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

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

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


( A ) Immunofluorescence microscopy for POMC ( green ) and Cre ( red ; n = 4 ) , ( B ) POMC ( green ) and Atg7 ( red ; n = 4 ) and ( C ) POMC ( green ) and p62 ( red ; n = 6 ) in MBH sections from Con and Atg7F / F ‐ POMC ‐ Cre ( KO )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence microscopy for POMC (green) and Cre (red; n=4), (B) POMC (green) and Atg7 (red; n=4) and (C) POMC (green) and p62 (red; n=6) in MBH sections from Con and Atg7F/F‐POMC‐Cre (KO) mice


( D ) TUNEL ‐ positivity ( green ; n = 4 ) and ( E ) number of POMC ‐ positive neurons ( red ) in MBH sections from Con and KO mice ( n = 6 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . White arrows indicate Atg7 , Cre , p62 or POMC signal . Yellow arrows indicate merged signal . Con , control ; DAPI , 4 ′ ‐ 6 ‐ diamidino ‐ 2 ‐ phenylindole ; KO , knockout ; MBH , mediobasal hypothalamic ; NS , not statistically significant ; POMC , proopiomelanocortin ; TUNEL , TdT ‐ mediated dUTP nick end labelling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TUNEL‐positivity (green; n=4) and (E) number of POMC‐positive neurons (red) in MBH sections from Con and KO mice (n=6). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. White arrows indicate Atg7, Cre, p62 or POMC signal. Yellow arrows indicate merged signal. Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; KO, knockout; MBH, mediobasal hypothalamic; NS, not statistically significant; POMC, proopiomelanocortin; TUNEL, TdT‐mediated dUTP nick end labelling.


( A ) Body wt of 4 ‐ mo ‐ and 12 ‐ mo ‐ old Con and KO mice on RD ( n = 9 - 13 ) or HFD ( n = 5 - 7 ) . ( B ) Total body fat and ( C ) lean mass of 4 ‐ mo ‐ old Con and KO mice on RD ( n = 7 ) and 12 ‐ mo ‐ old Con and KO mice on HFD ( n = 6 - 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body wt of 4‐mo‐ and 12‐mo‐old Con and KO mice on RD (n=9-13) or HFD (n=5-7). (B) Total body fat and (C) lean mass of 4‐mo‐old Con and KO mice on RD (n=7) and 12‐mo‐old Con and KO mice on HFD (n=6-8).


( D ) eWAT wt , ( E ) fat pads and ( F ) liver wt of 4 ‐ mo ‐ old Con and KO mice on RD ( n = 6 - 8 ) .

O O O O O O O O O O O O O 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) eWAT wt, (E) fat pads and (F) liver wt of 4‐mo‐old Con and KO mice on RD (n=6-8).


( G ) Immunoblots for POMC and ACTH , and ( H ) NPY in MBH from RD ‐ fed Con and KO mice ( n = 3 - 4 ) .

O O O O O O O O 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) Immunoblots for POMC and ACTH, and (H) NPY in MBH from RD‐fed Con and KO mice (n=3-4).


( I ) Immunofluorescence microscopy for POMC ( green ) and α ‐ MSH ( red ) , and ( J ) α ‐ MSH ( green ) in MBH from Con and KO mice on RD ( n = 5 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . Yellow arrows indicate merged signal . ACTH , adrenocorticotrophic hormone ; Con , control ; DAPI , 4 ′ ‐ 6 ‐ diamidino ‐ 2 ‐ phenylindole ; eWAT , epididymal fat ; HDF , high ‐ fat diet ; KO , knockout ; MBH , mediobasal hypothalamic ; mo , month ; MSH , melanocyte ‐ stimulating hormone ; NPY , neuropeptide Y ; POMC , proopiomelanocortin ; RD , regular chow ; wt , weight .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence microscopy for POMC (green) and α‐MSH (red), and (J) α‐MSH (green) in MBH from Con and KO mice on RD (n=5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; eWAT, epididymal fat; HDF, high‐fat diet; KO, knockout; MBH, mediobasal hypothalamic; mo, month; MSH, melanocyte‐stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; RD, regular chow; wt, weight.


( A ) Percent loss of body fat and ( B ) lean mass in 24 ‐ h ‐ fasted Con and KO mice on RD or HFD ( n = 6 - 8 ) .

O O O O O O O O O O O O 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) Percent loss of body fat and (B) lean mass in 24‐h‐fasted Con and KO mice on RD or HFD (n=6-8).


( C ) Histology for eWAT from fed or 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 3 - 4 ) .

O O O O 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) Histology for eWAT from fed or 6‐h‐fasted Con and KO mice on RD (n=3-4).


( D ) Serum fatty acids and ( E ) glycerol from fed or 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 7 - 9 ) .

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

(D) Serum fatty acids and (E) glycerol from fed or 6‐h‐fasted Con and KO mice on RD (n=7-9).


( F ) Serum fatty acids and ( G ) glycerol after intraperitoneal isoproterenol ( Iso ) in 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 5 - 6 ) .

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

(F) Serum fatty acids and (G) glycerol after intraperitoneal isoproterenol (Iso) in 6‐h‐fasted Con and KO mice on RD (n=5-6).


( H ) Immunoblot for oxidized proteins in eWAT from RD ‐ fed Con and KO mice ( n = 3 ) . ( I ) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD ( n = 3 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Con , control ; eWAT , epididymal fat ; FACS , fluorescence ‐ activated cell sorting ; HDF , high ‐ fat diet ; Iso , isoproterenol ; KO , knockout ; POMC , proopiomelanocortin ; RD , regular chow ; Stv , fasted .

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

(H) Immunoblot for oxidized proteins in eWAT from RD‐fed Con and KO mice (n=3). (I) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD (n=3). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; eWAT, epididymal fat; FACS, fluorescence‐activated cell sorting; HDF, high‐fat diet; Iso, isoproterenol; KO, knockout; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.


( A ) Blood glucose levels in 6 mo ( n = 7 - 9 ) and ( B ) 12 mo Con and KO mice on RD ( n = 6 ) , and in ( C ) 12 ‐ mo ‐ old Con and KO mice on HFD ( n = 4 ) .

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(A) Blood glucose levels in 6 mo (n=7-9) and (B) 12 mo Con and KO mice on RD (n=6), and in (C) 12‐mo‐old Con and KO mice on HFD (n=4).


( D ) Serum insulin levels in 6 mo , ( n = 7 - 9 ) and ( E ) 12 ‐ mo ‐ old RD ‐ fed Con and KO mice that were fed or 6 h fasted ( n = 6 ) .

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(D) Serum insulin levels in 6 mo, (n=7-9) and (E) 12‐mo‐old RD‐fed Con and KO mice that were fed or 6 h fasted (n=6).


( F ) HOMA values from 6 ‐ h ‐ fasted 6 ‐ mo ‐ old ( n = 7 - 9 ) and ( G ) 12 ‐ mo ‐ old Con and KO mice ( n = 6 ) on RD . ( H ) Glucose tolerance tests in 6 ‐ h ‐ fasted 6 ‐ mo ‐ old mice on RD ( n = 4 ) and in ( I ) 6 ‐ h ‐ fasted 12 ‐ mo ‐ old Con and KO mice fed HFD for 10 mo ( n = 4 ) .

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(F) HOMA values from 6‐h‐fasted 6‐mo‐old (n=7-9) and (G) 12‐mo‐old Con and KO mice (n=6) on RD. (H) Glucose tolerance tests in 6‐h‐fasted 6‐mo‐old mice on RD (n=4) and in (I) 6‐h‐fasted 12‐mo‐old Con and KO mice fed HFD for 10 mo (n=4).


( J ) Insulin tolerance tests in RD ‐ fed 6 ‐ mo ‐ old ( n = 4 ) and ( K ) HFD ‐ fed 12 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

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(J) Insulin tolerance tests in RD‐fed 6‐mo‐old (n=4) and (K) HFD‐fed 12‐mo‐old Con and KO mice (n=4).


( L ) Insulin tolerance tests and ( M ) body weights of 1 ‐ mo ‐ old Con and KO mice on RD ( n = 3 - 5 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Con , control ; HDF , high ‐ fat diet ; HOMA , homeostasis model of insulin resistance ; KO , knockout ; mo , month ; POMC , proopiomelanocortin ; RD , regular chow ; Stv , fasted .

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(L) Insulin tolerance tests and (M) body weights of 1‐mo‐old Con and KO mice on RD (n=3-5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; HDF, high‐fat diet; HOMA, homeostasis model of insulin resistance; KO, knockout; mo, month; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.


( A ) Immunoblots for Atg7 in MBH from young ( 3 mo ) , middle ‐ aged ( 12 mo ) and aged ( 22 mo ) fed and 6 ‐ h ‐ fasted ( Stv ) mice ( n = 4 ) , and ( B ) LC3 ‐ II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors ( Inh ) ( n = 11 - 13 ) . Mean ± s . e . m values for ( B ) steady ‐ state LC3 ‐ II and ( C ) LC3 ‐ II flux are shown ( n = 11 - 13 ) .

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(A) Immunoblots for Atg7 in MBH from young (3 mo), middle‐aged (12 mo) and aged (22 mo) fed and 6‐h‐fasted (Stv) mice (n=4), and (B) LC3‐II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors (Inh) (n=11-13). Mean±s.e.m values for (B) steady‐state LC3‐II and (C) LC3‐II flux are shown (n=11-13).


( D ) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh ( n = 11 - 13 ) .

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(D) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh (n=11-13).


( E ) Immunofluorescence microscopy for POMC ( green ) and p62 ( red ) ( n = 4 ) , ( F ) immunoblots for POMC and ACTH ( n = 4 ) , and ( G ) immunofluorescence for α ‐ MSH in MBH from 3 mo and 22 mo mice on RD ( n = 4 ) .

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(E) Immunofluorescence microscopy for POMC (green) and p62 (red) (n=4), (F) immunoblots for POMC and ACTH (n=4), and (G) immunofluorescence for α‐MSH in MBH from 3 mo and 22 mo mice on RD (n=4).


( H ) Body wt and ( I ) eWAT wt of 3 ‐ mo ‐ and 22 ‐ mo ‐ old mice on RD ( n = 9 - 13 ) .

(H) Body wt and (I) eWAT wt of 3‐mo‐ and 22‐mo‐old mice on RD (n=9-13).


( J ) Serum fatty acids and ( K ) glycerol from fed or 6 ‐ h ‐ fasted ( Stv ) 3 mo and 22 mo mice ( n = 7 - 9 ) , and ( L , M ) after intraperitoneal Iso in 6 ‐ h ‐ fasted 3 mo and 22 mo mice ( n = 5 - 6 ) .

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(J) Serum fatty acids and (K) glycerol from fed or 6‐h‐fasted (Stv) 3 mo and 22 mo mice (n=7-9), and (L,M) after intraperitoneal Iso in 6‐h‐fasted 3 mo and 22 mo mice (n=5-6).


( N ) Immunoblots for adipose triglyceride lipase ( ATGL ) in eWAT from 3 mo and 22 mo mice . Values are mean ± s . e . m . P values are as compared with controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . Yellow arrows indicate merged signal . ACTH , adrenocorticotrophic hormone ; Con , control ; eWAT , epididymal fat ; Iso , isoproterenol ; MBH , mediobasal hypothalamic ; mo , months ; MSH , melanocyte ‐ stimulating hormone ; POMC , proopiomelanocortin ; RD , regular chow ; wt , weight .

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(N) Immunoblots for adipose triglyceride lipase (ATGL) in eWAT from 3 mo and 22 mo mice. Values are mean±s.e.m. P values are as compared with controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; eWAT, epididymal fat; Iso, isoproterenol; MBH, mediobasal hypothalamic; mo, months; MSH, melanocyte‐stimulating hormone; POMC, proopiomelanocortin; RD, regular chow; wt, weight.


Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with drugs at the indicated concentrations for six days.


Human spheroids established from BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) DLD1 cells , were incubated with 1 . 25 µM olaparib or 0 . 5 µM chlorambucil over the indicated period of time .

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Human spheroids established from BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) DLD1 cells, were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time.


Dose - dependent viability assays of BRCA1 - proficient ( + BRCA1 ) or - deficient ( - BRCA1 ) human RPE1 - hTERT cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) human RPE1-hTERT cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA1 - proficient ( + BRCA1 ) or - deficient ( - BRCA1 ) TP53 - deleted cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) TP53-deleted cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of Brca1 + / + and Brca1 - / - mouse mammary tumour - derived cell lines treated with drugs at the indicated concentrations for six days .

Dose-dependent viability assays of Brca1+/+ and Brca1-/- mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA2 - deficient ( Capan - 1 ) or - proficient ( MIA PaCa - 2 ) human pancreatic carcinoma - derived cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-deficient (Capan-1) or -proficient (MIA PaCa-2) human pancreatic carcinoma-derived cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA2 - deficient ( PEO1 ) or - proficient ( C4 - 2 ) human ovarian tumour - derived cells treated with drugs at the indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells treated with drugs at the indicated concentrations for six days.


BRCA2 - deficient ( PEO1 ) or - proficient ( C4 - 2 ) human ovarian tumour - derived cells were infected with lentiviruses expressing control or CHD4 shRNAs , followed by selection with puromycin for 72 hours . Dose - dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days .

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BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 hours. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days.


BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells were incubated with 1 µM chlorambucil ( Chl ) . Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown . GAPDH was used as a loading control .

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BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control.


Quantification of chromosome aberrations and chromatid / chromosome break frequencies in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h . Data were obtained from three independent experiments and normalised to untreated controls . A minimum of 60 Giemsa - stained metaphases were analysed for each sample . Cis , cisplatin ; Chl , chlorambucil .

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Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil.


Quantification of G2 / M cell frequency relative to solvent control , using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours . Cis , cisplatin ; Chl , chlorambucil .

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Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil.


Human H1299 cells were treated with control ( CTR ) or indicated siRNAs two days before drugs were added to the media for dose - dependent viability assays . Cell extracts prepared at the time of drug addition were immunoblotted as indicated . SMC1 was used as a loading control .

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Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.


Nude mice ( nu / nu ) were injected subcutaneously with 5 x 106 human DLD1 cells , BRCA2 - proficient ( A ) or BRCA2 - deficient ( B ) . Tumour - bearing mice were treated with 3 mg / kg daily chlorambucil administered intraperitoneally ( i . p . ) for a total of ten days . Tumour weight was determined on the indicated days after initiation of the treatment .

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Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment.


PDTCs derived from breast cancer samples as previously described ( Bruna et al , 2016 ) were treated with chlorambucil at the indicated doses . Cell survival is represented relative to DMSO control . AB521 , ER - negative tumour , no BRCA1 alteration ; STG201 , tumour with BRCA1 promoter methylation and loss of BRCA1 expression ; VHIO179 , tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation ( olaparib - resistant )

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PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant)


CB17 / SCID mice were injected intramuscularly with 5 x 106 human BRCA2 - deficient HCT116 cells . Tumour - bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally ( i . p . ) , or talazoparib administered orally ( o . s . ) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment ( day 1 ) . Scale bar , 40 µm . Each experimental group included n = 5 mice . Error bars represent SEM . Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining .

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CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm. Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining.


Wild type Balb / c mice were injected intra - peritoneally with solvent ( daily ) or 3 mg / kg chlorambucil ( daily for 5 days ) or 3 . 3 mg / kg cisplatin ( daily for 3 days ) . Uptake of the apoptosis tracer 99mTc - Duramycin 2 h after intra - venous injection was quantified in selected organs using SPECT imaging in the indicated organs . Representative maximum intensity partial projections showing tracer distribution are shown .

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Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.


Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A . Scale bar , 25 µm .

Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A. Scale bar, 25 µm.


B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and / or Chloramphenicol ( Chl ) . After fixation , AF647 - azide ( magenta ) was used for click reaction ( see A ) , and cells were immunostained for TOM20 as mitochondrial marker ( green ) . Representative epifluorescent microscopy images are shown . Magenta images shown in the presence of only Chl and in the absence of both inhibitors ( none ) are presented at lower brightness level than the other panels , duo to their very strong intensities . An inset of Harr panel is zoomed in the panel below for better visualization . Met , methionine . Scale bars , 50 µm and 10 µm for the zoom panel .

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B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and/or Chloramphenicol (Chl). After fixation, AF647-azide (magenta) was used for click reaction (see A), and cells were immunostained for TOM20 as mitochondrial marker (green). Representative epifluorescent microscopy images are shown. Magenta images shown in the presence of only Chl and in the absence of both inhibitors (none) are presented at lower brightness level than the other panels, duo to their very strong intensities. An inset of Harr panel is zoomed in the panel below for better visualization. Met, methionine. Scale bars, 50 µm and 10 µm for the zoom panel.


A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths ( heat map ) and cells immunostained for TOM20 ( gray ) . Representative confocal images are shown for each time point . The same 5 - minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row . Scale bars , 10 µm . Mitochondrial protein synthesis was quantified over time . 30 cells from N = 3 independent experiments were analyzed for each time point ( Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values . Error bars , + / - SEM ) . A hyperbolic graph was fitted to the data . R - squared , 0 . 97 .

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A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths (heat map) and cells immunostained for TOM20 (gray). Representative confocal images are shown for each time point. The same 5-minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row. Scale bars, 10 µm. Mitochondrial protein synthesis was quantified over time. 30 cells from N = 3 independent experiments were analyzed for each time point (Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values. Error bars, +/- SEM). A hyperbolic graph was fitted to the data. R-squared, 0.97.


B As in A , Mitochondrial translation products were labeled for increasing time ( 10 and 20 minutes ) in HeLa cells . The number of analyzed cells was increased in the plots from 25 to 300 per time point . 150 or 300 random cells from N = 3 independent experiments were analyzed . Kruskal - Wallis followed by Fisher ' s test . * * * * p < 0 . 0001 . Error bars , + / - SEM .

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B As in A, Mitochondrial translation products were labeled for increasing time (10 and 20 minutes) in HeLa cells. The number of analyzed cells was increased in the plots from 25 to 300 per time point. 150 or 300 random cells from N = 3 independent experiments were analyzed. Kruskal-Wallis followed by Fisher's test. ****p < 0.0001. Error bars, +/- SEM.


C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 ( magenta ) and cells immunostained for TOM20 ( green ) in fibroblasts , iPSCs - derived cardiomyocytes , and hippocampal neurons . Representative epifluorescent microscopy images are shown . The correlation of HPG and TOM20 channels is represented in fire lookup table , after calculating the Pearson Correlation values over each pixel of the images . Scale bars , 25 µm .

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C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 (magenta) and cells immunostained for TOM20 (green) in fibroblasts, iPSCs-derived cardiomyocytes, and hippocampal neurons. Representative epifluorescent microscopy images are shown. The correlation of HPG and TOM20 channels is represented in fire lookup table, after calculating the Pearson Correlation values over each pixel of the images. Scale bars, 25 µm.


A Mitochondrial translation products were labeled using a FUNCAT approach and [ 35S ] Met labeling in control and TSFM patient fibroblasts . Representative epifluorescent microscopy and the autoradiography images are shown . Scale bars , 50 µm . Changes in mitochondrial protein synthesis were quantified for both approaches . For microscopy image analysis , > 300 cells from N = 3 independent experiments were analyzed for each cell type . Mann - Whitney U test , * * * * p < 0 . 0001 . For radiolabeling analyses , N = 3 independent samples were quantified for each cell type . Student t test , * * p < 0 . 01 . Error bars , + / - SEM . B As in A using control and COA6 KO HEK cells . Scale bars , 25 µm . For microscopy image analysis , * * * * p < 0 . 0001 , and for radiolabeling experiments , * p < 0 . 05 were calculated . Error bars , + / - SEM .

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A Mitochondrial translation products were labeled using a FUNCAT approach and [35S]Met labeling in control and TSFM patient fibroblasts. Representative epifluorescent microscopy and the autoradiography images are shown. Scale bars, 50 µm. Changes in mitochondrial protein synthesis were quantified for both approaches. For microscopy image analysis, >300 cells from N = 3 independent experiments were analyzed for each cell type. Mann-Whitney U test, ****p < 0.0001. For radiolabeling analyses, N =3 independent samples were quantified for each cell type. Student t test, **p < 0.01. Error bars, +/- SEM. B As in A using control and COA6 KO HEK cells. Scale bars, 25 µm. For microscopy image analysis, ****p < 0.0001, and for radiolabeling experiments, *p < 0.05 were calculated. Error bars, +/- SEM.


A Mitochondrial translation products were labeled with HPG in hippocampal neurons , immunostained for ANK - G ( initial axon segment ) , and plasma membrane was stained with DiO . Representative epifluorescent images and zoomed insets of neural branches are shown . In both axon ( ANK - G positive , arrow ) and dendrite ( star ) , HPG incorporation revealing mitochondrial protein synthesis was detected . Scale bars , 20 µm .

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A Mitochondrial translation products were labeled with HPG in hippocampal neurons, immunostained for ANK-G (initial axon segment), and plasma membrane was stained with DiO. Representative epifluorescent images and zoomed insets of neural branches are shown. In both axon (ANK-G positive, arrow) and dendrite (star), HPG incorporation revealing mitochondrial protein synthesis was detected. Scale bars, 20 µm.


B Representative confocal images of a dendrite ( top ) and an axon ( bottom ) in which mitochondrial translation products were labeled with HPG ( magenta ) . HOMER1 and VGLUT1 were used as post - and presynapse markers , respectively ( green ) and DiO stained randomly neurites ( gray ) . In each neurite , an exemplary region was selected and the DiO was outlined to mark the outer edge of the region . As a control , cells were treated with Chloramphenicol ( Chl ) to inhibit mitochondrial translation . Scale bars , 5 µm .

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B Representative confocal images of a dendrite (top) and an axon (bottom) in which mitochondrial translation products were labeled with HPG (magenta). HOMER1 and VGLUT1 were used as post- and presynapse markers, respectively (green) and DiO stained randomly neurites (gray). In each neurite, an exemplary region was selected and the DiO was outlined to mark the outer edge of the region. As a control, cells were treated with Chloramphenicol (Chl) to inhibit mitochondrial translation. Scale bars, 5 µm.


Confirmation of peptide entry into neurons . Cultures were incubated with Bio - TMyc ( 25 µM , 1 h ) ( panel b ) or left untreated ( panel a ) . Arrowheads highlight peptide permeability , detected by fluorescein Avidin D ( green ) , into neurons labeled with neuronal - specific antibody NeuN ( red ) . Peptide is not detected in some neurons ( arrow ) and non - neuronal cells ( asterisk ) . Confocal microscopy images correspond to single sections and are representative of five independent experiments . Scale bar , 10 μm .

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Confirmation of peptide entry into neurons. Cultures were incubated with Bio-TMyc (25 µM, 1 h) (panel b) or left untreated (panel a). Arrowheads highlight peptide permeability, detected by fluorescein Avidin D (green), into neurons labeled with neuronal-specific antibody NeuN (red). Peptide is not detected in some neurons (arrow) and non-neuronal cells (asterisk). Confocal microscopy images correspond to single sections and are representative of five independent experiments. Scale bar, 10 μm.


Effect of TMyc on neuronal survival . Primary cultures were incubated with TMyc ( 5 , 15 or 25 μM , 30 min ) and subjected to treatment with NMDA ( 100 µM ) and glycine ( 10 µM ) for 4 h . Specific neuronal viability was established and expressed relative to values in cultures with no treatment . Means ± SEM are represented ( n = 8 ) and statistical analysis was performed by ANOVA test followed by post - hoc Tukey HSD test .

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Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.


Effect of TFL457 , TFL482 and TFL541 on TrkB - FL levels . Cultures preincubated with TrkB - FL peptides or TMyc ( 25 µM , 30 min ) , followed by chronic NMDA treatment ( 2 h ) , were compared to cells without peptide . Levels of full - length ( FL ) TrkB were established with panTrkB , an antibody for the extracellular domain that also detects the truncated forms ( tTrkB ) . Quantitation of peptide interference of TrkB - FL downregulation . Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA . Means ± SEM are represented and analysis was performed by ANOVA test followed by post - hoc Tukey HSD test ( * p = 0 . 02 ; n = 4 ) .

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Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB). Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4).


Effect of TFL457 , TFL482 and TFL541 on neuronal viability after chronic excitotoxicity . Cultures were preincubated with peptides and treated with NMDA for 0 - 6 h as before . Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA . Mean ± SEM of eleven ( TFL457 ) , three ( TFL482 ) and seven ( TFL541 ) independent experiments is given .

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Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given.


Effect of TFL518 on TrkB - FL levels . Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457 .

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Effect of TFL518 on TrkB-FL levels. Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457.


TFL518 effect on neuronal viability . Cultures were treated and analyzed as above . No significant differences were found for TFL518 while neuroprotection by TFL457 was

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TFL518 effect on neuronal viability. Cultures were treated and analyzed as above. No significant differences were found for TFL518 while neuroprotection by TFL457 was confirmed


Effect of TFL457 on neuronal viability after acute excitotoxicity . Cells were induced with NMDA ( 50 μM ) and glycine ( 10 μM ) for 1 h and , after agonists removal , culture proceeded for 20 h with DL - AP5 ( 200 μM ) and TMyc or TFL457 ( 15 μM ) . Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented .

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Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented.


Effect of TFL457 on Y515 and Y816 phosphorylation . Cultures preincubated with TMyc or TFL457 ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) and analyzed with panTrkB or phosphospecific antibodies . Quantitation of pY515 and pY816 . Mean ± SEM of normalized pY515 ( n = 7 ) or pY816 ( n = 4 ) levels obtained after NMDA - treatment ( 2 h ) are represented relative to those found in cells with the same peptide but no NMDA . Statistical analysis was performed by unpaired Student t test ( * p = 0 . 046 ; n . s . = non - significant ) .

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Effect of TFL457 on Y515 and Y816 phosphorylation. Cultures preincubated with TMyc or TFL457 (25 μM, 30 min) were treated with NMDA (0-6 h) and analyzed with panTrkB or phosphospecific antibodies. Quantitation of pY515 and pY816. Mean ± SEM of normalized pY515 (n = 7) or pY816 (n = 4) levels obtained after NMDA-treatment (2 h) are represented relative to those found in cells with the same peptide but no NMDA. Statistical analysis was performed by unpaired Student t test (*p = 0.046; n.s. = non-significant).


Analysis by immunoprecipitation of TFL457 effect on pY816 levels . Cultures preincubated and treated as before with NMDA ( 2 h ) , were immunoprecipitated with antibodies specific for phosphorylated tyrosine ( pY ) . pY816 was analyzed by WB in immunoprecipates .

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Analysis by immunoprecipitation of TFL457 effect on pY816 levels. Cultures preincubated and treated as before with NMDA (2 h), were immunoprecipitated with antibodies specific for phosphorylated tyrosine (pY). pY816 was analyzed by WB in immunoprecipates.


TFL457 effects on neuronal viability after inhibition of TrkB - FL signaling . Cultures were preincubated ( 30 min ) with inhibitors specific for PI3K ( Wortmannin , 100 nM ) , MAPK / ERK ( UO126 , 300 nM ) or PLCγ ( U - 73122 , 5 μM ) before incubation with TMyc or TFL457 ( 25 μM , 30 min ) . Viability was established 4 h after NMDA treatment . Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post - hoc Tukey HSD test ( * * * p = 0 . 0001 , * * p = 0 . 009 , * p = 0 . 02 , n . s . = non - significant , respectively for TMyc vs TFL457 in untreated or Wortmannin , UO126 or U - 73122 - treated cells ; * * p = 0 . 008 for untreated vs U - 73122 - treated cells preincubated with TFL457 ; n = 5 ) .

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TFL457 effects on neuronal viability after inhibition of TrkB-FL signaling. Cultures were preincubated (30 min) with inhibitors specific for PI3K (Wortmannin, 100 nM), MAPK/ERK (UO126, 300 nM) or PLCγ (U-73122, 5 μM) before incubation with TMyc or TFL457 (25 μM, 30 min). Viability was established 4 h after NMDA treatment. Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post-hoc Tukey HSD test (***p = 0.0001, **p = 0.009, *p = 0.02, n.s. = non-significant, respectively for TMyc vs TFL457 in untreated or Wortmannin, UO126 or U-73122-treated cells; **p = 0.008 for untreated vs U-73122-treated cells preincubated with TFL457; n = 5).


Dose - dependent TFL457 effects . Cultures incubated with TMyc o TFL457 ( 5 , 15 o 25 μM , 30 min ) were treated with NMDA ( 2 h ) and compared to cells without peptide or NMDA .

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Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA.


Time - course TFL457 effects . Cells preincubated with peptides ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) .

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Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h).


Quantitation of peptide effects . Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA . Means ± SEM are given . Results were analyzed by a two - way ANOVA test followed by post - hoc Bonferroni test , comparing TMyc or TFL457 - treated cells for each time - point ( TrkB - FL , * * * p = 0 . 0009 and * p = 0 . 02 ; CREB , * p = 0 . 0149 and * p = 0 . 0148 ; pCREB , * * * p = 0 . 0007 and * * p = 0 . 0012 ; MEF2D , * * p = 0 . 0023 and * * p = 0 . 0047 . All values are respectively for 2 or 4 h ; n = 6 ) .

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Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).


Effect of TFL457 on MEF2 - promoter activity . Cultures transfected with pMEF2 ( two minimal wild type MEF2 elements ) or pMEF2mut ( mutant ) were preincubated with CPPs ( 25 μM , 30 min ) and treated with NMDA ( 100 µM , 2 h ) . Means ± SEM of luciferase activities obtained in excitotoxicity , relative to values found in cells treated with same peptide and no NMDA , are presented .

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Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented.


Effect on CRE - promoter activity . pCRE contains two minimal CREs . Peptide preincubation was as above with or without KG - 501 ( 10 µM ) . Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG - 501 .

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Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501.


Effect on NMDAR - subunit promoters . Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A ( pGluN1 , * p = 0 . 016 ; pGluN2A , * p = 0 . 029 ; n = 14 ) .

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Effect on NMDAR-subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A (pGluN1, *p = 0.016; pGluN2A, *p = 0.029; n = 14).


Effects on BDNF promoter III or TrkB promoter . Cells transfected with pIIIBDNF ( n = 9 ) , pTrkB ( n = 7 ) or pCRE ( n = 5 ) , as a control , were processed as above but using 20 µM NMDA for 4 h . Data are presented and analyzed as in panel A ( pCRE , * * p = 0 . 0098 ; pIIIBDNF , * p = 0 . 029 ; pTrkB , * p = 0 . 033 ) .

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Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF (n = 9), pTrkB (n = 7) or pCRE (n = 5), as a control, were processed as above but using 20 µM NMDA for 4 h. Data are presented and analyzed as in panel A (pCRE, **p = 0.0098; pIIIBDNF, *p = 0.029; pTrkB, *p = 0.033).


Effects of TFL457 on mRNA levels of CREB / MEF2 - regulated genes . Total RNA was extracted from cultures preincubated with CPPs ( 25 μM , 30 min ) and treated or not with NMDA ( 100 µM , 4 h ) . Levels of mRNA were normalized to NSE ( genes expressed in neurons , left panel ) or GAPDH ( neuronal and glial expression , right panel ) . Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented . Differences found in excitotoxicity were analyzed by ANOVA test followed by post - hoc Tukey HSD test ( GluN1 , * * * p = 0 . 00096 ; GluN2A , p = 0 . 98 ; TrkB - FL , * * * p = 0 . 00037 ; BDNF , * p = 0 . 045 ; n = 8 ) .

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Effects of TFL457 on mRNA levels of CREB/MEF2-regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 µM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (GluN1, ***p = 0.00096; GluN2A, p = 0.98; TrkB-FL, ***p = 0.00037; BDNF, *p = 0.045; n = 8).


TFL457 interference of RIP and calpain - processing in excitotoxicity . Cultures were preincubated with peptides ( 25 µM , 30 min ) or left untreated before NMDA addition ( 2 h ) . Different exposures are shown to facilitate visualization of FL , f42 / 39 or f32 . Quantitation of normalized f32 and f42 / 39 levels . Means ± SEM are shown relative to cultures treated with NMDA and no peptide .

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TFL457 interference of RIP and calpain-processing in excitotoxicity. Cultures were preincubated with peptides (25 µM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39 or f32. Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide.


Effect of TFL457 on neuronal viability after calpain inhibition . After 30 min preincubation with calpeptin ( Calp , 10 µM ) and calpain inhibitor III ( CiIII , 10 µM ) , cultures were treated with CPPs and NMDA ( 4 h ) as before . Means ± SEM relative to those obtained in untreated cultures are given .

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Effect of TFL457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 µM) and calpain inhibitor III (CiIII, 10 µM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given.


Effect of TFL457 on neuronal viability after metalloproteinase inhibition . Cultures preincubated with GM6001 ( 10 µM , 30 min ) were treated with CPPs and NMDA as before . Analysis was performed as above ( without GM6001 , * * * p = 0 . 0007 ; with GM6001 , * * * p = 0 . 0006 ; n = 4 ) .

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Effect of TFL457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 µM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, ***p = 0.0007; with GM6001, ***p = 0.0006; n = 4).


Effect of NMDA on total and cell - surface TrkB - FL levels . Cultures were incubated with NMDA ( 100 µM ) or BDNF ( 100 ng / ml ) for 1 h . Membrane proteins were labeled and purified and compared to corresponding total lysates . TrkB - FL levels are expressed relative to untreated cells .

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Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells.


Effect of TFL457 on the decrease of TrkB - FL surface levels induced by NMDA . Analysis was performed as before in cells incubated with CPPs ( 25 μM , 30 min ) and NMDA - treated ( 1 h ) . Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA .

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Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA.


Effect of endocytosis inhibition on TrkB - FL downregulation . The infarcted area of the ipsilateral hemisphere ( I ) was compared to the corresponding region of the contralateral area ( C ) . Results from representative mice injected with dynasore or vehicle are shown . Different exposures are presented to facilitate visualization of dynasore effects on TrkB - FL and f32 . Quantitation of TrkB - FL and TrkB - T1 in the infarcted area . Normalized protein levels are expressed relative to those of the corresponding contralateral region .

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Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32. Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region.


Confirmation of Bio - TFL457 delivery to mice cortex . Biotinylated TFL457 ( Bio - TFL457 , 4 nMole / g , retro - orbitally injected ) was detected in coronal sections stained with NeuN and DAPI . Representative confocal microscopy images of cortical areas correspond to single sections . Scale bar , 10 µm .

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Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.


Representative analysis of TrkB - FL at 3 h of damage . Comparison of the infarcted area ( I ) and the corresponding contralateral region ( C ) is shown . Quantitation of TrkB - FL levels . Means ± SEM of normalized TrkB - FL levels in the infarcted area relative to the corresponding contralateral

Representative analysis of TrkB-FL at 3 h of damage. Comparison of the infarcted area (I) and the corresponding contralateral region (C) is shown. Quantitation of TrkB-FL levels. Means ± SEM of normalized TrkB-FL levels in the infarcted area relative to the corresponding contralateral region


Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457 .

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Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457.


Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume . Means ± SEM are given . Differences were analyzed by Student ' s t - test ( * * * p = 0 . 00008 ; n = 14 ) . Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc .

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Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume. Means ± SEM are given. Differences were analyzed by Student's t-test (***p = 0.00008; n = 14). Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc.


Evaluation of balance and motor coordination . Number of contralateral hind paw slips were measured after 24 h of damage . Data spread is presented by box and whisker plots showing interquartile range , median , minimum and maximum values .

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Evaluation of balance and motor coordination. Number of contralateral hind paw slips were measured after 24 h of damage. Data spread is presented by box and whisker plots showing interquartile range, median, minimum and maximum values.


( B ) Migration efficiency ( % of cells that migrated through filter pores in each round ) of A375 cells during 5 μm constricted migration and 12 μm migration ( left panels ) . Biological replicates shown . Phase contrast imaging of the final cell populations after the 10 rounds of migration , cells cultured on 2D plastic ( right panels , Scale bar = 50 µm ) .

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(B) Migration efficiency (% of cells that migrated through filter pores in each round) of A375 cells during 5 μm constricted migration and 12 μm migration (left panels). Biological replicates shown. Phase contrast imaging of the final cell populations after the 10 rounds of migration, cells cultured on 2D plastic (right panels, Scale bar = 50 µm).


( C ) A375 nuclei stained with DAPI ( blue ) , Lamin A / C ( green ) and H3K9me3 ( red ) in the indicated migration conditions . Images shown represent a maximum projection of z - stacks ( Scale bar = 5 µm ) .

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(C) A375 nuclei stained with DAPI (blue), Lamin A/C (green) and H3K9me3 (red) in the indicated migration conditions. Images shown represent a maximum projection of z-stacks (Scale bar= 5 µm).


( D ) Cumulative distribution plot of Lamin A / C coefficient of variation in all indicated A375 conditions ( Control ( n = 59 ) , Top - 5 ( n = 64 ) , Bottom - 5 ( n = 62 ) nuclei ) . Kolmogorov - Smirnov test p - values indicated in plot .

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(D) Cumulative distribution plot of Lamin A/C coefficient of variation in all indicated A375 conditions (Control (n = 59), Top-5 (n = 64), Bottom-5 (n = 62) nuclei). Kolmogorov-Smirnov test p-values indicated in plot.


( E ) Radial intensity distribution of H3K9me3 signal in the maximum projected nuclei of Control ( n = 59 ) , Top - 5 ( n = 63 ) , Bottom - 5 ( n = 64 ) and Bottom - 12 ( n = 80 ) nuclei ( * * p = 0 . 0069 , two - tailed t - test ) . Error bars = mean ± SD .

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(E) Radial intensity distribution of H3K9me3 signal in the maximum projected nuclei of Control (n = 59), Top-5 (n = 63), Bottom-5 (n = 64) and Bottom-12 (n = 80) nuclei (** p = 0.0069, two-tailed t-test). Error bars = mean ± SD.


( F ) Maximum projection confocal images of Top - 5 and Bottom - 5 cells embedded in collagen and stained with DAPI ( blue ) and Phalloidin ( red ) . ( Inset ) Zoom in of nucleus of Bottom - 5 cell . Top - 5 and Bottom - 5 zoomed inset scale = 5 μm ; Bottom - 5 zoomed out image scale = 20 μm .

(F) Maximum projection confocal images of Top-5 and Bottom-5 cells embedded in collagen and stained with DAPI (blue) and Phalloidin (red). (Inset) Zoom in of nucleus of Bottom-5 cell. Top-5 and Bottom-5 zoomed inset scale = 5 μm; Bottom-5 zoomed out image scale = 20 μm.


( G ) Fraction of Control , Top - 5 and Bottom - 5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices . Each biological replicate is connected by a line .

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(G) Fraction of Control, Top-5 and Bottom-5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices. Each biological replicate is connected by a line.


( H ) Nuclei of Top - 5 ( left ) and Bottom - 5 cells ( right ) embedded in 3D collagen matrix and stained with DAPI ( blue ) and Lamin A / C ( green ) . Scale = 5μm . For 3D reconstruction visualization , see Movies EV5 and EV6 .

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(H) Nuclei of Top-5 (left) and Bottom-5 cells (right) embedded in 3D collagen matrix and stained with DAPI (blue) and Lamin A/C (green). Scale = 5μm. For 3D reconstruction visualization, see Movies EV5 and EV6.


( I ) Percent of cells embedded in collagen that had elongated nuclei ( Aspect Ratio > 2 . 5 ) for A375 Control ( n = 54 ) , Top - 5 ( n = 52 ) and Bottom - 5 ( n = 53 ) .

(I) Percent of cells embedded in collagen that had elongated nuclei (Aspect Ratio > 2.5) for A375 Control (n = 54), Top-5 (n = 52) and Bottom-5 (n = 53).


( J ) Cumulative distribution plot of Lamin A / C coefficient of variation for Control ( n = 70 ) , Top - 5 ( n = 78 ) and Bottom - 5 ( n = 109 ) cells embedded in 3D collagen matrices . Kolmogorov - Smirnov test p - values indicated in plot .

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(J) Cumulative distribution plot of Lamin A/C coefficient of variation for Control (n = 70), Top-5 (n = 78) and Bottom-5 (n = 109) cells embedded in 3D collagen matrices. Kolmogorov-Smirnov test p-values indicated in plot.


( E ) Metaphase spread labeled with chromosome specific FISH probes for chr5 ( green ) and chr13 ( red ) . Bottom graphs represent frequency of t ( 5 ; 13 ) quantified by FISH ( left ) and copy number quantification for chr5 and chr13 ( right ) . n = 1328 Control and 2370 metaphases analyzed for Bottom - 5 . ( Scale = 10 μm )

(E) Metaphase spread labeled with chromosome specific FISH probes for chr5 (green) and chr13 (red). Bottom graphs represent frequency of t(5;13) quantified by FISH (left) and copy number quantification for chr5 and chr13 (right). n = 1328 Control and 2370 metaphases analyzed for Bottom-5. (Scale = 10 μm)


A . Expression profiling of differentially expressed circRNAs between the tumor and normal groups . Rows represent circRNAs , and columns represent samples . Rows were ordered by fold change , and columns were ordered by their group . The sample of N8 was not included due to low sequencing library size .

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A. Expression profiling of differentially expressed circRNAs between the tumor and normal groups. Rows represent circRNAs, and columns represent samples. Rows were ordered by fold change, and columns were ordered by their group. The sample of N8 was not included due to low sequencing library size.


B . Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups . Both the row and column were unsupervised and clustered with the hierarchical clustering method .

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B. Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups. Both the row and column were unsupervised and clustered with the hierarchical clustering method.


C . The 4 of 22 differentially expressed circRNAs were confirmed by qRT - PCR , which were retained after marker selection procedure . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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C. The 4 of 22 differentially expressed circRNAs were confirmed by qRT-PCR, which were retained after marker selection procedure. **P < 0.01, Student's t-test, mean ± SD.


E . Time - dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset . P - values are shown for the indicated comparison of AUC between each marker and cirScore . Student ' s t - test , AUC = area under the curve .

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E. Time-dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset. P-values are shown for the indicated comparison of AUC between each marker and cirScore. Student's t-test, AUC=area under the curve.


A - F . Kaplan - Meier curves of DFS ( A ) and OS ( B ) in 249 patients in the training set . Kaplan - Meier curves of DFS ( C ) and OS ( D ) in 122 patients in the internal validation data set . Kaplan - Meier curves of DFS ( E ) and OS ( F ) in 180 patients in the external independent validation data set . Hazard ratios ( HRs ) were calculated with a univariate Cox regression analysis , P - values were calculated with the log - rank test .

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A-F. Kaplan-Meier curves of DFS (A) and OS (B) in 249 patients in the training set. Kaplan-Meier curves of DFS (C) and OS (D) in 122 patients in the internal validation data set. Kaplan-Meier curves of DFS (E) and OS (F) in 180 patients in the external independent validation data set. Hazard ratios (HRs) were calculated with a univariate Cox regression analysis, P-values were calculated with the log-rank test.


A . RT - PCR products with divergent and convergent primers showing circularization of has _ circ _ 0079480 and has _ circ _ 0087391 . cDNA , complementary DNA ; gDNA , genomic DNA .

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A. RT-PCR products with divergent and convergent primers showing circularization of has_circ_0079480 and has_circ_0087391. cDNA, complementary DNA; gDNA, genomic DNA.


B . qRT - PCR evaluated the knockdown efficiency of has _ circ _ 0079480 and and has _ circ _ 0087319 in SW620 and HCT116 cells transfected with two unique shRNAs ( # 1 , # 2 ) . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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B. qRT-PCR evaluated the knockdown efficiency of has_circ_0079480 and and has_circ_0087319 in SW620 and HCT116 cells transfected with two unique shRNAs (#1, #2). **P < 0.01, Student's t-test, mean ± SD.


C . Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs .

C. Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs.


D . The relative fold - change of the transwell migration for indicated knockdown cells over those of control cells . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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D. The relative fold-change of the transwell migration for indicated knockdown cells over those of control cells. **P < 0.01, Student's t-test, mean ± SD.


E - F . Representative hematoxylin and eosin ( H & E ) staining and statistical results of the micro - metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days . N = 8 per group . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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E-F. Representative hematoxylin and eosin (H&E) staining and statistical results of the micro-metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days. N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


G - H . Representative H & E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days . ( Five sections evaluated per lung ) . N = 8 per group . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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G-H. Representative H&E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days. (Five sections evaluated per lung). N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


C DNA breaks were measured using alkaline comet assay in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with 2 µM pyridostatin ( PDS ) for 16 hours and released into fresh medium without pyridostatin . Representative images are shown . Scale bar represents 100 µm . D Quantification of DNA breaks shown in ( C ) . Graph and error bars represent the mean and SEM of n = 3 independent experiments . A minimum of 50 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; NS , P > 0 . 5 . E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in ( C ) . A minimum of 200 cells were analysed per condition per experiment . Graph and error bars represent the mean and SEM of n = 3 independent experiments . P values were calculated using an unpaired two - tailed t - test . * * * , P ≤ 0 . 001 ; NS , P > 0 . 5 .

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

C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm. D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5. E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.


F BRCA2 - deficient ( - BRCA2 ) human DLD1 cells were treated with 2 µM pyridostatin ( PDS ) for 24 hours . Next , pyridostatin was removed and cells were released in a medium containing 250 nM NU - 7441 ( NU ) . Whole - cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown . SMC1 was used as a loading control . KAP1 phosphorylation site is indicated in red .

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 B-CONTROLLED_VAR 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 O O O O O O O O O O O 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 BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.


G Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with pyridostatin ( PDS ) and NU - 7441 at the indicated concentrations for six days . Graphs represent average values obtained from of n = 3 independent experiments , each performed in technical triplicates .

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G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.


Quantitative RT - PCR of cells grown as in ( A ) and treated with 10 µM pyridostatin ( PDS ) for 3 days was performed using primers specific for the indicated genes . mRNA levels are expressed relative to GAPDH and to - DOX ( + BRCA2 ) cells . Error bars represent the SEM of n = 3 independent experiments , each performed in technical triplicate . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 .

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

Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001.


BRCA2 + / + and BRCA2 - / - RPE - 1 cells were treated with 10 μM pyridostatin ( PDS ) for 2 days . Whole - cell extracts were immunoblotted as indicated . KAP1 , IRF3 and STAT1 phosphorylation sites are shown in red . Tubulin and GAPDH were used as loading controls .

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 O O O O O O O O O O

BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls.


H1299 + shBRCA2DOX cells treated as in ( B ) were fixed and prepared for immunofluorescence with antibody against cGAS . DNA was counterstained with DAPI . Scale bar represents 20 µm . Quantification of cGAS - positive micronuclei per cells shown in ( E ) . Graph and error bars represent the mean and SEM of of n = 3 independent experiments . A minimum of 250 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; NS , P > 0 . 5 .

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H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm. Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.


FVB female mice were injected intramuscularly with ( A ) BRCA1 - proficient KP3 . 33 ( Brca1 + / + ) or ( B ) BRCA1 / 53BP1 - deficient KB1PM5 ( Brca1 - / - , Tp53bp1 - / - ) mouse mammary tumour cells . Pyridostatin ( PDS ) was administered intravenously ( i . v . ; 7 . 5 mg / kg / day ) and talazoparib was administered orally ( p . o . ; 0 . 33 mg / kg / day ) , over the indicated periods of time . Vertical dotted line indicates end of treatment . Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment . Each experimental group included n = 5 mice . Error bars represent SEM . P values were calculated between treated and untreated tumours at day 16 , using an unpaired two - tailed t - test . * * * * , P ≤ 0 . 0001 ; NS , P > 0 . 5 .

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

FVB female mice were injected intramuscularly with (A) BRCA1-proficient KP3.33 (Brca1+/+) or (B) BRCA1/53BP1-deficient KB1PM5 (Brca1-/-, Tp53bp1-/-) mouse mammary tumour cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 16, using an unpaired two-tailed t-test. ****, P ≤ 0.0001; NS, P > 0.5.


BRCA1 + / + , BRCA1 - / - and BRCA1 - / - / TP53BP1 - / - RPE - 1 cells were treated with 10 μM pyridostatin ( PDS ) or 2 μM olaparib for 2 days . Whole - cell extracts were immunoblotted as indicated . KAP1 , IRF3 and STAT1 phosphorylation sites are shown in red . SMC1 and GAPDH were used as loading controls .

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

BRCA1+/+, BRCA1-/- and BRCA1-/-/TP53BP1-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) or 2 μM olaparib for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.