E . Summary of Biot - Aβ1 - 42 binding throughout 8 membranes , color indicates the mean between membranes and the size of the outline the standard deviation .

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 O O O O O O O O O

E. Summary of Biot-Aβ1-42 binding throughout 8 membranes, color indicates the mean between membranes and the size of the outline the standard deviation.


A - C . ThT kinetics of 10μM rAβ1 - 42 alone or in presence ( 1 : 1 ) of 3 homologue peptides ( full screen on Appendix Figure S3 , S4 , n = 2 independent experiments with 4 repeats ) . Reused images in Appendix figure

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

A-C. ThT kinetics of 10μM rAβ1-42 alone or in presence (1:1) of 3 homologue peptides (full screen on Appendix Figure S3,S4, n= 2 independent experiments with 4 repeats). Reused images in Appendix figure S3


I . Representative AFM height images of Aβ fibrils alone or in a 1 : 1 mixture with P3 , P5 , P8 and P12 peptides are show in the top row . The boxes indicate the magnified regions shown in the second row . Arrows indicate the locations of representative individual fibrils shown in magnified detail , each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data . The scale bar for each row are shown to the left , with both the 3D model and the straightened image data representing 10 nm . The colour scale of the 3D models from blue to yellow indicate the distance ( from low to high ) between the fibril surface and fibril centre axis to demonstrate their twist patterns . The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single , not fragmented , amyloid fibrils are shown in the bottom row .

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

I. Representative AFM height images of Aβ fibrils alone or in a 1:1 mixture with P3, P5, P8 and P12 peptides are show in the top row. The boxes indicate the magnified regions shown in the second row. Arrows indicate the locations of representative individual fibrils shown in magnified detail, each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data. The scale bar for each row are shown to the left, with both the 3D model and the straightened image data representing 10 nm. The colour scale of the 3D models from blue to yellow indicate the distance (from low to high) between the fibril surface and fibril centre axis to demonstrate their twist patterns. The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single, not fragmented, amyloid fibrils are shown in the bottom row.


A . Overrepresentation of Aβ APRs in amyloid plaques of AD brains ( statistics : hypergeometric test with Bonferroni correction ) * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . The original data was taken from Xiong et al and included three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.


G . Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains . ( statistics : hypergeometric test with Bonferroni correction ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 The original data was taken from Xiong et al and included three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains.(statistics: hypergeometric test with Bonferroni correction). * P ≤0.05, ** P≤0.01 The original data was taken from Xiong et al and included three biological replicates.


A . Overrepresentation of Aβ APRs in amyloid plaques of APP / PS1 mouse brains ( mean ± SD ) from two biological replicates . ( statistics : hypergeometric test with Bonferroni correction )

O O O O O O O O O O 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. Overrepresentation of Aβ APRs in amyloid plaques of APP/PS1 mouse brains (mean ±SD) from two biological replicates. (statistics: hypergeometric test with Bonferroni correction)


D . No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions ( α - synuclein aggregates ) . ( statistics : hypergeometric test with Bonferroni correction )

O O O O O O O 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. No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions (α-synuclein aggregates). (statistics: hypergeometric test with Bonferroni correction)


D . Representative images of 3 proteins that can induce aggregation of Aβ1 - 42 in biosensor cell . Increase aggregation is observed in cells expressing the construct but not GFP alone ( left panels ) . Scale bar : 100μm . FRAP of the resulting aggregates shows no recovery ( in detail at Appendix Figure S11 ) . Scale bar : 2 . 5μm Removal of the homologue regions resulting in reduced aggregation ( right panels ) . ( n = 3 independent experiments ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments).


E . Quantification of number of spots per cell in cells expressing / not expressing the construct ( identified by GFP , transfection reporter ) ( n = 3 independent experiments , statistics : ordinary one - way Anova with Dunnett T3 multiple comparison correction , unpaired t - test for transfected / non - transfected cells ) . Bar plot : mean with 95 % CI . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of number of spots per cell in cells expressing/not expressing the construct (identified by GFP, transfection reporter) (n=3 independent experiments, statistics: ordinary one-way Anova with Dunnett T3 multiple comparison correction, unpaired t-test for transfected/non-transfected cells). Bar plot: mean with 95%CI. * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001


F - G . Quantification of Spots per cell for construct6 and construct6 control ( 6Ctrl , removal of homologue region ) ( F ) and construct10 and construct10 control ( 10ctrl , removal of homologue region ) ( G ) . ( n = 4 independent experiments , statistics : ordinary one - way Anova ) . Bar plot : mean with 95 % CI . * * * * P ≤ 0 .

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

F-G. Quantification of Spots per cell for construct6 and construct6 control (6Ctrl, removal of homologue region) (F) and construct10 and construct10 control (10ctrl, removal of homologue region) (G). (n=4 independent experiments, statistics: ordinary one-way Anova). Bar plot: mean with 95%CI. **** P≤0.0001


H - I ) Confocal images of colocalization of protein of interest with Aβ aggregates . ( Contrast of images were enhanced to 0 . 1 % saturated pixels using Fiji ) . 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

H-I) Confocal images of colocalization of protein of interest with Aβ aggregates. (Contrast of images were enhanced to 0.1% saturated pixels using Fiji). Scale bars: 22μm


B . Beta - hydroxybutyrate ( ketone body ) concentrations , plasma .

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

B. Beta-hydroxybutyrate (ketone body) concentrations, plasma.


C . Creatine kinase ( CK ) concentrations , plasma .

C. Creatine kinase (CK) concentrations, plasma.


D . Myoglobin concentrations , plasma . Dashed line : upper limit of control range . The individual patients are shown separately , and controls as mean with standard deviation ( n = 10 ) . Arrow : mAD endpoint in PEO patients ; lines under graphs : duration of mAD .

D. Myoglobin concentrations, plasma. Dashed line: upper limit of control range. The individual patients are shown separately, and controls as mean with standard deviation (n=10). Arrow: mAD endpoint in PEO patients; lines under graphs: duration of mAD.


E - G . PEO patient ( P1 ) muscle before diet : E . Ultrathin section of abnormal muscle fiber ( arrowhead ) with subsarcolemmal accumulations of mitochondria . Scale bar 50 µmF . Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria ( same cell as in E ) . G . Enlargement of the area marked in F ; mitochondria with paracrystalline " parking lot " inclusions ( arrow ) and concentric cristae ( asterisk ) . Scale bar 1µmH - J . PEO patient ( P1 ) muscle after mAD ( H - J ) : H . Ultrathin section , lytic muscle fiber with centrally located organellar debris ( arrow ) . Scale bar 50 µmI . Electron micrograph of a degrading muscle fiber with an invaded macrophage ( arrow ) ( same cell as in H ) . J . Enlargement of the fiber in I : paracrystalline inclusions within mitochondria ( asterisk ) . Scale bar 1µmK . Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections . n . a . , not available .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. PEO patient (P1) muscle before diet:E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µmF. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).G. Enlargement of the area marked in F; mitochondria with paracrystalline "parking lot" inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µmH-J. PEO patient (P1) muscle after mAD (H-J):H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µmI. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µmK. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.


A - B . Cleaved caspase - 3 positive muscle fibers ( * ) . A . PEO patient ( P3 ) on normal diet ( inset : caspase staining in healthy control individual ) B . PEO patient ( P3 ) after modified Atkins diet . Arrowhead : caspase negative necrotic fiber .

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

A-B. Cleaved caspase-3 positive muscle fibers (*).A. PEO patient (P3) on normal diet (inset: caspase staining in healthy control individual)B. PEO patient (P3) after modified Atkins diet. Arrowhead: caspase negative necrotic fiber.


C . Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation ; left , blot ; right , signal quantification in each patient .

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

C. Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation; left, blot; right, signal quantification in each patient.


D - E . Immunohistochemistry analysis of p62 and mitochondrial mass marker , consecutive sections . Asterisks indicate p62 - positive fibers and corresponding affected ragged - red fibers . F - G . p62 and mitochondrial mass after mAD ; consecutive sections . Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass .

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

D - E. Immunohistochemistry analysis of p62 and mitochondrial mass marker, consecutive sections. Asterisks indicate p62-positive fibers and corresponding affected ragged-red fibers.F - G. p62 and mitochondrial mass after mAD; consecutive sections. Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass.


H . p62 and LC3 positive ragged - red fibers ( * ) in PEO patient ( P2 ) , normal diet . I . Muscle fibers showing granular LC3 - positivity , PEO patient ( P1 ) , after mAD . Immunohistochemistry on frozen sections ; bars : 50 µm , except for H , 100 µm .

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

H. p62 and LC3 positive ragged-red fibers (*) in PEO patient (P2), normal diet.I. Muscle fibers showing granular LC3-positivity, PEO patient (P1), after mAD. Immunohistochemistry on frozen sections; bars: 50 µm, except for H, 100 µm.


A - C . Muscle strength and performance of PEO patients ( P1 - P4 ) on normal diet ( ND ) and after modified Atkins diet ( mAD ) ( one and six months and 2 . 5 years after initiation of diet ) .

O O O O O O O O O O O O O O O O O O O O O O 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. Muscle strength and performance of PEO patients (P1-P4) on normal diet (ND) and after modified Atkins diet (mAD) (one and six months and 2.5 years after initiation of diet).


D - G . Spiroergometry results from PEO patients and control subjects on normal diet ( ND ) , as well as one month and six months after mAD initiation . PEO patients had higher serumlactate levels , and attenuated respiratory response to exercise . These effects were further enhanced after mAD and remained after 6 months of the study . mAD increased patients ' serumammonium ( NH4 + ) levels . Data presented as mean and SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. Spiroergometry results from PEO patients and control subjects on normal diet (ND), as well as one month and six months after mAD initiation. PEO patients had higher serumlactate levels, and attenuated respiratory response to exercise. These effects were further enhanced after mAD and remained after 6 months of the study. mAD increased patients' serumammonium (NH4+) levels. Data presented as mean and SD.


A . PEO patients , relative amino acid levels on normal diet ( PEO ) , immediately after finishing mAD ( PEO _ mAD ) , and one month after mAD . B . Control subjects , relative amino acid levels on normal diet ( Control ) and immediately after finishing mAD ( Control _ mAD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. PEO patients, relative amino acid levels on normal diet (PEO), immediately after finishing mAD (PEO_mAD), and one month after mAD.B. Control subjects, relative amino acid levels on normal diet (Control) and immediately after finishing mAD (Control_mAD).


C . Relative values of plasma metabolites in creatine pathway , compared to controls on ND . Right : illustration of major metabolites and pathway of creatine synthesis .

C. Relative values of plasma metabolites in creatine pathway, compared to controls on ND. Right: illustration of major metabolites and pathway of creatine synthesis.


D . Relative values of plasma purine and pyrimidine degradation pathway intermediates , compared to controls on ND . Right : illustration of major metabolites and synthesis of pyrimidine and purines .

O O O O O O O O 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. Relative values of plasma purine and pyrimidine degradation pathway intermediates, compared to controls on ND. Right: illustration of major metabolites and synthesis of pyrimidine and purines.


Gel shift assay of biotin - labeled mascRNA and 5S rRNA with the cytosol ( Cyto ) and the postnuclear membrane lysate ( Mem ) .

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

Gel shift assay of biotin-labeled mascRNA and 5S rRNA with the cytosol (Cyto) and the postnuclear membrane lysate (Mem).


Northern blotting of mascRNA ( top panel ) and western blotting of β - Tubulin and Mortalin ( bottom two panels ) in the cytosolic fractions and the postnuclear membrane fractions of HEK293 and HepG2 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

Northern blotting of mascRNA (top panel) and western blotting of β-Tubulin and Mortalin (bottom two panels) in the cytosolic fractions and the postnuclear membrane fractions of HEK293 and HepG2 cells.


SDS - PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples . Biotin - labeled mascRNA ( S ) and the antisense RNA ( AS ) were used to pull down the RNA - protein complexes . Representive data of mass spectrometry analysis of the RNA pulldown samples . Proteins most enriched in mascRNA ( S ) RNA pulldown sample are listed .

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

SDS-PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples. Biotin-labeled mascRNA (S) and the antisense RNA (AS) were used to pull down the RNA-protein complexes.   Representive data of mass spectrometry analysis of the RNA pulldown samples. Proteins most enriched in mascRNA (S) RNA pulldown sample are listed.


Western blots of the proteins in mascRNA S and AS pulldown samples ( top 4 panels ) , and mascRNA and menRNA pulldown samples ( Bottom 2 panels ) . β - tubulin was used as a loading control .

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

Western blots of the proteins in mascRNA S and AS pulldown samples (top 4 panels), and mascRNA and menRNA pulldown samples (Bottom 2 panels). β-tubulin was used as a loading control.


RT - PCR analysis of RNAs extracted from ARS immunoprecipitation ( RIP ) samples ( erro bars : mean ± SD ; biological replicates n = 3 ) . Eight complex - forming ARSs ( DARS , EPRS , IARS , KARS , LARS , MARS , QARS and RARS ) and GARS tagged with FLAG were overexpressed in HEK cells . ARS - RNA complexes were imminoprecipitated with anti - FLAG Magnetic Beads . The cell line constructed with PQCXIP vector ( PQ ) was used as a negative control . GAPDH was used as an internal control for qRT - PCR .

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

RT-PCR analysis of RNAs extracted from ARS immunoprecipitation (RIP) samples (erro bars: mean ± SD; biological replicates n = 3). Eight complex-forming ARSs (DARS, EPRS, IARS, KARS, LARS, MARS, QARS and RARS) and GARS tagged with FLAG were overexpressed in HEK cells. ARS-RNA complexes were imminoprecipitated with anti-FLAG Magnetic Beads. The cell line constructed with PQCXIP vector (PQ) was used as a negative control. GAPDH was used as an internal control for qRT-PCR.


Northern blot validation of mascRNA in the RIP samples . 5S rRNA from the RIP samples was used as an internal control .

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

Northern blot validation of mascRNA in the RIP samples. 5S rRNA from the RIP samples was used as an internal control.


Northern blot ( top 2 panels ) and western blot ( bottom 3 panels ) analysis of ARS immunoprecipitation samples ( RIP ) . RIP was performed using EPRS , QARS and GARS antibodies .

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

Northern blot (top 2 panels) and western blot (bottom 3 panels) analysis of ARS immunoprecipitation samples (RIP). RIP was performed using EPRS, QARS and GARS antibodies.


Gel shift assay of biotin - labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody .

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

Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.


Biotin - labeled mascRNA ( sense ( S ) and antisense ( AS ) ) and the acceptor stem mutants ( Mut1 - 8 ) were used to pulldown the RNA - protein complexes . Top panel : western blotting of QARS in the pulldown samples . β - Tubulin was used as an cytosolic input control for the pulldown . Bottom panel : Biotin detection of the biotin - labeled mascRNA and the mutants . Biotin - labeled mascRNA ( sense ( S ) and antisense ( AS ) ) and the anticodon stem - loop mutants ( Anti - Mut1 - 9 ) were used to pulldown the RNA - protein complexes . Top panel : western blotting of QARS in the pulldown samples . β - Tubulin was used as an cytosolic input control for the pulldown . Bottom panel : Biotin detection of the biotin - labeled mascRNA and the mutants .

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

Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the acceptor stem mutants (Mut1-8) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.   Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the anticodon stem-loop mutants (Anti-Mut1-9) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.


Gel shift assay of biotin - labeled mascRNA or the mutants ( Anti - Mut1 and Mut1 ) with the cytosol .

Gel shift assay of biotin-labeled mascRNA or the mutants (Anti-Mut1 and Mut1) with the cytosol.


Northern blot analysis of mascRNA in mascRNA overexpressing HEK cells ( mascRNA ) and the cells expressing the scrambled RNA ( Scramble ) . 5S rRNA was used as an internal control .

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

Northern blot analysis of mascRNA in mascRNA overexpressing HEK cells (mascRNA) and the cells expressing the scrambled RNA (Scramble). 5S rRNA was used as an internal control.


qRT - PCR analysis of MALAT1 ( total RNAs used as templates ) in mascRNA overexpressing ( mascRNA ) and the scrambled RNA expressing ( Scramble ) 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

qRT-PCR analysis of MALAT1 (total RNAs used as templates) in mascRNA overexpressing (mascRNA) and the scrambled RNA expressing (Scramble) cells.


Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA ( mascRNA ) or the scrambled RNA ( Scramble ) . Left : biotin detection of the labeled nascent proteins ; right : coomassie staining of total proteins . Quantification of the levels of the labeled nascent proteins in Panel C .

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

Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel C .


Northern blot analysis of mascRNA in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA . 5S rRNA was used as an internal control .

Northern blot analysis of mascRNA in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. 5S rRNA was used as an internal control.


Detection of the newly synthesized proteins in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA . Left : biotin detection of the labeled nascent proteins ; right : coomassie staining of total proteins . Quantification of the levels of the labeled nascent proteins in Panel F .

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

Detection of the newly synthesized proteins in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel F.


Northern blot analysis of mascRNA in mascRNA overexpressing ( mascRNA ) , and the mutants ( Anti - Mut1 and Mut1 ) or the scrambled RNA ( Scramble ) expressing HEK cells . 5S rRNA was used as an internal control .

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

Northern blot analysis of mascRNA in mascRNA overexpressing (mascRNA), and the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble) expressing HEK cells. 5S rRNA was used as an internal control.


Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA ( mascRNA ) , the mutants ( Anti - Mut1 and Mut1 ) or the scrambled RNA ( Scramble ) . Left : biotin detection of the labeled nascent proteins ; right : coomassie staining of total proteins . Quantification of the levels of the labeled nascent proteins in Panel I .

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

Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel I.


Decay of the pulse Biotin - labeled proteins in HEK cells stably expressing mascRNA ( mascRNA ) or the scrambed RNA ( Scramble )

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

Decay of the pulse Biotin-labeled proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambed RNA (Scramble)


Western blot detection of ARS proteins in mascRNA overexpressing ( mascRNA ) or the scrambled RNA expressing ( Scramble ) cells . β - Tubulin was used as a loading control . Quantification of the protein levels in Panel A .

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

Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells. β-Tubulin was used as a loading control.   Quantification of the protein levels in Panel A.


Western blot detection of ARS proteins in HEK cells stably expressing mascRNA ( mascRNA ) , the mutants ( Anti - Mut1 and Mut1 ) or the scrambled RNA ( Scramble ) . β - Tubulin was used as a loading control .

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

Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control.


Western blot detection of ARS proteins in HEK cells stably expressing mascRNA ( mascRNA ) , the mutants ( Anti - Mut1 and Mut1 ) or the scrambled RNA ( Scramble ) . β - Tubulin was used as a loading control . Quantification of the protein levels in Panel C .

Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control. Quantification of the protein levels in Panel C.


qRT - PCR analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells .

qRT-PCR analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells.


Northern blot analysis of mascRNA in MALAT1 knockdown and the control cells . 5S rRNA was used as an internal control .

Northern blot analysis of mascRNA in MALAT1 knockdown and the control cells. 5S rRNA was used as an internal control.


Western blot detection of ARS proteins in MALAT1 knockdown and the control cells . Quantification of the protein levels in Panel G .

Western blot detection of ARS proteins in MALAT1 knockdown and the control cells.   Quantification of the protein levels in Panel G.


qRT - PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing ( mascRNA ) or the scrambled RNA expressing ( Scramble ) 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

qRT-PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells.


Western blot detection of ARS proteins in mascRNA overexpressing ( mascRNA ) or the scrambled RNA expressing ( Scramble ) cells , before and after emetine and cycloheximide treatment . β - Tubulin was used as a loading control . Quantification of the relative QARS protein levels in Panel J . Quantification of the relative GARS protein levels in Panel J .

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

Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells, before and after emetine and cycloheximide treatment. β-Tubulin was used as a loading control.   Quantification of the relative QARS protein levels in Panel J.   Quantification of the relative GARS protein levels in Panel J.


Western blot detection of endogenous and FLAG - tagged ARS proteins in HEK cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . β - Tubulin was used as a loading control .

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

Western blot detection of endogenous and FLAG-tagged ARS proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.


Detection of the newly synthesized proteins in HEK cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins . Quantification of the labeled nascent protein levels in Panel B .

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

Detection of the newly synthesized proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel B.


Western blot detection of FLAG - tagged ARS proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . β - Tubulin was used as a loading control .

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

Western blot detection of FLAG-tagged ARS proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.


Detection of the newly synthesized proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins . Quantification of the labeled nascent protein levels in Panel E .

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

Detection of the newly synthesized proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel E.


Western blot detection of FLAG - tagged ARS proteins in mascRNA anticodon stem - loop mutant Anti - Mut1 expressing cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . β - Tubulin was used as a loading control .

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

Western blot detection of FLAG-tagged ARS proteins in mascRNA anticodon stem-loop mutant Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.


Detection of the newly synthesized proteins in mascRNA Anti - Mut1 expressing cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins .

Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.


Detection of the newly synthesized proteins in mascRNA Anti - Mut1 expressing cells transiently transfected with PQCXIP vector ( PQ ) , QARS expressing ( QARS ) or GARS expressing ( GARS ) plasmid . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins . Quantification of the labeled nascent protein levels in Panel H .

Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins. Quantification of the labeled nascent protein levels in Panel H.


Western blot detection of FLAG - tagged ARS proteins in MALAT1 knockdown cells with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) . β - Tubulin was used as a loading control .

Western blot detection of FLAG-tagged ARS proteins in MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). β-Tubulin was used as a loading control.


Detection of the newly synthesized proteins in wild - type HEK cells ( WT ) and MALAT1 knockdown cells ( sh - MALAT1 ) , or MALAT1 knockdown cells with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins .

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

Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.


Detection of the newly synthesized proteins in wild - type HEK cells ( WT ) and MALAT1 knockdown cells ( sh - MALAT1 ) , or MALAT1 knockdown cells with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins . Quantification of the labeled nascent protein levels in Panel K .

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

Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins. Quantification of the labeled nascent protein levels in Panel K.


Detection of the newly synthesized proteins in QARS ( shQARS ) or GARS ( shGARS ) knockdown cells and the control cells stably overexpressing mascRNA ( mascRNA ) or expressing the scrambled RNA ( Scramble ) . Left , biotin detection of the labeled nascent proteins ; right , coomassie staining of total proteins . Quantification of the labeled nascent protein levels in Panel M .

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

Detection of the newly synthesized proteins in QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the scrambled RNA (Scramble). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel M.


Northern blotting of mascRNA and 5S rRNA in the lysates of HEK cells before and after FBS starvation .

Northern blotting of mascRNA and 5S rRNA in the lysates of HEK cells before and after FBS starvation.


Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation .

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

Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation.


Soft agar colony formation assay of HEK , HeLa or MCF - 7 cells overexpressing mascRNA or expressing the the scrambled RNA . Quantification of stained colones in Panel C .

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

Soft agar colony formation assay of HEK, HeLa or MCF-7 cells overexpressing mascRNA or expressing the the scrambled RNA.   Quantification of stained colones in Panel C.


Soft agar colony formation assay of HEK cells overexpressing mascRNA , or expressing the mutants ( Anti - Mut1 and Mut1 ) or the the scrambled RNA ( Scramble ) .

Soft agar colony formation assay of HEK cells overexpressing mascRNA, or expressing the mutants (Anti-Mut1 and Mut1) or the the scrambled RNA (Scramble).


Soft agar colony formation assay of QARS ( shQARS ) or GARS ( shGARS ) knockdown cells and the control cells stably overexpressing mascRNA ( mascRNA ) or expressing the the scrambled RNA ( Scramble ) .

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

Soft agar colony formation assay of QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the the scrambled RNA (Scramble).


Soft agar colony formation assay of MALAT1 knockdown cells ( shMALAT1 ) and the control cells ( Control ) with stable QARS ( QARS ) or GARS ( GARS ) overexpression or without ( PQ ) .

Soft agar colony formation assay of MALAT1 knockdown cells (shMALAT1) and the control cells (Control) with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ).


A , B , Western blotting analysis of proteins co - precipitated with anti - FLAG antibody from testis extracts of E15 . 5 wild - type embryos and transgenic embryos expressing FLAG - tagged NANOS2 with or without RNase ( A ) or from extracts of HeLa cells transfected with FLAG - tagged DND1 and with or without HA - tagged NANOS2 ( B ) .

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

A, B, Western blotting analysis of proteins co-precipitated with anti-FLAG antibody from testis extracts of E15.5 wild-type embryos and transgenic embryos expressing FLAG-tagged NANOS2 with or without RNase (A) or from extracts of HeLa cells transfected with FLAG-tagged DND1 and with or without HA-tagged NANOS2 (B).


Precipitates were analyzed with the indicated antibodies . C , GST pull - down assay using E . coli extracts expressing GST , GST - fused NANOS2 , or NANOS3 mixed with MBP - tagged LacZ or DND1 . Precipitates were analyzed by Coomassie Brilliant Blue ( CBB ) staining or western blotting with an anti - MBP antibody . An arrowhead indicates MBP - DND1 co - precipitated with GST - tagged NANOS2 or NANOS3 .

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

Precipitates were analyzed with the indicated antibodies. C, GST pull-down assay using E. coli extracts expressing GST, GST-fused NANOS2, or NANOS3 mixed with MBP-tagged LacZ or DND1. Precipitates were analyzed by Coomassie Brilliant Blue (CBB) staining or western blotting with an anti-MBP antibody. An arrowhead indicates MBP-DND1 co-precipitated with GST-tagged NANOS2 or NANOS3.


D , E , Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with FLAG - tagged NANOS2 , NANOS2 ( ΔN10 ) , NANOS2 ( ΔC10 ) , NANOS2 ( C61A , C96A ) or NANOS3 , and HA - tagged DND1 ( D )

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

D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D)


D , E , Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with FLAG - tagged NANOS2 , NANOS2 ( ΔN10 ) , NANOS2 ( ΔC10 ) , NANOS2 ( C61A , C96A ) or NANOS3 , and HA - tagged DND1 ( D ) or with FLAG - tagged NANOS2 , NANOS2 ( C61A ) , NANOS2 ( C96A ) or NANOS2 ( C61A , C96A ) , and HA - tagged DND1 ( E ) .

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

D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D) or with FLAG-tagged NANOS2, NANOS2 (C61A), NANOS2 (C96A) or NANOS2 (C61A, C96A), and HA-tagged DND1 (E).


Precipitates were analyzed with the indicated antibodies . F , GST pull - down assay using E . coli extracts expressing GST - fused DND1 mixed with MBP - tagged LacZ , NANOS2 , NANOS2 ( C61A , C96A ) or NANOS2 ( 60 - 114 ) . Arrowheads indicate MBP - tagged NANOS2 ( lane 2 ) or NANOS2 ( 60 - 114 ) ( lane 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Precipitates were analyzed with the indicated antibodies. F, GST pull-down assay using E. coli extracts expressing GST-fused DND1 mixed with MBP-tagged LacZ, NANOS2, NANOS2 (C61A, C96A) or NANOS2 (60-114). Arrowheads indicate MBP-tagged NANOS2 (lane 2) or NANOS2 (60-114) (lane 4).


Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with HA - tagged NANOS1 , NANOS2 or NANOS3 , and FLAG - tagged DND1 ( A )

Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A)


Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with HA - tagged NANOS1 , NANOS2 or NANOS3 , and FLAG - tagged DND1 ( A ) or with FLAG - tagged PUMILIO1 or PUMILIO2 , and HA - tagged NANOS1 , NANOS2 or NANOS3 with or without MYC - tagged DND1 ( B ) .

Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A) or with FLAG-tagged PUMILIO1 or PUMILIO2, and HA-tagged NANOS1, NANOS2 or NANOS3 with or without MYC-tagged DND1 (B).


C , Characterization of antibodies against PUMILIO1 and PUMILIO2 . Western blotting analyses of Flag - tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG - tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG , PUMILIO1 or PUMILIO2 . Note that the antibodies against PUMILIO1 specifically recognized FLAG - tagged PUMILIO1 while the anti - PUMILIO2 antibody could detect both Flag - tagged PUMILIO1 and PUMILIO2 but less actively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Characterization of antibodies against PUMILIO1 and PUMILIO2. Western blotting analyses of Flag-tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG-tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG, PUMILIO1 or PUMILIO2. Note that the antibodies against PUMILIO1 specifically recognized FLAG-tagged PUMILIO1 while the anti-PUMILIO2 antibody could detect both Flag-tagged PUMILIO1 and PUMILIO2 but less actively.


D , FLAG - tagged NANOS2 was immunoprecipitated with an anti - FLAG antibody from E15 . 5 testicular extracts from transgenic mice expressing FLAG - tagged NANOS2 . Precipitates were analyzed by western blotting with the indicated antibodies . Note that co - precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co - precipitated .

D, FLAG-tagged NANOS2 was immunoprecipitated with an anti-FLAG antibody from E15.5 testicular extracts from transgenic mice expressing FLAG-tagged NANOS2. Precipitates were analyzed by western blotting with the indicated antibodies. Note that co-precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co-precipitated.


Figure 3 | DND1 co - localizes with NANOS2 in P - bodies . A − C , Sections of male gonads from E15 . 5embryos were immunostained with antibodies against DND1 ( green ) and DCP1a ( red ) . D − F , Squash preparation of a male gonocyte from E16 . 5embryoimmunostained with antibodies against DND1 ( red ) and DCP1a ( green ) . Arrowheads indicate co - localization of DND1 and DCP1a . G − I , Sections of male gonads from E15 . 5embryos were immunostained with antibodies against DND1 ( red ) and NANOS2 ( green ) . Arrowheads indicate co - localization of DND1 and NANOS2 . J − M , NIH3T3 cells transfected with HA - tagged Dnd1 and FLAG - tagged Nanos2 were then immunostained with antibodies against DND1 ( J ) ( magenta ) , NANOS2 ( K ) ( red ) , and DCP1a ( L ) ( green ) . N − Y , Biomolecular fluorescence complementation assay . NIH3T3 cells transfected with VENUS - C - fused Dnd1 and VENUS - N - fused Nanos2 ( N − U ) or Nanos2 ( C61A , C96A ) ( V - γ ) were immunostained with antibodies against DND1 ( N , S , V ) , NANOS2 ( O , W , α ) or DDX6 ( R , Z ) . Then , the signals of VENUS fusion protein were visualized ( P , T , X , β ) . Arrowheads in Z indicate P - bodies . DNA was labeled with DAPI ( blue ) . Scale bars : 50 m in A for A − C ; 10 m in D for D - F , G for G − I , J for J − M , N for N − γ . Insets in A - C and J - γ show an enlarged vision of each picture to better depict localization of each protein . See also Fig . EV2T - Y .

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

Figure 3 | DND1 co-localizes with NANOS2 in P-bodies. A−C, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (green) and DCP1a (red). D−F, Squash preparation of a male gonocyte from E16.5embryoimmunostained with antibodies against DND1 (red) and DCP1a (green). Arrowheads indicate co-localization of DND1 and DCP1a. G−I, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (red) and NANOS2 (green). Arrowheads indicate co-localization of DND1 and NANOS2. J−M, NIH3T3 cells transfected with HA-tagged Dnd1 and FLAG-tagged Nanos2 were then immunostained with antibodies against DND1 (J) (magenta), NANOS2 (K) (red), and DCP1a (L) (green). N−Y, Biomolecular fluorescence complementation assay. NIH3T3 cells transfected with VENUS-C-fused Dnd1 and VENUS-N-fused Nanos2 (N−U) or Nanos2 (C61A, C96A) (V-γ) were immunostained with antibodies against DND1 (N, S, V), NANOS2 (O, W, α) or DDX6 (R, Z). Then, the signals of VENUS fusion protein were visualized (P, T, X, β). Arrowheads in Z indicate P-bodies. DNA was labeled with DAPI (blue). Scale bars: 50 m in A for A−C; 10 m in D for D-F, G for G−I, J for J−M, N for N−γ. Insets in A-C and J-γ show an enlarged vision of each picture to better depict localization of each protein. See also Fig. EV2T-Y.


A , Western blotting analyses of proteins in testes from E13 . 5 to E16 . 5 embryos of Dnd1flox / flox or Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) each administered with tamoxifen at E13 . 5 and E15 . 5 embryos of Nanos2 + / - or Nanos2 - / - with the indicated antibodies .

A, Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1flox/flox or Dnd1flox/flox_Tg(Oct4PE-CreERT2) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2+/- or Nanos2-/- with the indicated antibodies.


B - P , Sections of testes from Dnd1flox / flox ( B , E , H , K , N ) , Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) ( C , F , I , L , O ) or Nanos2 - / - ( D , G , J , M , P ) embryos were prepared at E16 . 5 and then immunostained with antibodies against pH3 ( B - D ) , STRA8 ( E - G ) , SYCP3 ( H - J ) , activated - Caspase3 ( K - M ) or LAMININ ( N - P ) ( green ) . Germ cells were immunostained with TRA98 ( B - D ) or DAZL ( N - P ) ( red ) , and DNA was labeled with DAPI ( blue ) . Tamoxifen was administered at E13 . 5 . Scale bars : 50 m in B for B − D and K - P , 50 m in E for E − J . See also Fig . EV3 .

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

B-P, Sections of testes from Dnd1flox/flox (B, E, H, K, N), Dnd1flox/flox_Tg(Oct4PE-CreERT2) (C, F, I, L, O) or Nanos2-/- (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B-D), STRA8 (E-G), SYCP3 (H-J), activated-Caspase3 (K-M) or LAMININ (N-P) (green). Germ cells were immunostained with TRA98 (B-D) or DAZL (N-P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in B for B−D and K-P, 50 m in E for E−J. See also Fig. EV3.


A - L , Sections of testes from Dnd1flox / flox ( A , B , C , G , H , I , M , N , O ) and Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) ( D , E , F , J , K , L , P , Q , R ) embryos were prepared at E16 . 5 and then immunostained with antibodies against NANOS2 ( A , D ) ( green ) , DCP1a ( G , J ) ( green ) , DDX6 ( M , P ) ( green ) and TRA98 ( B , E , H , K , N , Q ) . DNA was labeled with DAPI ( blue ) . Tamoxifen was administered at E13 . 5 . Scale bars : 50 m in A for A − F ; 50 m in G for G - L ; 50 m in M for M - R . Insets show an enlarged vision of each picture to better depict localization of NANOS2 , DCP1a and DDX6 .

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

A-L, Sections of testes from Dnd1flox/flox (A, B, C, G, H, I, M, N, O) and Dnd1flox/flox_Tg(Oct4PE-CreERT2) (D, E, F, J, K, L, P, Q, R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in A for A−F; 50 m in G for G-L; 50 m in M for M-R. Insets show an enlarged vision of each picture to better depict localization of NANOS2, DCP1a and DDX6.


S - V , Immunoprecipitation with an anti - FLAG antibody from E15 . 5 male gonadal extracts of wild - type and the transgenic mouse line expressing FLAG - tagged DND1 ( S , T ) , or with an anti - NANOS2 antibody from E15 . 5 male gonadal extracts of Dnd1flox / flox mice with or without Oct4PE - CreERT2 ( U , V ) . Precipitates were analyzed by western blotting ( S , U ) or by RT - qPCR ( T , V ) . The RT - qPCR data are shown as average relative mRNA levels ± SEs ( n = 3 ) .

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

S-V, Immunoprecipitation with an anti-FLAG antibody from E15.5 male gonadal extracts of wild-type and the transgenic mouse line expressing FLAG-tagged DND1 (S, T), or with an anti-NANOS2 antibody from E15.5 male gonadal extracts of Dnd1flox/flox mice with or without Oct4PE-CreERT2 (U, V). Precipitates were analyzed by western blotting (S, U) or by RT-qPCR (T, V). The RT-qPCR data are shown as average relative mRNA levels ± SEs (n=3).


W , X , Western blotting analyses of proteins in NANOS2 - depleted ( W ) or DND1 - depleted ( X ) testis extracts from E15 . 5 embryos . Proteins were analyzed with the indicated antibodies .

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

W, X, Western blotting analyses of proteins in NANOS2-depleted (W) or DND1-depleted (X) testis extracts from E15.5 embryos. Proteins were analyzed with the indicated antibodies.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) or control followed by western blot analysis ( A ) The basal levels of p53 normalized to GAPDH were arbitrarily set at 1 . 0 and the fold change is shown in ( ii ) ( n = 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 O O O O O O O O O O O O O O O O O O O O O O

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I) or control followed by western blot analysis (A) The basal levels of p53 normalized to GAPDH were arbitrarily set at 1.0 and the fold change is shown in (ii) (n=4).


Treatment of HCT116 cells with siRNA targeting Ku70 or control ( A - I ) combined or not with a treatment with doxorubicin ( Doxo ) ( positive control for phosphorylation of ATM and p53 ) ( B ) followed by western blot analysis .

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

Treatment of HCT116 cells with siRNA targeting Ku70 or control (A-I) combined or not with a treatment with doxorubicin (Doxo) (positive control for phosphorylation of ATM and p53) (B) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , ASF ( C ) ( positive control for y - H2AX activation [ 29 ] ) or control ( A - I ) followed by western blot 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 O O O

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), ASF (C) (positive control for y-H2AX activation [29])or control (A-I) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with wortmannin ( WTN ) ( D ) followed by western blot 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with wortmannin (WTN) (D) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with NU - 7441 ( E , F ) followed by western blot 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with NU-7441 (E,F) followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , XRCC4 ( F ) or control ( A - I ) combined or not with a treatment with NU - 7441 ( E , F ) , followed by western blot 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), XRCC4 (F) or control (A-I) combined or not with a treatment with NU-7441 (E,F), followed by western blot analysis.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) followed by RT - qPCR analysis ( G ) . In RT - qPCR analysis ( G ) , mRNA levels were standardized against HPRT mRNA ( n = 3 ) . The two panels of Fig . 1C are partion of the same gel . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ) . All error bars reflect SEM .

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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) followed by RT-qPCR analysis (G). In RT-qPCR analysis (G), mRNA levels were standardized against HPRT mRNA (n=3). The two panels of Fig. 1C are partion of the same gel. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01). All error bars reflect SEM.


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with MG132 ( H ) followed by western blot analysis ( A - F , H - I ) .

O O O O 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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with MG132 (H) followed by western blot analysis (A-F, H-I).


Treatment of HCT116 cells with siRNA targeting Ku70 ( A - I ) , or control ( A - I ) combined or not with a treatment with cycloheximide ( CHX ) ( I ) followed by western blot analysis ( A - F , H - I ) .

O O O O 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

Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with cycloheximide (CHX) (I) followed by western blot analysis (A-F, H-I).


( A ) RNA affinity chromatography using the p53 5 ' UTR ( 5 ' p53 ) or a portion of the p47 5 ' UTR ( 5 ' p47 ) ( depicted in ( i ) ) and HeLa cytoplasmic extracts ( CE ) , followed by western blot analysis ( ii ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNA affinity chromatography using the p53 5'UTR (5'p53) or a portion of the p47 5'UTR (5'p47) (depicted in (i)) and HeLa cytoplasmic extracts (CE), followed by western blot analysis (ii).


( B ) IP of in cellulo RNA - protein complexes ( RIP ) in HCT116 cells , followed either by RT - qPCR analysis . Bar plots represent the mean and the data of two independent experiments ( i ) or by western blot analysis ( ii ) . The relative p53 mRNA levels for each IP sample were normalized to the corresponding IP IgG and to the corresponding input sample and were plotted relatively to the HPRT mRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IP of in cellulo RNA-protein complexes (RIP) in HCT116 cells, followed either by RT-qPCR analysis. Bar plots represent the mean and the data of two independent experiments (i) or by western blot analysis (ii). The relative p53 mRNA levels for each IP sample were normalized to the corresponding IP IgG and to the corresponding input sample and were plotted relatively to the HPRT mRNA.


( C ) UV cross - linking of recombinant ( Ku70 / Ku80 ) with the in vitro transcribed 5 ' p53 with alpha 32P - GTP or alpha 32P - UTP ( i ) . UV cross - linking of recombinant Ku70 / Ku80 with the in vitro transcribed 5 ' p53 and the Ctrl ( 5 ' p53 antisense ) with alpha 32PGTP ( ii ) .

O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) UV cross-linking of recombinant (Ku70/Ku80) with the in vitro transcribed 5'p53 with alpha 32P-GTP or alpha 32P-UTP (i). UV cross-linking of recombinant Ku70/Ku80 with the in vitro transcribed 5'p53 and the Ctrl (5'p53 antisense) with alpha 32PGTP (ii).


( D , E ) RNA affinity chromatography using the different RNA constructs depicted in ( i ) , as described in A .

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

(D,E) RNA affinity chromatography using the different RNA constructs depicted in (i), as described in A.


( A ) Non polysomal ( NP ) and polysomal ( P ) fractions were extracted from HCT116 cells transfected with the siRNA Ku70 and quantitative RT - qPCR was performed using specific primers for p53 and HPRT mRNAs . The p53 mRNA levels in P and NP fractions were normalized to the input ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Non polysomal (NP) and polysomal (P) fractions were extracted from HCT116 cells transfected with the siRNA Ku70 and quantitative RT-qPCR was performed using specific primers for p53 and HPRT mRNAs. The p53 mRNA levels in P and NP fractions were normalized to the input (n=3)


( B ) Western blot from H1299 ( p53 - null ) cells treated with siRNA Ku70 or control ( ii ) , followed by transfections of RNA constructs depicted in ( i ) . p53 protein levels from the WT and the ΔB2 reporters depicted in ( i ) normalized to firefly luciferase ( transfection control reporter ) ( iii , n = 3 ) .

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

(B) Western blot from H1299 (p53-null) cells treated with siRNA Ku70 or control (ii), followed by transfections of RNA constructs depicted in (i). p53 protein levels from the WT and the ΔB2 reporters depicted in (i) normalized to firefly luciferase (transfection control reporter) (iii, n=3).


( C ) AnnexinV / PI staining and flow cytometry experiments performed as in ( B ) to assess cell death ( n = 3 ) .

O O O 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) AnnexinV/PI staining and flow cytometry experiments performed as in (B) to assess cell death (n=3).


( D ) RT - qPCR analysis of Bax , TP53I3 , BTG2 , GADD45α and p21 after siRNA - mediated Ku or control depletion in HCT116p53 + / + or HCT116p53 - / - ( n = 3 ) . Relative mRNA levels were standardized against GAPDH mRNA . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 NS , non significant ) . All error bars reflect SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR analysis of Bax, TP53I3, BTG2, GADD45α and p21 after siRNA-mediated Ku or control depletion in HCT116p53+/+ or HCT116p53-/- (n=3). Relative mRNA levels were standardized against GAPDH mRNA. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001 NS, non significant). All error bars reflect SEM.


( A ) Western blot analysis after treatment with Ku70 or control siRNA , followed by incubation with DMSO , etoposide ( ETO ) or bleomycin ( BLEO ) for 16 h ( i ) . p53 levels after Ku70 depletion normalized to siRNA control for each condition ( ii , n = 3 ) . All the experiments were performed with HCT116 cells . GAPDH : loading control in A , B , C . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

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

(A) Western blot analysis after treatment with Ku70 or control siRNA, followed by incubation with DMSO, etoposide (ETO) or bleomycin (BLEO) for 16 h (i). p53 levels after Ku70 depletion normalized to siRNA control for each condition (ii, n=3). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( B ) Western blot analysis as in A , except that tricostatin A ( TSA ) was used after Ku / control knockdown ( n = 4 ) . All the experiments were performed with HCT116 cells . GAPDH : loading control in A , B , C . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Western blot analysis as in A, except that tricostatin A (TSA) was used after Ku/control knockdown (n=4). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( C ) Western blot in cells treated either with DMSO , TSA , or ETO for 16 or 24 h . All the experiments were performed with HCT116 cells . GAPDH : loading control in A , B , C . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

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

(C) Western blot in cells treated either with DMSO, TSA, or ETO for 16 or 24 h. All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( D ) RNA affinity chromatography using the 5 ' p53RNA and total extracts from cells treated with TSA , ETO or BLEO for 24 h , followed by western blot analysis of Ku70 quantified and normalized to the input ( ii , n = 3 ) . All the experiments were performed with HCT116 cells . Statistical analysis by unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS , non significant ) . All error bars reflect SEM .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNA affinity chromatography using the 5'p53RNA and total extracts from cells treated with TSA, ETO or BLEO for 24 h, followed by western blot analysis of Ku70 quantified and normalized to the input (ii, n=3). All the experiments were performed with HCT116 cells. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.


( E ) RNA affinity chromatography using the 5 ' p53RNA and total extracts from cells transfected or not with PCAF encoding plasmid for 48 h , followed by western blot analysis . All the experiments were performed with HCT116 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 O O

(E) RNA affinity chromatography using the 5'p53RNA and total extracts from cells transfected or not with PCAF encoding plasmid for 48 h, followed by western blot analysis. All the experiments were performed with HCT116 cells.


( F ) RNA affinity chromatography using the 5 ' p53RNA or a dsDNA and total extracts from cells transfected with HA - tagged Ku70 wild - type ( WT ) or mutated ( Mut6E ) Ku70 [ 25 ] for 48 h , followed by western blot analysis . All the experiments were performed with HCT116 cells .

(F) RNA affinity chromatography using the 5'p53RNA or a dsDNA and total extracts from cells transfected with HA-tagged Ku70 wild-type (WT) or mutated (Mut6E) Ku70 [25] for 48 h, followed by western blot analysis. All the experiments were performed with HCT116 cells.


( G ) RNA affinity chromatography as in F , but with two additional Ku mutants ( K282 / 338Q and K282 / 338R ) , followed by western blot of Ku70 quantified and normalized to the input and to the loading control , PTB ( ii , n = 3 ) All the experiments were performed with HCT116 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 O O O O O O O O O O O O O O O O O O O O O

(G) RNA affinity chromatography as in F, but with two additional Ku mutants (K282/338Q and K282/338R), followed by western blot of Ku70 quantified and normalized to the input and to the loading control, PTB (ii, n=3) All the experiments were performed with HCT116 cells.


C , Comparison of wild - type colonies with phytochrome and hox - deletion strains in the presence of 0 . 12 mM menadion ( MD ) , 7 mM H2O2 or 2 mM tert - butyl hydroperoxide ( tBooH ) . The strains were incubated for 5 days at 28 ° C in the dark .

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 B-CONTROLLED_VAR I-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

C, Comparison of wild-type colonies with phytochrome and hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.


E , Overlay of normalized ( at 705 nm ) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO

E, Overlay of normalized (at 705 nm) difference spectra plus a difference spectrum of the photosensory domain in the presence of the bacterial HO BphO


B , HoxA - GFP and CitA - GFP ( ca . 170 amino acids from the N - terminus of citrate synthase ) were expressed over night at 30 ° C in minimal medium with 2 % threonine and 0 . 2 % glucose . Mitochondria were incubated with 100 μg / ml proteinase K ( PK ) for 20 min on ice . Crude extract ( CE ) , supernatant 1 ( S1 ) containing mitochondria , pellet 1 ( P1 ) , supernatant 2 ( S2 ) and pellet 2 ( P2 ) with mitochondria were analyzed by Western Blot using anti GFP antibodies . CitA - GFPc = cytoplasmic form ; CitA - GFPi = imported version .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HoxA-GFP and CitA-GFP (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2% threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.


C , D Role of the C - terminal anchor in HoxA . Microscopy was done as in A using a fluorescent microscope without the Airyscan technology . C , Expression of GFP - HoxA lacking the C - terminal anchor ( ∆ CTA ) in A . alternata . D , Expression of GFP fused to the CTA motif .

C, D Role of the C-terminal anchor in HoxA. Microscopy was done as in A using a fluorescent microscope without the Airyscan technology. C, Expression of GFP-HoxA lacking the C-terminal anchor (∆CTA) in A. alternata. D, Expression of GFP fused to the CTA motif.


A , BiFC ( split - YFP ) analysis of HOs and phytochrome . Strains ( SChS29 - 33 ) were grown over night at 28 ° C minimal medium with 2 % threonine and 0 . 2 % glucose . MitoTracker Red CMOXRos was used for mitochondrial staining . The mitotracker signal was observed in the RFP and the split - YFP signal in the YFP channel of the fluorescent microscope . Pictures in both channels were overlaid ( merge ) . The left pictures show the same hyphae in differential interference contrast ( DIC ) . The scale bar represents 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, BiFC (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2% threonine and 0.2% glucose. MitoTracker Red CMOXRos was used for mitochondrial staining. The mitotracker signal was observed in the RFP and the split-YFP signal in the YFP channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast (DIC). The scale bar represents 10 µm.


C , Co - expression of the HOXs along with the photosensory domain of A . nidulans FphA ( PGP ) over night at 20 ° C . PGP was purified via the Strep - tag system and the eluate concentrated ( upper panel ) . The lanes were loaded as follows : negative control before induction ( - ) , 20 h after induction ( 20 h ) crude extract ( CE ) , supernatant ( S ) , pellet ( P ) , flow through ( F ) and eluate ( E ) . The fractions were analyzed by Western Blot using the Anti - His antibody ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot using the Anti-His antibody (lower panel).


A , B , Biolayer interferometry analysis with Apo - PGP A and holo - PGP B . Proteins were immobilized at a streptavidin sensor tip . Free binding sites were blocked with biotin . Association and dissociated kinetics were recorded at the indicated HoxA concentrations . Kd values were calculated with the manufacturers software using a global fit for both

A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment


( A ) Immunofluorescent staining of ABCA12 ( green ) and Insulin ( red ) in mouse islets showing extensive co - localisation in β - cells as well as expression in other islet populations ( arrowheads , scale bar = 25μm ) . ( B ) Co - expression of Abca12 ( green ) and Insulin ( red ) in MIN6 cells ( scale bar = 10μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescent staining of ABCA12 (green) and Insulin (red) in mouse islets showing extensive co-localisation in β-cells as well as expression in other islet populations (arrowheads, scale bar=25μm). (B) Co-expression of Abca12 (green) and Insulin (red) in MIN6 cells (scale bar = 10μm).


( C ) LacZ staining ( blue ) to detect gene expression of the knocked - in LacZ cassette from the Abca12 locus in islets ( islet outlined , scale bar = 25μm ) .

O O O O O O O O O O O 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) LacZ staining (blue) to detect gene expression of the knocked-in LacZ cassette from the Abca12 locus in islets (islet outlined, scale bar = 25μm).


( D , E ) ABCA12 immunostaining and the development of hyperkeratotic skin disease in E18 . 5 Abca12tm1a / tm1a mouse embryos ( brackets indicate thickened epidermis , dashed line indicates epidermal basement membrane , scale bar = 50μm ) .

(D,E) ABCA12 immunostaining and the development of hyperkeratotic skin disease in E18.5 Abca12tm1a/tm1a mouse embryos (brackets indicate thickened epidermis, dashed line indicates epidermal basement membrane, scale bar = 50μm).


( F , G ) Immunohistochemical detection of cre expression ( green ) in pancreatic islets from conditional mouse lines showing co - expression with Insulin ( red ) ( scale bar = 50μm ) .

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

(F, G) Immunohistochemical detection of cre expression (green) in pancreatic islets from conditional mouse lines showing co-expression with Insulin (red)(scale bar = 50μm).


( H ) Detection of recombination of the Abca12 locus by PCR , indicating different alleles ( tm1c , wild type and tm1d ) .

O 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) Detection of recombination of the Abca12 locus by PCR, indicating different alleles (tm1c, wild type and tm1d).


( I - K ) Detection of ABCA12 expression ( green ) and deletion in pancreas sections from mice of indicated genotypes ( islets outlined , scale bar = 25μm ) .

O O O O O O O O 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-K) Detection of ABCA12 expression (green) and deletion in pancreas sections from mice of indicated genotypes (islets outlined, scale bar = 25μm).


( L ) Quantitation of ABCA12 expression by densitometry of Western blots from purified pancreatic islets ( values relative to wild type , n = 3 - 8 animals per genotype , mean ± SEM , * p = < 0 . 05 , Students t - test ) , showing representative blot with Actin loading control ( right ) .

(L) Quantitation of ABCA12 expression by densitometry of Western blots from purified pancreatic islets (values relative to wild type, n=3-8 animals per genotype, mean±SEM, *p=<0.05, Students t-test), showing representative blot with Actin loading control (right).


Average daily food intake of conditionally modified mice ( n = 4 - 10 animals / genotype , mean ± SEM ) .

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

Average daily food intake of conditionally modified mice (n=4-10 animals/genotype, mean±SEM).


( N ) animal body weight ( n = 4 - 10 animals / genotype , mean ± SEM ) .

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

(N) animal body weight (n=4-10 animals/genotype, mean±SEM).


( A - F ) Oral and intraperitoneal glucose tolerance testing in Abca12tm1d mice at 6 ( A , B ) , 8 ( C , D ) and 12 weeks ( E , F ) ( mean ± SEM , n = 4 - 9 mice per group , * p = < 0 . 05 , * * p < 0 . 01 Students 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-F) Oral and intraperitoneal glucose tolerance testing in Abca12tm1d mice at 6 (A,B), 8 (C, D) and 12 weeks (E,F) (mean±SEM, n=4-9 mice per group, * p=<0.05, **p<0.01 Students t-test).


( G ) Insulin tolerance in 12 week old mice ( mean ± SEM , n = 4 - 9 mice per group ) .

(G) Insulin tolerance in 12 week old mice (mean±SEM, n=4-9 mice per group).


( H ) Whole blood Hb1Ac levels in 16 week old Abca12tm1d mice ( mean ± SEM , n = 4 - 9 mice per group , * * p < 0 . 01 Students t - test ) .

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

(H) Whole blood Hb1Ac levels in 16 week old Abca12tm1d mice (mean±SEM, n=4-9 mice per group, **p<0.01 Students t-test).


( I ) Acute phase insulin secretion after intraperitoneal injection of glucose ( mean ± SEM , n = 4 - 9 animals per group , white / grey / black = Abca12 + / + , cre / + and Abca12tm1d respectively , mice at 8 weeks of age , * * p < 0 . 01 Students 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 O O 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) Acute phase insulin secretion after intraperitoneal injection of glucose (mean±SEM, n=4-9 animals per group, white/grey/black = Abca12+/+, cre/+ and Abca12tm1d respectively, mice at 8 weeks of age, **p<0.01 Students t-test).


( J ) Silencing of ABCA12 in MIN6 cells as assessed by Western blot of ABCA12 immuno - precipitate ( top panel ) or confocal microscopy in cells transfected with Dy547 - siRNAABCA12 ( lower panel , scale bar = 10μm ) .

O O O O O O O O O O O O O O O O O O O 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) Silencing of ABCA12 in MIN6 cells as assessed by Western blot of ABCA12 immuno-precipitate (top panel) or confocal microscopy in cells transfected with Dy547-siRNAABCA12 (lower panel, scale bar = 10μm).


( K ) Glucose stimulated insulin secretion ( GSIS ) from MIN6 cells after silencing of ABCA12 ( mean ± SEM , n = 4 replicates from assays in quadruplicate , * * * p < 0 . 0001 , Students t - test ) .

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

(K) Glucose stimulated insulin secretion (GSIS) from MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate, ***p<0.0001, Students t-test).


( L ) Cellular insulin content in MIN6 cells after silencing of ABCA12 ( mean ± SEM , n = 4 replicates from assays in quadruplicate ) .

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

(L) Cellular insulin content in MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate).


( A - C ) ( A ) ABCA1 , ( B ) ABCG1 and ( C ) LXRβ expression in islets of 8 week old mice assessed by Western blotting ( mean ± SEM , n = 3 - 6 mice per genotype , relative to β - actin controls , * p < 0 . 05 , * * p = < 0 . 01 , Students 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 O O O O O O O O O O O O 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) (A) ABCA1, (B) ABCG1 and (C) LXRβ expression in islets of 8 week old mice assessed by Western blotting (mean±SEM, n=3-6 mice per genotype, relative to β-actin controls, *p<0.05, **p=<0.01, Students t-test).


( D ) Western blot of cell lysates of MIN6 cells after silencing of Abca12 , probed with antibodies against ABCA1 , ABCG1 , SR - B1 and LXRβ .

O O O O O O 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) Western blot of cell lysates of MIN6 cells after silencing of Abca12, probed with antibodies against ABCA1, ABCG1, SR-B1 and LXRβ.


( E ) Total cholesterol ( COH ) or cholesteryl esters ( CE ) levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β - cells ( mean ± SEM n = 9 , 5 and 5 mice per genotype respectively ) .

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

(E) Total cholesterol (COH) or cholesteryl esters (CE) levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=9, 5 and 5 mice per genotype respectively).


( F ) Ceramides levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β - cells ( mean ± SEM n = 10 , 4 and 5 mice per genotype respectively ) .

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

(F) Ceramides levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=10, 4 and 5 mice per genotype respectively).


( G ) Lipid levels in MIN6 cells in which ABCA12 had been depleted by siRNA treatment ( mean ± SEM , n = 5 ( biological replicates assayed in triplicate ) , FC = free cholesterol , CE = cholesterol ester , Cer = ceramide , SM = sphingomyelin , TG = triacylglycerol , PC = phosphatidylcholine , * p < 0 . 05 , * * p = < 0 . 01 , Students 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-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(G) Lipid levels in MIN6 cells in which ABCA12 had been depleted by siRNA treatment (mean±SEM, n=5 (biological replicates assayed in triplicate), FC = free cholesterol, CE = cholesterol ester, Cer = ceramide, SM = sphingomyelin, TG = triacylglycerol, PC = phosphatidylcholine, *p<0.05, **p=<0.01, Students t-test).


( H ) Cholesterol efflux to lipid - free apoA - I ( 30 μg / ml , 2 h ) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 ( 4 μM ) ( mean ± SEM , n = 4 , ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students 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 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

(H) Cholesterol efflux to lipid-free apoA-I (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM)(mean±SEM, n=4, (biological replicates assayed in triplicate), *p=<0.05, Students t-test).


( I ) Cholesterol efflux to HDL ( 30 μg / ml , 2 h ) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 ( 4 μM ) ; ( mean ± SEM , n = 4 ( biological replicates assayed in triplicate ) ) .

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

(I) Cholesterol efflux to HDL (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM); (mean±SEM, n=4 (biological replicates assayed in triplicate)).


( J ) The effect of overexpression of ABCA1 on GSIS from MIN6 cells with ABCA12 deficiency ( mean ± SEM , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - test ) , inset shows a Western blot of cell lysates probed with anti - ABCA1 antibody in which the order of bands corresponds to the order of 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

(J) The effect of overexpression of ABCA1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test), inset shows a Western blot of cell lysates probed with anti-ABCA1 antibody in which the order of bands corresponds to the order of bars).


( K ) The effect of overexpression of ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency ( mean ± SEM , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - test , inset shows a Western blot of cell lysate probed with anti - ABCG1 anitbodies in which the order of bands corresponds to the order of bars ) .

(K) The effect of overexpression of ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test, inset shows a Western blot of cell lysate probed with anti-ABCG1 anitbodies in which the order of bands corresponds to the order of bars).


( L ) The effect of overexpression of ABCA1 and ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency ( mean ± SEM , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students 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 O O O O O O O O O O O

(L) The effect of overexpression of ABCA1 and ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).


( M ) The effect of activation of cells with TO901317 ( 4 μM ) on GSIS from MIN6 cells with ABCA12 deficiency ( mean ± SEM , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - test ) .

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

(M) The effect of activation of cells with TO901317 (4 μM) on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).


( N ) The effect of overexpression of LXRβ on GSIS from MIN6 cells with ABCA12 deficiency ( mean ± SEM , n = 4 ( biological replicates ) , * p = < 0 . 05 , Students t - test , inset shows a Western blot of cell lysates probed with anti - LXRβ antibody in which the order of bands corresponds to the order of bars ) .

(N) The effect of overexpression of LXRβ on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test, inset shows a Western blot of cell lysates probed with anti-LXRβ antibody in which the order of bands corresponds to the order of bars).


( B ) Scatter plot and heat map of differential gene expression between cre control and Abca12tm1d mice at 8 weeks of age . Legend indicates Log2 fold change values .

O O O O O O O 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) Scatter plot and heat map of differential gene expression between cre control and Abca12tm1d mice at 8 weeks of age. Legend indicates Log2 fold change values.


( C ) Circulating glucose levels after administration of intraperitoneal L - arginine in mice at 8 weeks of age , mean ± SEM , n = 3 mice per genotype , * p = < 0 . 05 of cre relative to tm1d , Students t - test ) .

O O O O B-MEASURED_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

(C) Circulating glucose levels after administration of intraperitoneal L-arginine in mice at 8 weeks of age, mean±SEM, n =3 mice per genotype, *p=<0.05 of cre relative to tm1d, Students t-test).


( D ) Representative images of insulin granule fusion events ( yellow circles ) in mice of indicated genotypes in response to administration of 15mM glucose ( scale bar = 10μm ) . Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel ( mean ± SEM , 8 - 10 islets / mouse , > 4 mice / genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 of cre relative to tm1d , Students 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 15mM glucose (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01, ***p=<0.001 of cre relative to tm1d, Students t-test).


( E ) Representative images of insulin granule fusion events ( yellow circles ) in mice of indicated genotypes in response to administration of 20mM KCl ( scale bar = 10μm ) . Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the right panel ( mean ± SEM , 8 - 10 islets / mouse , > 4 mice / genotype , * * p = < 0 . 01 of cre relative to tm1d , Students 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 20mM KCl (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the right panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01 of cre relative to tm1d, Students t-test).


( F ) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old animals . Fluorescence responses were corrected for basal fill ( F / F0 ) . ( mean ± SEM , each data point represents the average of 10 cells / coverslip for 13 - 16 coverslips for each of 4 wild type and 5 Abca12tm1d mice )

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

(F) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old animals. Fluorescence responses were corrected for basal fill (F/F0). (mean±SEM, each data point represents the average of 10 cells/coverslip for 13-16 coverslips for each of 4 wild type and 5 Abca12tm1d mice)


( G ) Expression of GLUT2 in isolated mouse islets at 8 weeks of age . The upper panel depicts a representative Western blot and the lower graph the quantitation of protein levels from 3 mice of each genotype , normalised to β - actin ( mean ± SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of GLUT2 in isolated mouse islets at 8 weeks of age. The upper panel depicts a representative Western blot and the lower graph the quantitation of protein levels from 3 mice of each genotype, normalised to β-actin (mean±SEM).


( H ) Insulin content of islets of Abca12tm1d mice ( mean ± SEM , n = 3 mice of each genotype , normalised to total protein content ) .

(H) Insulin content of islets of Abca12tm1d mice (mean±SEM, n=3 mice of each genotype, normalised to total protein content).


( A - C ) Transmission electron microscopy of β - cells from Abca12tm1d and control mice at 8 weeks of age . Panels A ' - C ' depict higher resolution images from the boxed regions in A - C ( scale bar = 1μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Transmission electron microscopy of β-cells from Abca12tm1d and control mice at 8 weeks of age. Panels A'-C' depict higher resolution images from the boxed regions in A-C (scale bar = 1μm).


( D ) Quantitation of the area of 3000 granules from each genotype ( mean ± SEM , n = 4 , 3 and 3 animals respectively , * p = < 0 . 05 of cre relative to tm1d , Students t - test ) . ( E ) Relative frequency distribution of granule areas from ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitation of the area of 3000 granules from each genotype (mean±SEM, n=4, 3 and 3 animals respectively, *p=<0.05 of cre relative to tm1d, Students t-test). (E) Relative frequency distribution of granule areas from (D).


( F ) Assessment of total cholesterol ( COH ) by mass spectroscopy in mice fed normal chow or a high cholesterol diet ( HCD ) ( mean ± SEM , n = 5 and 4 animals respectively , * * p = < 0 . 01 , Students t - test ) .

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

(F) Assessment of total cholesterol (COH) by mass spectroscopy in mice fed normal chow or a high cholesterol diet (HCD)(mean±SEM, n=5 and 4 animals respectively, **p=<0.01, Students t-test).


( G , H ) Quantification of change in mean granule area in Abca12tm1d on chow diet and Abca12tm1d on high cholesterol diet islets assessed by transmission electron microscopy ( n = 3000 granules / genotype , mean ± SEM , n = 4 , 3 , 3 and 4 animals respectively , * p = < 0 . 05 , Students t - test ) as an average and as a relative frequency distribution .

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

(G, H) Quantification of change in mean granule area in Abca12tm1d on chow diet and Abca12tm1d on high cholesterol diet islets assessed by transmission electron microscopy (n=3000 granules/genotype, mean±SEM, n=4, 3, 3 and 4 animals respectively, *p=<0.05, Students t-test) as an average and as a relative frequency distribution.