA target and non-target TEs is in Fig EV1E. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.


Boxplot showing DamID - seq signals of piRNA target or non - target TEs upon EGFP ( control ) - or Piwi - KD . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test .

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Boxplot showing DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Piwi-KD. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.


Oligo - FISH images for the representative piRNA target region described in Fig 4A , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin immunofluorescence staining . Scale bar , 5μm . Ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Oligo - FISH images for changes in the intra - TAD interaction at the TAD described in ( C ) , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , green and magenta indicates FISH signal at the different positions of TADs ( TAD - 1 and TAD - 2 ) . Details of TAD probe design is described in Fig EV2C . Scale bar , 5μm . Distance between two FISH signals is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Boxplots showing fold changes in the histone mark association of piRNA target or non - target TEs based on ChIP - seq upon Piwi - KD ( Definition of piRNA target and non - target TEs are described in Fig EV1E ) . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated by Wilcoxon rank - sum test .

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Boxplots showing fold changes in the histone mark association of piRNA target or non-target TEs based on ChIP-seq upon Piwi-KD (Definition of piRNA target and non-target TEs are described in Fig EV1E). Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated by Wilcoxon rank-sum test.


ChIP ‐ qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN - Nxf2 expression vector or control EGFP expression vector . Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid . Error bars indicate SD ( n = 3 ) , * = p - value < 0 . 01 ( t - test ) . .

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ChIP‐qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN-Nxf2 expression vector or control EGFP expression vector. Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid. Error bars indicate SD (n = 3), * = p-value<0.01 (t-test)..


Immunoprecipitation ( IP ) from OSC lysate using anti - Lam , or myc antibody , followed by WB using indicated antibodies . Mouse immunoglobulin G ( n . i . ) was used for control IP . Note that OSCs used at the right panel is a line which stably expresses myc - Nxf2 .

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Immunoprecipitation (IP) from OSC lysate using anti-Lam, or myc antibody, followed by WB using indicated antibodies. Mouse immunoglobulin G (n.i.) was used for control IP. Note that OSCs used at the right panel is a line which stably expresses myc-Nxf2.


Density plots for DamID - seq signals over consensus sequences from piRNA target ( gypsy and mdg1 , orange ) and non - target TEs ( roo , blue ) in EGFP ( control ) - or Nxf2 - KD cells .

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Density plots for DamID-seq signals over consensus sequences from piRNA target (gypsy and mdg1, orange) and non-target TEs (roo, blue) in EGFP (control)- or Nxf2-KD cells.


Oligo - FISH images for the reporter of the tethering system , at 0h ( control ) , 48h hours , 96 hours after transfection of λN - Nxf2 expression vector . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin staining . Scale bar , 5μm . The ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Oligo-FISH images for the reporter of the tethering system, at 0h (control), 48h hours, 96 hours after transfection of λN-Nxf2 expression vector. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin staining. Scale bar, 5μm. The ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in intra - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 ) Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in inter - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 )

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Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05)


Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours . Scale bar : 10µm Morphological parameters quantification within 48 hours after LPS

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Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours. Scale bar: 10µm Morphological parameters quantification within 48 hours after LPS injection


Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection . Scale bar :

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Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection. Scale bar: 20µm


Morphological parameters quantification for 5 and 15 months old mice after LPS injection


Two - photon representative images of wild - type and Nlrp3 - / - mice ( 5 and 15 months old ) . Scale bar : 20µm . Quantification of morphological parameters for wild - type and Nlpr3 - / - mice after LPS

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Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm. Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection


Representative cortical images of MXO4 staining for APP and APP / Nlrp3 - / - 15 months old mice . Scale bar : 50µm .

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Representative cortical images of MXO4 staining for APP and APP/Nlrp3-/- 15 months old mice. Scale bar: 50µm.


Cortical amyloid plaque number and size quantification , and Amyloid - beta1 - 40 and 1 - 42 ELISA quantification for APP and APP / Nlrp3 - / - mice ( 5 and 15 month old )

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Cortical amyloid plaque number and size quantification, and Amyloid-beta1-40 and 1-42 ELISA quantification for APP and APP/Nlrp3-/- mice (5 and 15 month old)


Two - photon images of microglia ( e - GFP ) cells clustering around amyloid plaque for APP and APP / Nlrp3 - / - ( 5 and 15 months old ) . Scale bar : 20µm b ) Quantification of morphological parameters in ( a ) ( mean of 5 - 6 ± SEM ) .

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Two-photon images of microglia (e-GFP) cells clustering around amyloid plaque for APP and APP/Nlrp3-/- (5 and 15 months old). Scale bar: 20µm b) Quantification of morphological parameters in (a) (mean of 5-6±SEM).


Flow cytometry plots from APP and APP / Nlrp3 - / - mice ( 15 months old ) , cells were gated on CD11b and MXO4 after microglia isolation . d ) Relative Aβ microglia uptake

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Flow cytometry plots from APP and APP/Nlrp3-/- mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. d) Relative Aβ microglia uptake quantification


Two - photon microglia ( e - GFP ) images from APP and APP / Nlrp3 - / - in areas free of plaque ( 5 and 15 months old mice ) . Scale bar : 20µm Quantification of APP and APP / Nlrp3 - / - morphological parameters for microglia in areas free of

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Two-photon microglia (e-GFP) images from APP and APP/Nlrp3-/- in areas free of plaque (5 and 15 months old mice). Scale bar: 20µm Quantification of APP and APP/Nlrp3-/- morphological parameters for microglia in areas free of plaque


Iba - 1 ( green ) , CD169 ( red ) and MXO4 staining in plaque - associated areas ( cortex and hippocampus ) of 5 and 15 months old of APP and APP / Nlrp3 - / - . Note the colocalization between Iba - 1 and CD169 ( white arrows ) in APP 15 - month - old mice 2 days post LPS injection . Scale bar :

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Iba-1 (green), CD169 (red) and MXO4 staining in plaque-associated areas (cortex and hippocampus) of 5 and 15 months old of APP and APP/Nlrp3-/-. Note the colocalization between Iba-1 and CD169 (white arrows) in APP 15-month-old mice 2 days post LPS injection. Scale bar: 20µm


Iba - 1 , Ki67 and DAPI staining in the cortex and hippocampus of wild - type and Nlrp3 - / - ( 5 and 15 months old ) . Microglia proliferates upon LPS injection ( white arrows ) . Non - microglia cells proliferation was observed as well ( yellow arrows ) Scale bar : 20µm . Quantification of microglial proliferation in cortex ( left panel ) and hippocampus ( right panel ) ( mean of 5 ± SEM ; two - way ANOVA followed by Tukey ' s post hoc test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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Iba-1, Ki67 and DAPI staining in the cortex and hippocampus of wild-type and Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows) Scale bar: 20µm. Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).


Iba - 1 , Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP / Nlrp3 - / - ( 5 and 15 months old ) . Microglia proliferates upon LPS injection ( white arrows ) . Non - microglia cells proliferation was observed as well ( yellow arrows ) . Scale bar : 20µm Quantification of microglial proliferation in cortex ( left panel ) and hippocampus ( right panel )

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Iba-1, Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP/Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20µm Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel)


( A ) Summary of HMCES recovery upon TAP - MS analyses performed with 293T cells stably expressing SFB - PCNA . Cells were treated with CPT : camptothecin ( 1 μg / ml for 6 h ) ; HU : hydroxyurea ( 2 mM for 16 h ) , UV : ultraviolet light irradiation ( 10 mJ / cm2 harvested after 6 h post - irradiation ) or Thymidine ( 100 mM for 6 h ) . Recovered peptide - spectrum matches ( PSMs ) for PCNA and HMCES are indicated for each condition .

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(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition.


( C ) Immunoprecipitation ( IP ) of SFB - tagged full - length ( FL ) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction .

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(C) Immunoprecipitation (IP) of SFB-tagged full-length (FL) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction.


( D ) GST - PCNA pulldown assay was used to evaluate the interaction between PCNA and full - length HMCES or indicated HMCES deletion mutants .

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(D) GST-PCNA pulldown assay was used to evaluate the interaction between PCNA and full-length HMCES or indicated HMCES deletion mutants.


( E ) aniPOND - Western blotting analysis revealed the presence of HMCES on replication fork . Cells were pulsed with EdU for 10 mins , and click - reaction was performed . In thymidine chase samples , cells were incubated with thymidine for another 30 mins prior to click - reaction .

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(E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction.


( F ) Analysis of HMCES level throughout the cell cycle . Cells were synchronized by double thymidine block and released for the indicated time points . CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases .

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(F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.


( A ) Western blotting analysis confirmed HMCES knockout in 293A cells ( 293A HMCES KO ) . Blots also included knockout cells reconstituted with WT SFB - HMCES .

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(A) Western blotting analysis confirmed HMCES knockout in 293A cells (293A HMCES KO). Blots also included knockout cells reconstituted with WT SFB- HMCES.


( B , C ) Colony - forming capacity of HMCES KO cells was compared to that of control wild - type 293A cells . ( C ) Box plot showing colony forming efficiencies of 293A wild - type control and HMCES KO cells . Box limits represent 25th and 75th percentile , centerline shows the median and whiskers extend minimum to maximum . Data include more than three independent repeats performed in duplicates . Student ' s t - test were performed for statistical analysis ( * * * * , p < 0 . 0001 ) .

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(B, C) Colony-forming capacity of HMCES KO cells was compared to that of control wild-type 293A cells. (C) Box plot showing colony forming efficiencies of 293A wild-type control and HMCES KO cells. Box limits represent 25th and 75th percentile, centerline shows the median and whiskers extend minimum to maximum. Data include more than three independent repeats performed in duplicates. Student's t-test were performed for statistical analysis (****, p<0.0001).


( D ) Colonogenic survival assay of 293A wild - type control and HMCES KO cells treated with various concentrations of hydroxyurea ( HU ) was shown . Seeding density with respect to control was indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate .

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(D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate.


( E ) Quantification showing survival fraction of wild - type control ( WT ) and HMCES KO cells upon HU treatment . Data are presented as mean ± s . e . m ( n = 3 , each performed in duplicates ) and student ' s t - test were performed for statistical analysis ( ns , not significant ; * , p < 0 . 05 ) .

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(E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05).


( F ) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation ( IR ) . Data are presented as mean ± s . e . m . Quantifications are from three biological repeats performed in duplicates . Student ' s t - test was performed for statistical analysis ( * , p < 0 . 05 ; * * , p < 0 . 01 ) .

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(F) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation (IR). Data are presented as mean±s.e.m. Quantifications are from three biological repeats performed in duplicates. Student's t-test was performed for statistical analysis (*, p<0.05; **, p<0.01).


( G , H ) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony - forming assay upon exposure to H2O2 . Experiments were repeated three independent times , each time performed in duplicates and data are presented as mean ± s . e . m . Statistical significance for the corresponding experiment was analyzed by student ' s t - test ( * , p < 0 . 05 ) .

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(G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05).


( I ) Bar graph showing sensitivity of WT , HMCES KO and stably reconstituted WT cells towards H2O2 treatment . Experiment was repeated two independent times with technical repeats .

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(I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.


( A , B ) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild - type ( WT ) 293A cells and HMCES knockout ( HMCES KO ) cells ( A ) and quantification of results were shown ( B ) . Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug - dose response curve . Seeding density with respect to control is indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate . ( B ) Data are presented as mean ± s . e . m . and student ' s t - test was performed for statistical analysis ( * * * * , p < 0 . 0001 ) .

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(A, B) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild-type (WT) 293A cells and HMCES knockout (HMCES KO) cells (A) and quantification of results were shown (B). Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug-dose response curve. Seeding density with respect to control is indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate. (B) Data are presented as mean±s.e.m. and student's t-test was performed for statistical analysis (****, p<0.0001).


( C ) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs .

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(C) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs.


( D ) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs .

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(D) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs.


( E ) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III ( APEi ) . Results are presented from two independent experiments and data are presented as mean ± s . e . m .

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(E) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III (APEi). Results are presented from two independent experiments and data are presented as mean±s.e.m.


( F ) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi . Data are presented as mean ± s . e . m ( n = 2 each with technical repeats ) .

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(F) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi. Data are presented as mean±s.e.m (n=2 each with technical repeats).


( G , H ) Western blotting revealed levels of POLH ( G ) and REV1 ( H ) in two independent clones upon selection following transfection with indicated shRNA .

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(G, H) Western blotting revealed levels of POLH (G) and REV1 (H) in two independent clones upon selection following transfection with indicated shRNA.


( I ) Colony formation assay was conducted to reveal proliferation of WT , POLH KD , REV1 KD , HMCES KO , HMCES KO + POLH KD and HMCES KO + REV1 KD cells .

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(I) Colony formation assay was conducted to reveal proliferation of WT, POLH KD, REV1 KD, HMCES KO, HMCES KO + POLH KD and HMCES KO +REV1 KD cells.


( J ) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA . Experiment was repeated two independent times in duplicates .

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(J) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA. Experiment was repeated two independent times in duplicates.


( K ) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector - based assay system . Experiments were repeated three independent times and data are presented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * , p < 0 . 01 ) .

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(K) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector-based assay system. Experiments were repeated three independent times and data are presented as mean±s.e.m. Student's t-test was used to calculate statistical significance (**, p<0.01).


( B ) Volcano plot showing DDR CRISPR - CAS9 screen for HMCES KO cells . - log10 ( p - value ) is plotted against log2 ( HMCES KO / WT ) fold change . Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled .

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(B) Volcano plot showing DDR CRISPR-CAS9 screen for HMCES KO cells. -log10(p-value) is plotted against log2(HMCES KO/ WT) fold change. Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled.


( C ) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library .

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(C) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library.


Western blots showing levels of CtIP ( D ) upon shRNA mediated knockdown in 293A cells .

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Western blots showing levels of CtIP (D) upon shRNA mediated knockdown in 293A cells.


Western blots showing levels of BRCA2 ( E ) upon shRNA mediated knockdown in 293A cells .

Western blots showing levels of BRCA2 (E) upon shRNA mediated knockdown in 293A cells.


Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP ( F ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

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Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP (F) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 ( G ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 (G) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( A ) CD4 + T cells were isolated from the spleen by negative selection before incubating with anti - CD3 / CD28 antibodies for the indicated time periods . Levels of expression of EGR1 , EGR2 , EGR3 and EGR4 were measured by qPCR .

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T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (A) CD4+ T cells were isolated from the spleen by negative selection before incubating with anti-CD3/CD28 antibodies for the indicated time periods. Levels of expression of EGR1, EGR2, EGR3 and EGR4 were measured by qPCR.


T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( B ) WBC were collected from WT , EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition ( gating strategies in Appendix Fig S1 ) . Subclasses of CD8 + , CD4 + and Treg populations were determined as shown .

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T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (B) WBC were collected from WT, EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition (gating strategies in Appendix Fig S1). Subclasses of CD8+, CD4+ and Treg populations were determined as shown.


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . A Cells were incubated under unstimulated conditions or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies for 0 to 5 days ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. A Cells were incubated under unstimulated conditions or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies for 0 to 5 days (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . B Dose dependence of EGR4 - dependent differences in proliferation were also determined in both CD4 + and CD8 + cells aft er treatment with anti - TCRβ ( E , F ) or anti - CD3 / CD28 ( G , H ) at the indicated concentrations before FACS analysis ( Appendix Fig S2 ) ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. B Dose dependence of EGR4-dependent differences in proliferation were also determined in both CD4+ and CD8+ cells aft er treatment with anti-TCRβ (E,F) or anti-CD3/CD28 (G,H) at the indicated concentrations before FACS analysis (Appendix Fig S2) (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).


WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . C , D Generation analysis was then assessed in WT , EGR1 - / - and EGR4 - / - CD4 + ( C ) or CD8 + ( D ) T cells after a 4 day incubation , under unstimulated conditions , or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies . Generation analysis was determined using FlowJo as depicted in Appendix figure S3 ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) . . EGR4 deletion significantly shifted the number of generations as determined by two - way ANOVA ; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions ( p < 0 . 0001 ) .

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WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. C,D Generation analysis was then assessed in WT, EGR1-/- and EGR4-/- CD4+ (C) or CD8+ (D) T cells after a 4 day incubation, under unstimulated conditions, or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies. Generation analysis was determined using FlowJo as depicted in Appendix figure S3 (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).. EGR4 deletion significantly shifted the number of generations as determined by two-way ANOVA; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions (p < 0.0001).


CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( A - F ) Cytokine secretion was measured by ELISA after incubating CD4 + ( A , C , E ) and CD8 + ( B , D , F ) T cells for 48 hrs in resting conditions ( A , B ) or stimulated with either anti - TCRβ ( C , D ) or anti - CD3 / CD28 antibodies ( E , F ) .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (A-F) Cytokine secretion was measured by ELISA after incubating CD4+ (A,C,E) and CD8+ (B,D,F) T cells for 48 hrs in resting conditions (A,B) or stimulated with either anti-TCRβ (C,D) or anti-CD3/CD28 antibodies (E,F).


CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR4KO mice by cell sorting . ( G ) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4 + and CD8 + T cells .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR4KO mice by cell sorting. (G) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4+ and CD8+ T cells.


CD4 + and CD8 + T cells were isolated from the spleens of WT , and EGR4KO mice by cell sorting . ( H ) Comparison of IFNγ levels in WT vs . EGR4 - / - T cells .

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CD4+ and CD8+ T cells were isolated from the spleens of WT, and EGR4KO mice by cell sorting. (H) Comparison of IFNγ levels in WT vs. EGR4-/- T cells.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( I ) Representative FACS plots of CD4 + T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL - 17 antibodies .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (I) Representative FACS plots of CD4+ T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL-17 antibodies.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( J ) Comparison of the number of Th1 polarized cells between WT , EGR1 - / - and EGR4 - / - CD4 + T cells .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (J) Comparison of the number of Th1 polarized cells between WT, EGR1-/- and EGR4-/- CD4+ T cells.


CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( K ) CD4 + T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA .

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CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (K) CD4+ T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA.


( A ) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4 + and CD8 + T cells isolated by negative selection from WT , EGR1KO and EGR4KO mice . Cells were stimulated with anti - CD3 / CD28 antibodies for the indicated time periods before fixation and staining . Nuclear localization of NFATc1 in CD4 + and CD8 + T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis ( mean ± SEM ; data are based upon 13 to 20 cells / data point from one experiment ; experiment was repeated 3 times ) . EGR4 - mediated differences in NFATc1 localization were determined by two - way ANOVA . NFAT localization was dependent upon EGR4 expression in both CD4 + ( p = 0 . 025 ) and CD8 + ( p = 0 . 039 ) T cells . Post - hoc analysis revealed EGR4 - dependent differences at specific timepoints * p < 0 . 05 ; * * * p < 0 . 001 .

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 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 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 O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4+ and CD8+ T cells isolated by negative selection from WT, EGR1KO and EGR4KO mice. Cells were stimulated with anti-CD3/CD28 antibodies for the indicated time periods before fixation and staining. Nuclear localization of NFATc1 in CD4+ and CD8+ T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis (mean±SEM; data are based upon 13 to 20 cells/data point from one experiment; experiment was repeated 3 times). EGR4-mediated differences in NFATc1 localization were determined by two-way ANOVA. NFAT localization was dependent upon EGR4 expression in both CD4+ (p=0.025) and CD8+ (p=0.039) T cells. Post-hoc analysis revealed EGR4-dependent differences at specific timepoints * p < 0.05; *** p < 0.001.


( B ) WT , EGR1KO and EGR4KO CD4 + T cells were isolated from the spleen by negative selection before plating on poly - lysine ( control ) or CD3 / CD28 and loading with fura2 . Ca2 + levels shown are from representative single cells measured from multiple experiments . ( C ) Scatter plots showing area under the curve ( AUC ) and variance for each cell measured under all conditions .

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(B) WT, EGR1KO and EGR4KO CD4+ T cells were isolated from the spleen by negative selection before plating on poly-lysine (control) or CD3/CD28 and loading with fura2. Ca2+ levels shown are from representative single cells measured from multiple experiments. (C) Scatter plots showing area under the curve (AUC) and variance for each cell measured under all conditions.


CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( A ) CD4 + and CD8 + T cells were loaded with Fura - 2AM and plated on poly - D - lysine ( 2 hrs ) before treating with biotinylated anti - CD3ε , followed by streptavidin where indicated . The average response of the majority of CD4 + ( WT 74 . 2 ± 3 . 2 % ; EGR4 - / - 77 . 8 ± 2 . 9 % ) and CD8 + ( WT 79 . 4 ± 3 . 5 % ; EGR4 - / - 78 . 2 ± 2 . 5 % ) cells that responded to streptavidin crosslinking of CD3 ( mean ± SEM ; 40 - 60 cells / coverslip ; 7 to 8 separate experiments ) .

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

CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (A) CD4+ and CD8+ T cells were loaded with Fura-2AM and plated on poly-D-lysine (2 hrs) before treating with biotinylated anti-CD3ε, followed by streptavidin where indicated. The average response of the majority of CD4+ (WT 74.2±3.2%; EGR4-/- 77.8±2.9%) and CD8+ (WT 79.4±3.5%; EGR4-/- 78.2±2.5%) cells that responded to streptavidin crosslinking of CD3 (mean±SEM; 40-60 cells/coverslip; 7 to 8 separate experiments).


CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( B ) Cells were plated on anti - CD3 / CD28 antibodies for 2 hrs ; BTP2 ( 10 μM ) was added where marked by the arrow .

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

CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (B) Cells were plated on anti-CD3/CD28 antibodies for 2 hrs; BTP2 (10 μM) was added where marked by the arrow.


CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( C ) CD4 + T cells were incubated with anti - CD3 / CD28 antibodies for 6 hrs before measuring expression of IFNγ , IL - 2 , IL - 17 and IL - 4 by FACS analysis . Data are mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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CD4+ T cells were isolated by negative selection from WT or EGR4-/-mice. (C) CD4+ T cells were incubated with anti-CD3/CD28 antibodies for 6 hrs before measuring expression of IFNγ, IL-2, IL-17 and IL-4 by FACS analysis. Data are mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( A ) WT or EGR4 - / - cells were loaded with Fura - 2AM and plated on anti - CD3 / CD28 antibodies for 2 hrs . Senicapoc ( 4 μM ) or Tram - 34 ( 5 μM ) were added where marked by the arrow .

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

CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( B ) CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice and incubated with anti - CD3 / CD28 antibodies for 6 hrs in the presence or absence of either senicapoc , TRAM - 34 or Shk - Dap22 before measuring expression of IFNγ levels by FACS analysis . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates . * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001.


CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( C ) To measure the effect of KCa3 . 1 inhibition of cell proliferation , WT and EGR4 - / - CD4 + T cells were isolated from the spleen by cell sorting and stained with CFSE . Cells were incubated with anti - TCRβ antibodies in the presence of vehicle , Senicapoc ( 5 µM ) or TRAM - 34 ( 5 µM ) for 4 days and then collected for FACS analysis . ( D ) Generation analysis was completed for all data using FlowJo software . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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

CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( A ) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections.


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( B - D ) Mice were sacrificed on day 20 and tumors were counted . Data is presented as total / mouse ( B ) , N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (B-D) Mice were sacrificed on day 20 and tumors were counted. Data is presented as total/mouse (B), N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . Mice were sacrificed on day 20 and tumors were counted . ( B ) , lung tumors ( C ) and metastatic tumors ( all tumors found outside of the lungs ; D ) . N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. Mice were sacrificed on day 20 and tumors were counted. (B), lung tumors (C) and metastatic tumors (all tumors found outside of the lungs; D). N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . WT lung tumors were 5 . 407 ± 1 . 843 % CD45 + , while EGR4KO mice were 12 . 445 ± 2 . 239 % CD45 + . WT spleens were 83 . 92 ± 3 . 344 % CD45 + , while EGR4KO mice were 95 . 13 ± 1 . 856 % CD45 + . Relative distributions of T and myeloid cells within the CD45 + populations were determined by flow cytometric analysis . ( E , F ) Distributions of T and myeloid cells found within lung tumors ( E ) vs . the spleen ( F ) .

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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. WT lung tumors were 5.407 ± 1.843% CD45+, while EGR4KO mice were 12.445 ± 2.239% CD45+. WT spleens were 83.92 ± 3.344% CD45+, while EGR4KO mice were 95.13 ± 1.856% CD45+. Relative distributions of T and myeloid cells within the CD45+ populations were determined by flow cytometric analysis. (E,F) Distributions of T and myeloid cells found within lung tumors (E) vs. the spleen (F).


B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . ( G ) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and

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 B-CONTROLLED_VAR 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

B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. (G) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and F


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( A ) CD4 + and CD8 + T cells were stimulated in vitro with ionomycin / PMA before intracellular staining for IFNγ , IL - 17 and IL - 9 and FACS analysis .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( B , C ) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to D


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( D ) Percentages of each population .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( E ) Gating strategies .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( F ) FR4 and PD1 levels in WT vs . EGR4 - / - T cells .

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

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.


a - c , Expression of CD45RO and CCR7 ( a ) , CD45RO and CD27 ( b ) , CD27 and CD28 ( c ) on peripheral blood CD8 T cells of three participants [ P1 ( heterozygous mutation ) , P2 ( homozygous mutation ) and P3 ( heterozygous ) for the c . 192G > C mutation in the DJ - 1 gene ] . d - g , Expression of CD57 ( d ) , PD - 1 ( e ) , EOMES ( f ) and T - bet ( g ) on peripheral blood CD8 T cells of three participants . The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population .

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

a-c, Expression of CD45RO and CCR7 (a), CD45RO and CD27 (b), CD27 and CD28 (c) on peripheral blood CD8 T cells of three participants [P1 (heterozygous mutation), P2 (homozygous mutation) and P3 (heterozygous) for the c.192G>C mutation in the DJ-1 gene]. d-g, Expression of CD57 (d), PD-1 (e), EOMES (f) and T-bet (g) on peripheral blood CD8 T cells of three participants. The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population. h


b - c , Percentages of CD44low CD62Lhigh ( Tn ) ( b ) and CD44high CD62Llow ( Tem ) ( c ) cells among total CD8 T cells of spleen and pLNs from young and 45 - wk - old Dj - 1 KO and WT littermates ( young KO , n = 5 ; young WT , n = 5 ; 45 - wk - old KO , n = 8 ; 45 - wk - old WT , n = 6 ; for 45 - wk - old mice , data pooled from 2 independent experiments ) .

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b-c, Percentages of CD44low CD62Lhigh (Tn) (b) and CD44high CD62Llow (Tem) (c) cells among total CD8 T cells of spleen and pLNs from young and 45-wk-old Dj-1 KO and WT littermates (young KO, n=5; young WT, n=5; 45-wk-old KO, n=8; 45-wk-old WT, n=6; for 45-wk-old mice, data pooled from 2 independent experiments).