( B ) ChIP experiments were performed in SF10 wild - type , nat4 ∆ , NET1 - TAP and NET1 - TAP nat4 ∆ strains using an anti - IgG antibody . The enrichment from the antibody was normalized to input . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * * p ≤ 0 . 01 compared nat4 ∆ to WT values .

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(B) ChIP experiments were performed in SF10 wild-type, nat4∆, NET1-TAP and NET1-TAP nat4∆ strains using an anti-IgG antibody. The enrichment from the antibody was normalized to input. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared nat4∆ to WT values.


( C ) Quantitative real - time PCR analysis of rDNA copy number for young and old cells in BY4741 wild - type , nat4Δ and fob1Δ strains . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * * p ≤ 0 . 01 compared to corresponding WT 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 O O O O O O O O O O O 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) Quantitative real-time PCR analysis of rDNA copy number for young and old cells in BY4741 wild-type, nat4Δ and fob1Δ strains. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared to corresponding WT values.


( D ) Polysome profiling analysis performed in BY4741 wild - type and nat4Δ strains . The plots are representative of two independent experiments .

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

(D) Polysome profiling analysis performed in BY4741 wild-type and nat4Δ strains. The plots are representative of two independent experiments.


( C ) Gene expression analysis of selected upregulated stress - response genes ( according to results in 3A ) in BY4741 wild - type and nat4 ∆ strains grown under NCR and CR conditions . Expression levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM of 3 independent experiments . p - values were obtained comparing results to wild - type under NCR conditions , and calculated by unpaired two - tailed Student ' s t - test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene expression analysis of selected upregulated stress-response genes (according to results in 3A) in BY4741 wild-type and nat4∆ strains grown under NCR and CR conditions. Expression levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained comparing results to wild-type under NCR conditions, and calculated by unpaired two-tailed Student's t-test: * p ≤ 0.05; ** p ≤ 0.01.


( D ) Gene expression analysis showing fold induction levels ( Log2 ) of selected genes comparing BY4741 wild - type and STE5p - NAT4 strains under NCR ( 2 % glucose ) and CR ( 0 . 1 % glucose ) conditions . Expression levels were normalized to TAF10 , whose expression remains unchanged .

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

(D) Gene expression analysis showing fold induction levels (Log2) of selected genes comparing BY4741 wild-type and STE5p-NAT4 strains under NCR (2% glucose) and CR (0.1% glucose) conditions. Expression levels were normalized to TAF10, whose expression remains unchanged.


( A ) RLS analysis for BY4741 wild - type and nat4 - E186A strains . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

(A) RLS analysis for BY4741 wild-type and nat4-E186A strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: *** p ≤ 0.001; **** p ≤ 0.0001.


( B ) Mean RLS for YSC5106 wild - type , nat4 ∆ and H4S1D strains .

(B) Mean RLS for YSC5106 wild-type, nat4∆ and H4S1D strains.


( C ) Gene expression analysis of the indicated stress - induced genes in YSC5106 wild - type , nat4 ∆ and H4S1D strains . Expression levels were normalized to RPP0 whose expression remains unchanged . Error bars , SEM of 3 independent experiments . p - values were obtained by comparing nat4 ∆ and H4S1D to wild - type values and calculated by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene expression analysis of the indicated stress-induced genes in YSC5106 wild-type, nat4∆ and H4S1D strains. Expression levels were normalized to RPP0 whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained by comparing nat4∆ and H4S1D to wild-type values and calculated by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01.


( D ) Gene expression analysis of the indicated stress - induced genes in BY4742 wild - type , nat4 ∆ and nat4 ∆ [ hNAA40 ] strains . Expression levels were normalized to ACT1 , whose expression remains unchanged .

O O O O O O O O O O O O O O O O O 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) Gene expression analysis of the indicated stress-induced genes in BY4742 wild-type, nat4∆ and nat4∆[hNAA40] strains. Expression levels were normalized to ACT1, whose expression remains unchanged.


( A ) RLS analysis for YSC5106 wild - type , nat4 ∆ , H4R3K and double mutant strains . Values in parenthesis indicate mean lifespan . Statistical significance was determined by one - way ANOVA test : * * * * p ≤ 0 . 0001 .

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

(A) RLS analysis for YSC5106 wild-type, nat4∆, H4R3K and double mutant strains. Values in parenthesis indicate mean lifespan. Statistical significance was determined by one-way ANOVA test: **** p ≤ 0.0001.


( B ) Gene expression analysis of the indicated stress - induced genes in YSC5106 WT , nat4 ∆ , H4R3K and double mutant strains . Expression levels were normalized to RPP0 , whose expression remains unchanged .

O O O O O O O O O O O O O O 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) Gene expression analysis of the indicated stress-induced genes in YSC5106 WT, nat4∆, H4R3K and double mutant strains. Expression levels were normalized to RPP0, whose expression remains unchanged.


( A ) Gene expression analysis of the indicated stress - induced genes in young and old cells isolated from YSC5106 WT , nat4 ∆ and H4R3K nat4 ∆ strains . Expression levels were normalized to ACT1 , whose expression remains unchanged . Error bars , SEM of 3 independent experiments . P - values were calculated by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 compared to young wild - type ; arrowhead p ≤ 0 . 05 compared to old wild - type .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene expression analysis of the indicated stress-induced genes in young and old cells isolated from YSC5106 WT, nat4∆ and H4R3K nat4∆ strains. Expression levels were normalized to ACT1, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. P-values were calculated by unpaired two-tailed Student's t test: * p ≤ 0.05 compared to young wild-type; arrowhead p ≤ 0.05 compared to old wild-type.


( B ) RLS analysis for WT , nat4 ∆ , pnc1 ∆ and double mutant strains in BY4741 genetic background . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ns = non - significant .

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

(B) RLS analysis for WT, nat4∆, pnc1∆ and double mutant strains in BY4741 genetic background. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. ns = non-significant.


( C ) Mean RLS in BY4741 wild - type , msn2 ∆ msn4 ∆ double and msn2 ∆ msn4 ∆ nat4 ∆ triple mutant strains .

O O O 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) Mean RLS in BY4741 wild-type, msn2∆ msn4∆ double and msn2∆ msn4∆ nat4∆ triple mutant strains.


( D ) Mean RLS in BY4741 wild - type , nat4 ∆ , fob1 ∆ sir2 ∆ double and fob1 ∆ sir2 ∆ nat4 ∆ triple mutant strains .

O O O O O O 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) Mean RLS in BY4741 wild-type, nat4∆, fob1∆ sir2∆ double and fob1∆ sir2∆ nat4∆ triple mutant strains.


( E ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown under NCR and CR conditions . Chromatin was immunoprecipitated using antibodies against N - acH4 and Histone H4 , and analyzed by qRT - PCR with the indicated primers . PNC1 - P ( Promoter ) , PNC1 - S ( Transcriptional Start ) , PNC1 - E ( End of gene ) . The enrichment from the antibody was normalized to Histone H4 .

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(E) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown under NCR and CR conditions. Chromatin was immunoprecipitated using antibodies against N-acH4 and Histone H4, and analyzed by qRT-PCR with the indicated primers. PNC1-P (Promoter), PNC1-S (Transcriptional Start), PNC1-E (End of gene). The enrichment from the antibody was normalized to Histone H4.


( left ) representative photomicrographs of H & E stained tibialis anterior muscle sections from control and iNOS KO mice injected with or without LPS . Scale bars = 100 μm . ( right ) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the tibialis anterior muscles from saline or LPS treated ( top ) WT control and ( bottom ) iNOS KO mice ( n = 4 ) . A total of 300 fibers per muscle were used for the analysis . Statistical comparisons , mean , and standard deviation of the mean are shown in the histogram legend .

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(left) representative photomicrographs of H&E stained tibialis anterior muscle sections from control and iNOS KO mice injected with or without LPS. Scale bars = 100 μm. (right) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the tibialis anterior muscles from saline or LPS treated (top) WT control and (bottom) iNOS KO mice (n = 4). A total of 300 fibers per muscle were used for the analysis. Statistical comparisons, mean, and standard deviation of the mean are shown in the histogram legend.


( left ) Western blot analysis of pThr172 - AMPK ( pAMPK ) and total AMPK ( AMPK ) in quadriceps muscle . ( right ) Quantification of the pAMPK to AMPK ratio . Ratios are expressed relative to the saline treated controls .

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

(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle. (right) Quantification of the pAMPK to AMPK ratio. Ratios are expressed relative to the saline treated controls.


Relative concentrations of metabolites involved in glycolysis and TCA cycle . Relative concentration of arginine .

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

Relative concentrations of metabolites involved in glycolysis and TCA cycle. Relative concentration of arginine.


( D - G ) Weight of tibialis anterior ( D ) , gastrocnemius ( E ) , quadricep ( F ) and soleus ( G ) muscle normalized to initial body weight .

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(D-G) Weight of tibialis anterior (D), gastrocnemius (E), quadricep (F) and soleus (G) muscle normalized to initial body weight.


( left ) Western blot analysis of pThr172 - AMPK ( pAMPK ) and total AMPK ( AMPK ) in quadriceps muscle ( n = 3 ) . ( right ) Quantification of the pAMPK to AMPK ratio relative to the saline treated control ( 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

(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle (n = 3). (right) Quantification of the pAMPK to AMPK ratio relative to the saline treated control (n = 3).


Relative concentrations of metabolites involved in glycolysis . Relative concentrations of amino acids .

Relative concentrations of metabolites involved in glycolysis. Relative concentrations of amino acids.


Representative immunofluorescence imaging for myoglobin and myosin heavy chain ( MyHC ) in not treated ( NT ) controls and IFNγ / TNFα ( IT ) samples treated with or without GW . Nuclei were visualized with DAPI staining ( n = 4 ) .

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Representative immunofluorescence imaging for myoglobin and myosin heavy chain (MyHC) in not treated (NT) controls and IFNγ/TNFα (IT) samples treated with or without GW. Nuclei were visualized with DAPI staining (n=4).


Western blot analysis for pThr172 - AMPK ( pAMPK ) , total AMPK ( AMPK ) , pSer79 - ACC ( pACC ) , and total ACC ( ACC ) .

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Western blot analysis for pThr172-AMPK (pAMPK), total AMPK (AMPK), pSer79-ACC (pACC), and total ACC (ACC).


Western blot analysis for pThr389 - S6K ( pS6K ) , total S6K ( S6K ) , pSer235 / 236 - S6 ( pS6 ) , and total S6 ( S6 ) ( n = 5 ) .

Western blot analysis for pThr389-S6K (pS6K), total S6K (S6K), pSer235/236-S6 (pS6), and total S6 (S6) (n=5).


Glycolytic index of basal metabolism.


Western blot analysis for ETC protein complex subunits .

Western blot analysis for ETC protein complex subunits.


Male C57BL / 6 wildtype ( WT ) and iNOS knockout ( KO ) mice were intraperitoneally injected with 1mg kg - 1 LPS or an equivalent volume of carrier solution . Control WT , Control KO , and LPS - treated KO cohorts were pair - fed ( PF ) to the WT LPS - treated cohorts . After 18h , mice were euthanized , and tibialis anterior muscles were imaged by transmission electron microscopy . ( left ) Representative micrograph of tibialis anterior muscle ( n = 2 ) . Scale bar = 1 µm . ( right ) Zoomed section of representative image to highlight mitochondria . Scale bar = 0 . 5 µm .

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

Male C57BL/6 wildtype (WT) and iNOS knockout (KO) mice were intraperitoneally injected with 1mg kg-1 LPS or an equivalent volume of carrier solution. Control WT, Control KO, and LPS-treated KO cohorts were pair-fed (PF) to the WT LPS-treated cohorts. After 18h, mice were euthanized, and tibialis anterior muscles were imaged by transmission electron microscopy. (left) Representative micrograph of tibialis anterior muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria. Scale bar = 0.5 µm.


Male BALB / C mice were injected subcutaneously with C26 cells ( 1 . 25x106 cells ) or an equivalent volume of saline . After 5 days , and everyday thereafter , saline and C26 injected mice were treated with or without GW ( 5mg kg - 1 ) . After 16 days , mice were euthanized and gastrocnemius muscles were imaged by transmission electron microscopy . ( left ) Representative micrograph of gastrocnemius muscle ( n = 2 ) . Scale bar = 1 µm . ( right ) Zoomed section of representative image to highlight mitochondria mitochondria . Scale bar = 0 . 5 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 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

Male BALB/C mice were injected subcutaneously with C26 cells (1.25x106 cells) or an equivalent volume of saline. After 5 days, and everyday thereafter, saline and C26 injected mice were treated with or without GW (5mg kg-1). After 16 days, mice were euthanized and gastrocnemius muscles were imaged by transmission electron microscopy. (left) Representative micrograph of gastrocnemius muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria mitochondria. Scale bar = 0.5 µm.


B ) Protein classification of the total proteins ( 3209 , left panel ) and of the glycoproteins ( 571 , right panel ) detected in 3 out of 3 experiments . More than 90 % of the glycoproteins detected are classified as membrane proteins , as expected for the surface labeling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Protein classification of the total proteins (3209, left panel) and of the glycoproteins (571, right panel) detected in 3 out of 3 experiments. More than 90% of the glycoproteins detected are classified as membrane proteins, as expected for the surface labeling.


C ) Changes of the 3209 proteins detected on the cell surface of SEZ6KO neurons compared to WT . The average log2 fold change of SEZ6KO neurons over WT ( log2 SEZ6KO / WT ) is plotted against the negative log10 of each protein ' s p - value according to a one - sample t test . Blue labeled dots are considered as hits ( including GluK2 and GluK3 ) and are proteins with a p - value less than 0 . 05 ( neg . log10 ( 0 . 05 ) = 1 . 3 ; horizontal dotted line ) and a fold - change to less than - 0 . 5 or more than 0 . 5 ( vertical dotted lines ) . GluK2 and GluK3 are labeled in pink , GluA1 and GluA2 are indicated with green lines and dots and GluN1 and GluN2B are marked with red lines and dots .

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C) Changes of the 3209 proteins detected on the cell surface of SEZ6KO neurons compared to WT. The average log2 fold change of SEZ6KO neurons over WT (log2 SEZ6KO/WT) is plotted against the negative log10 of each protein's p-value according to a one-sample t test. Blue labeled dots are considered as hits (including GluK2 and GluK3) and are proteins with a p-value less than 0.05 (neg. log10(0.05)=1.3; horizontal dotted line) and a fold-change to less than -0.5 or more than 0.5 (vertical dotted lines). GluK2 and GluK3 are labeled in pink, GluA1 and GluA2 are indicated with green lines and dots and GluN1 and GluN2B are marked with red lines and dots.


A ) SEZ6KO and WT neurons were biotinylated with Sulfo - NHS - Biotin and surface proteins were enriched by streptavidin bead pull - down . The GluK2 / 3 antibody cannot discriminate the subunits 2 and 3 ( Lerma & Marques , 2013 ) , therefore the band is commonly indicated with the labeling GluK2 / 3 . GluK2 / 3 , GluA2 and GluN2B surface levels were quantified , normalized to SEZ6L2 surface levels ( negative control ) in the same sample ( GluK2 / 3 / SEZ6L2 ) and divided by the WT levels , with the ratio for WT being set to 1 . 0 ( plot shows mean ± S . E . M . , at least 10 replicates in 4 independent biological experiments , Mann - Whitney * * * * p - value < 0 . 005 ) .

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A) SEZ6KO and WT neurons were biotinylated with Sulfo-NHS-Biotin and surface proteins were enriched by streptavidin bead pull-down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma & Marques, 2013), therefore the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2 and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3 / SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± S.E.M., at least 10 replicates in 4 independent biological experiments, Mann-Whitney ****p-value<0.005).


B ) mRNA levels of GluA1 , GluA2 , GluK2 , GluK3 and GluN2B were quantified in SEZ6KO neurons , normalized to GAPDH and β - actin mRNA in the same sample and compared to the mRNA levels in WT neurons . No difference in GluK2 / 3 mRNA was detected in SEZ6KO neurons compared to WT ( plot shows mean ± SD , 6 replicates in 2 independent biological experiments , multiple t - test was used ) .

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B) mRNA levels of GluA1, GluA2, GluK2, GluK3 and GluN2B were quantified in SEZ6KO neurons, normalized to GAPDH and β-actin mRNA in the same sample and compared to the mRNA levels in WT neurons. No difference in GluK2/3 mRNA was detected in SEZ6KO neurons compared to WT (plot shows mean ± SD, 6 replicates in 2 independent biological experiments, multiple t-test was used).


C ) Editing of GluK2 mRNA was tested in SEZ6KO and WT neurons . The editing value was calculated as ( intensity of 376 [ edited ] / intensity of [ 376 ( edited ) + 269 ( unedited ) ] ) × 100 and normalized to the band at 76 bp . No difference was detected in SEZ6KO neurons compared to WT ( plot shows mean ± S . E . M . , 6 replicates in 2 independent biological experiments , Mann - Whitney test was used ) .

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C) Editing of GluK2 mRNA was tested in SEZ6KO and WT neurons. The editing value was calculated as (intensity of 376 [edited] / intensity of [376 (edited) + 269 (unedited)]) × 100 and normalized to the band at 76 bp. No difference was detected in SEZ6KO neurons compared to WT (plot shows mean ± S.E.M., 6 replicates in 2 independent biological experiments, Mann-Whitney test was used).


( A ) Left : Current trace resulting from whole - cell voltage - clamp recordings at a holding potential of - 70 mV in a CA1 pyramidal neuron in a WT mouse hippocampal slice ( P15 ) . The current was recorded in the presence of 10 µM bicuculline , 20 µM GYKI53655 and 30 µM APV in the ACSF . Perfusion of the recording chamber with 10 µM kainate and 100 µM 2 , 3 - dioxo - 6 - nitro - 1 , 2 , 3 , 4 - tetrahydrobenzo [ f ] quinoxaline - 7 - sulfonamide ( NBQX ) are indicated with green and blue bars , respectively . Right : Analogous experiment in a SEZ6KO mouse ( P15 ) . ( B ) Summary of the experiments in A . Bar graph illustrates mean charge with standard error of the mean carried by the inward currents ( measured as area under the curve , AUC ) for the two genotypes at P15 ( WT 46 . 6 ± 0 . 5 nC ( n = 10 cells ) , SEZ6KO 30 . 8 ± 0 . 5 nC ( n = 9 cells ) , * p = 0 . 0375 Mann - Whitney - U - test ) .

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(A) Left: Current trace resulting from whole-cell voltage-clamp recordings at a holding potential of -70 mV in a CA1 pyramidal neuron in a WT mouse hippocampal slice (P15). The current was recorded in the presence of 10 µM bicuculline, 20 µM GYKI53655 and 30 µM APV in the ACSF. Perfusion of the recording chamber with 10 µM kainate and 100 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) are indicated with green and blue bars, respectively. Right: Analogous experiment in a SEZ6KO mouse (P15). (B) Summary of the experiments in A. Bar graph illustrates mean charge with standard error of the mean carried by the inward currents (measured as area under the curve, AUC) for the two genotypes at P15 (WT 46.6 ± 0.5 nC (n=10 cells), SEZ6KO 30.8 ± 0.5 nC (n=9 cells), *p=0.0375 Mann-Whitney-U-test).


A ) In WT neurons , GluK2 / 3 was seen as two closely comigrating bands , whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons ( schematic representation on the right ) . When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H ( EndoH ) - which removes immature , but not mature sugars - the two closely comigrating bands were converted to three bands of a lower apparent molecular weight , consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands ( marked in light blue , red and green in the right panel ) . In SEZ6KO neurons the uppermost ( light blue ) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO . No difference in the glycosylation of GluA2 was detectable , pointing to a specific effect of SEZ6 on GluK2 / 3 .

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A) In WT neurons, GluK2/3 was seen as two closely comigrating bands, whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons (schematic representation on the right). When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H (EndoH) - which removes immature, but not mature sugars - the two closely comigrating bands were converted to three bands of a lower apparent molecular weight, consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands (marked in light blue, red and green in the right panel). In SEZ6KO neurons the uppermost (light blue) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO. No difference in the glycosylation of GluA2 was detectable, pointing to a specific effect of SEZ6 on GluK2/3.


B ) The running pattern of GluK2 / 3 in SEZ6KO and WT brains was compared upon EndoH digestion . Similar to primary neurons , GluK2 / 3 displayed an immature glycosylation pattern in SEZ6KO brains .

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B) The running pattern of GluK2/3 in SEZ6KO and WT brains was compared upon EndoH digestion. Similar to primary neurons, GluK2/3 displayed an immature glycosylation pattern in SEZ6KO brains.


C ) Total GluK2 / 3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates ( plot shows mean ± S . E . M . , 9 replicates in 3 independent biological experiments , Mann - Whitney * * p - value = 0 . 0012 ) . D ) No significant change in GluK2 / 3 total levels was detectable in SEZ6KO and WT adult brains ( plot shows mean ± S . E . M . , 6 replicates , Mann - Whitney test was used ) .

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C) Total GluK2/3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates (plot shows mean ± S.E.M., 9 replicates in 3 independent biological experiments, Mann-Whitney **p-value=0.0012). D) No significant change in GluK2/3 total levels was detectable in SEZ6KO and WT adult brains (plot shows mean ± S.E.M., 6 replicates, Mann-Whitney test was used).


E ) Synaptosomes from knock - out ( KO ) brains of single SEZ6 family members ( SEZ6 , SEZ6L , SEZ6L2 ) , triple knock - out ( TKO ) and respective controls ( WT and TWT - triple WT ) were digested with EndoH . GluK2 / 3 displayed the immature glycosylation in SEZ6KO and TKO brains , but not in SEZ6L and SEZ6L2 brains , demonstrating non - redundant functions for the SEZ6 family members . ( n = 4 )

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E) Synaptosomes from knock-out (KO) brains of single SEZ6 family members (SEZ6, SEZ6L, SEZ6L2), triple knock-out (TKO) and respective controls (WT and TWT - triple WT) were digested with EndoH. GluK2/3 displayed the immature glycosylation in SEZ6KO and TKO brains, but not in SEZ6L and SEZ6L2 brains, demonstrating non-redundant functions for the SEZ6 family members. (n=4)


F ) Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH . GluK2 / 3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice , suggesting that no compensation effect is occurring with aging .

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F) Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH. GluK2/3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice, suggesting that no compensation effect is occurring with aging.


A ) The glycome fingerprint of GluK2 / 3 was analyzed by lectin chip microarray ( LecChip ) . Lectin PHAL and PHAE ( indicated with asterisks ) detected reduced amounts of the oligosaccharide Galβ1 - 4GlcNAcβ1 on GluK2 / 3 in SEZ6KO neurons ( plot shows mean ± S . E . M . , 3 WT replicates and 4 SEZ6KO replicates were used , discoveries were determined using the FDR method of Benjamini and Hochberg , with Q = 2 % ) .

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A) The glycome fingerprint of GluK2/3 was analyzed by lectin chip microarray (LecChip). Lectin PHAL and PHAE (indicated with asterisks) detected reduced amounts of the oligosaccharide Galβ1-4GlcNAcβ1 on GluK2/3 in SEZ6KO neurons (plot shows mean ± S.E.M., 3 WT replicates and 4 SEZ6KO replicates were used, discoveries were determined using the FDR method of Benjamini and Hochberg, with Q = 2%).


B ) Immunoblot analysis revealed reduction of HNK - 1 epitope on GluK2 / 3 in SEZ6KO brains . Left panels : Comparable amounts of GluK2 / 3 were immunoprecipitated ( IP ) in WT and SEZ6KO brains using excess of brain homogenates compared to beads . Anti - HNK - 1 antibody was used for detection . HNK - 1 band on GluK2 / 3 was detected only in WT brains but not in SEZ6KO brains ( n = 6 ) . As a control , HNK - 1 modification of NCAM - 1 was unaltered , as revealed after IP of NCAM - 1 from WT and SEZ6KO brains ( n = 3 ) , followed by detection with anti - HNK - 1 antibody . Different isoforms of NCAM - 1 are annotated with arrowheads ( NCAM 180 , NCAM 140 and NCAM 120 ) . Mid panel : To prove the specificity of the HNK - 1 antibody , GluK2 / 3 was immunoprecipitated and digested with Peptide - N - Glycosidase F ( PNGase F ) , which removes N - linked oligosaccharides . Upon PNGase F digestion , HNK - 1 was not detectable in WT brains and the molecular weight of GluK2 / 3 was reduced ( n = 2 ) . Right panels : In total brain homogenates ( input ) , no difference of general HNK - 1 epitope or NCAM - 1 levels was detected .

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B) Immunoblot analysis revealed reduction of HNK-1 epitope on GluK2/3 in SEZ6KO brains. Left panels: Comparable amounts of GluK2/3 were immunoprecipitated (IP) in WT and SEZ6KO brains using excess of brain homogenates compared to beads. Anti-HNK-1 antibody was used for detection. HNK-1 band on GluK2/3 was detected only in WT brains but not in SEZ6KO brains (n=6). As a control, HNK-1 modification of NCAM-1 was unaltered, as revealed after IP of NCAM-1 from WT and SEZ6KO brains (n = 3), followed by detection with anti-HNK-1 antibody. Different isoforms of NCAM-1 are annotated with arrowheads (NCAM 180, NCAM 140 and NCAM 120). Mid panel: To prove the specificity of the HNK-1 antibody, GluK2/3 was immunoprecipitated and digested with Peptide-N-Glycosidase F (PNGase F), which removes N-linked oligosaccharides. Upon PNGase F digestion, HNK-1 was not detectable in WT brains and the molecular weight of GluK2/3 was reduced (n=2). Right panels: In total brain homogenates (input), no difference of general HNK-1 epitope or NCAM-1 levels was detected.


B , C ) The constitutively ER - retained SEZ6 mutant SEZ6 ∆ cytoER - HA ( B ) and full length wild - type SEZ6FL - HA ( C ) were co - transfected with SBP - GFP - GluK2 . Biotin was added to elicit release of SBP - GFP - GluK2 from the ER ( 0 min ) and cells were fixed at different time points ( 0 , 20 and 40 min ) . HA - tagged SEZ6FL and SEZ6 ∆ cytoER transgenes were labeled with anti - HA ( mouse ) and anti - mouse - Alexa594 antibodies . Size bars represent 5 µm .

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B,C) The constitutively ER-retained SEZ6 mutant SEZ6∆cytoER-HA (B) and full length wild-type SEZ6FL-HA (C) were co-transfected with SBP-GFP-GluK2. Biotin was added to elicit release of SBP-GFP-GluK2 from the ER (0 min) and cells were fixed at different time points (0, 20 and 40 min). HA-tagged SEZ6FL and SEZ6∆cytoER transgenes were labeled with anti-HA (mouse) and anti-mouse-Alexa594 antibodies. Size bars represent 5 µm.


D ) Scatter dot plot represents number of vesicles per cell in SBP - GFP - GluK2 RUSH experiment with SEZ6 ∆ cytoER - HA ( control ) or SEZ6FL - HA co - transfection . Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars ( SD ) are shown . Mann - Whitney test was used to compare HA - tagged SEZ6FL and SEZ6 ∆ cytoER at each time point . At 20 min * * * p - value = 0 . 0007 , at 40 min * * * * p - value < 0 . 0001 .

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D) Scatter dot plot represents number of vesicles per cell in SBP-GFP-GluK2 RUSH experiment with SEZ6∆cytoER-HA (control) or SEZ6FL-HA co-transfection. Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars (SD) are shown. Mann-Whitney test was used to compare HA-tagged SEZ6FL and SEZ6∆cytoER at each time point. At 20 min ***p-value=0.0007, at 40 min ****p-value<0.0001.


A ) Representative pictures of WT hippocampal neurons transfected with the SBP - mCherry - GluK2 RUSH construct , followed by treating neurons with biotin and anti - SBP antibodies ( mouse , green ) for 20 min or 40 min to live - label the GluK2 transported to the neuronal cell surface . As a control ( no biotin ) , neurons were incubated only with SBP antibody for 40 min . The surface / total ratio of SBP - mCherry - GluK2 constructs was estimated by calculating the ratio of SBP ( green ) / mCherry ( red ) fluorescence signals in the same area chosen for quantification . B ) Quantification results for A , indicating that incubation with biotin increases the surface / total ratio of SBP - mCherry - GluK2 over time . No Biotin n = 16 , 20 min n = 13 , 40 min n = 15 , all biological replicates . Graph shows mean ± SEM , 1 - way ANOVA p < 0 . 0001 and Dunnett ' s post hoc test , No Biotin vs 20 min * p = 0 . 0030 ; No Biotin vs 40 min * * * p < 0 . 0001 .

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A) Representative pictures of WT hippocampal neurons transfected with the SBP-mCherry-GluK2 RUSH construct, followed by treating neurons with biotin and anti-SBP antibodies (mouse, green) for 20 min or 40 min to live-label the GluK2 transported to the neuronal cell surface. As a control (no biotin), neurons were incubated only with SBP antibody for 40 min. The surface/total ratio of SBP-mCherry-GluK2 constructs was estimated by calculating the ratio of SBP (green)/ mCherry (red) fluorescence signals in the same area chosen for quantification. B) Quantification results for A, indicating that incubation with biotin increases the surface/total ratio of SBP-mCherry-GluK2 over time. No Biotin n=16, 20 min n=13, 40 min n=15, all biological replicates. Graph shows mean±SEM, 1-way ANOVA p<0.0001 and Dunnett's post hoc test, No Biotin vs 20 min *p=0.0030; No Biotin vs 40 min ***p<0.0001.


C - D ) SEZ6 flox / flox neurons were lentivirally infected with inactivated Cre ( Y324F , Ctrl ) or Cre recombinase ( SEZ6KO ) , followed by transfection with the SBP - mCherry - GluK2 construct and treated with biotin and anti - SBP antibodies ( mouse , green ) for 20 min . Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti - Cre antibody ( rabbit , blue ) . SEZ6KO neurons showed a significantly reduced surface / total ratio of SBP - mCherry - GluK2 ( ctl n = 25 , SEZ6KO n = 23 biological replicates . Graph shows mean ± SEM , unpaired t - test , * * p = 0 . 0068 ) .

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C-D) SEZ6 flox/flox neurons were lentivirally infected with inactivated Cre (Y324F, Ctrl) or Cre recombinase (SEZ6KO), followed by transfection with the SBP-mCherry-GluK2 construct and treated with biotin and anti-SBP antibodies (mouse, green) for 20 min. Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti-Cre antibody (rabbit, blue). SEZ6KO neurons showed a significantly reduced surface/total ratio of SBP-mCherry-GluK2 (ctl n=25, SEZ6KO n=23 biological replicates. Graph shows mean ± SEM, unpaired t-test, **p=0.0068).


B , SEZ6 constitutive KO neurons were transduced with SEZ6 constructs . SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature ( higher ) and immature ( lower ) forms of SEZ6 . No difference in the apparent molecular weight was detected because the high percentage of the gel used ( 12 % ) and the small C - terminal deletion ( 39 amino acids ) . SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF ( with HA and Flag - tags ) was detected at the expected low molecular weight . Additionally , the C - terminal fragment generated from SEZ6FL by BACE1 cleavage was detected . Due to its lack of an HA - tag , it had a lower molecular weight than SEZ6CTF . The C - terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight . GluK2 / 3 glycosylation was rescued when SEZ6FL , SEZ6Δcyto and SEZ6ecto were expressed in the neurons , but SEZ6CTF was not able to rescue the phenotype . Color coding for asterisks is the same as in figure 4A . Representative blot of 3 independent experiments .

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B, SEZ6 constitutive KO neurons were transduced with SEZ6 constructs. SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature (higher) and immature (lower) forms of SEZ6. No difference in the apparent molecular weight was detected because the high percentage of the gel used (12%) and the small C-terminal deletion (39 amino acids). SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF (with HA and Flag-tags) was detected at the expected low molecular weight. Additionally, the C-terminal fragment generated from SEZ6FL by BACE1 cleavage was detected. Due to its lack of an HA-tag, it had a lower molecular weight than SEZ6CTF. The C-terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight. GluK2/3 glycosylation was rescued when SEZ6FL, SEZ6Δcyto and SEZ6ecto were expressed in the neurons, but SEZ6CTF was not able to rescue the phenotype. Color coding for asterisks is the same as in figure 4A. Representative blot of 3 independent experiments.


C ) SEZ6ecto was present as the mature , secreted band in the conditioned medium in panel C .

C) SEZ6ecto was present as the mature, secreted band in the conditioned medium in panel C.


D ) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto . SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6 , as expected for a protein retained in the ER . SEZ6Δcyto , but not SEZ6ΔcytoER , was able to rescue GluK2 / 3 glycosylation , showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2 / 3 glycosylation ( n = 5 ) . Color coding for asterisks is the same as in figure 4A .

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D) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto. SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6, as expected for a protein retained in the ER. SEZ6Δcyto, but not SEZ6ΔcytoER, was able to rescue GluK2/3 glycosylation, showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2/3 glycosylation (n=5). Color coding for asterisks is the same as in figure 4A.


E ) HEK293T cells were co - transfected with a GluK2a plasmid and the SEZ6 constructs . Expression of SEZ6 constructs was analyzed in the total lysates ( " Input " ) and revealed a similar expression of the different proteins . GluK2a was immunoprecipitated with GluK2 / 3 antibody and the SEZ6 mutants were detected with the HA . 11 antibody ( " IP " ) . SEZ6FL , SEZ6Δcyto and SEZ6ecto , but not SEZ6CTF were co - immunoprecipitated with GluK2a , suggesting that SEZ6 extracellular domain is necessary for GluK2a binding . Representative blot of 2 independent experiments . * indicates the light chain of the antibody used for immunoprecipitation .

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E) HEK293T cells were co-transfected with a GluK2a plasmid and the SEZ6 constructs. Expression of SEZ6 constructs was analyzed in the total lysates ("Input") and revealed a similar expression of the different proteins. GluK2a was immunoprecipitated with GluK2/3 antibody and the SEZ6 mutants were detected with the HA.11 antibody ("IP"). SEZ6FL, SEZ6Δcyto and SEZ6ecto, but not SEZ6CTF were co-immunoprecipitated with GluK2a, suggesting that SEZ6 extracellular domain is necessary for GluK2a binding. Representative blot of 2 independent experiments. * indicates the light chain of the antibody used for immunoprecipitation.


A , B : Quantitative RT - PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes ( GV and MII ) , somatic tissues , ES cells ( A ) and preimplantation embryos ( B ) . n = 3 biological

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A, B: Quantitative RT-PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes (GV and MII), somatic tissues, ES cells (A) and preimplantation embryos (B). n = 3 biological replicates


C : Cumulative numbers of pups per female showing fertility of WT and Cnot6l - / - female mice . n = 5 females for each

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C: Cumulative numbers of pups per female showing fertility of WT and Cnot6l-/- female mice. n = 5 females for each genotype


D : Representative images results of oocytes collected from oviducts of WT and Cnot6l - / - mice at 16 h after hCG injection . scale bar , 100 μm . Arrows indicate polar body - 1 ( PB1 )

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D: Representative images results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 100 μm. Arrows indicate polar body-1 (PB1)


E : Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l - / - mice at 16 h after hCG injection . scale bar , 20 μm . Arrows indicate spindle

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E: Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 20 μm. Arrows indicate spindle poles


F : Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l - / - female mice . The numbers of analyzed oocytes were indicated ( n )

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F: Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l-/- female mice. The numbers of analyzed oocytes were indicated (n)


G : Quantification of preimplantation embryos derived from WT and Cnot6l - / - females that develop to the indicated stages after hCG administration and mated with adult WT males . The numbers of analyzed embryos are indicated ( n )

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G: Quantification of preimplantation embryos derived from WT and Cnot6l-/- females that develop to the indicated stages after hCG administration and mated with adult WT males. The numbers of analyzed embryos are indicated (n)


H : Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l - / - female at indicated time points after hCG administration . Scale bar , 100 μm . Data information : Error bars , s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non -

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H: Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l-/- female at indicated time points after hCG administration. Scale bar, 100 μm. Data information: Error bars, s.e.m. **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif


A : Rates of germinal vesicle breakdown ( GVBD ) and PB1 emission in oocytes cultured in vitro . Fully - grown GV oocytes were collected from PMSG - primed ( 44 h ) WT and Cnot6l - / - mice . PB1 : polar body - 1 . Error bars , s . e . m . The numbers of analyzed oocytes were indicated ( n )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)


B : Representative images of WT , Cnot6l - / - and Btg4 - / - oocytes showing PB1 emission at 16 h after culture . Arrows indicate PB1 . Scale bar , 100

B: Representative images of WT, Cnot6l-/- and Btg4-/- oocytes showing PB1 emission at 16 h after culture. Arrows indicate PB1. Scale bar, 100 μm


C : Representative images of chromosome spreads made from WT and Cnot6l - / - oocytes after 16 h of in vitro maturation culture . Immunofluorescent staining of topoisomerase II ( TOP2 ) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres , respectively . Numbers of paired sister chromatids are indicated . Scale bar , 5

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

C: Representative images of chromosome spreads made from WT and Cnot6l-/- oocytes after 16 h of in vitro maturation culture. Immunofluorescent staining of topoisomerase II (TOP2) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres, respectively. Numbers of paired sister chromatids are indicated. Scale bar, 5 μm


D : Percentage ( % ) of aneuploidy among in vitro cultured WT and Cnot6l - / - oocytes that have released PB1s . Error bars , s . e . m . * P < 0 . 05 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Percentage (%) of aneuploidy among in vitro cultured WT and Cnot6l-/- oocytes that have released PB1s. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


E : Rates of GVBD , PB1 emission , and normal spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes cultured in vitro . Error bars , s . e . m . * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

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E: Rates of GVBD, PB1 emission, and normal spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes cultured in vitro. Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


F : Confocal microscopy results showing spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes at metaphase I ( MI ) and metaphase II ( MII ) . Scale bar , 20

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F: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μm


G : Pericentrin immunofluorescence showing MTOCs in cultured WT , Cnot6l - / - and Btg4 - / - oocytes at MI and MII stages . Spindle and DNA were labeled by microtubule nucleation factor ( TPX2 ) and DAPI , respectively . Scale bar , 20

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G: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm


A : Live cell imaging results showing in vitro meiotic division of WT and Cnot6l - / - oocytes . Abbreviations : PM , prometaphase ; MI , metaphase I ; AI , anaphase I ; TI , anaphase I - metaphase II transition ; MII , metaphase II ; PB1 : polar body - 1 . Hours after released from GV arrest were indicated . Scale bar , 20

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A: Live cell imaging results showing in vitro meiotic division of WT and Cnot6l-/- oocytes. Abbreviations: PM, prometaphase; MI, metaphase I; AI, anaphase I; TI, anaphase I-metaphase II transition; MII, metaphase II; PB1: polar body-1. Hours after released from GV arrest were indicated. Scale bar, 20 μm


B : Data represent the mean and standard deviations of mCherry - Securin fluorescence intensity in WT and Cnot6l - / - oocytes at each time point . Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest . The numbers of analyzed oocytes were indicated ( n )

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B: Data represent the mean and standard deviations of mCherry-Securin fluorescence intensity in WT and Cnot6l-/- oocytes at each time point. Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest. The numbers of analyzed oocytes were indicated (n)


C : SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l - / - oocytes at 16 h after culture . Centromeres and DNA were labeled by CREST and DAPI , respectively . Scale bar , 5

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

C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μm


D : BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l - / - oocytes at 9 . 5 h after culture . More than 8 oocytes were observed for each genotype with similar results . Scale bar , 5

D: BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l-/- oocytes at 9.5 h after culture. More than 8 oocytes were observed for each genotype with similar results. Scale bar, 5 μm


E : PB1 emission rates in WT and Cnot6l - / - oocytes cultured with or without MPS1 inhibitor reversin ( 5 μm ) . Reversin is added at 9 hours after culture . Error bars , s . e . m . The numbers of analyzed oocytes were indicated ( n )

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E: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)


A : Relative mRNA copy number dynamics in WT and Cnot6l - / - samples at the indicated stages . Error bars indicate values of the two biological

O O O O 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: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicates


B : Box plot showing gene expression levels of WT , Cnot6l - / - and Btg4 - / - oocytes at the MII stage . Genes were divided into 10 bins according to their expression levels in the WT MII oocytes . The box indicates upper and lower quantiles , the thick line in the box indicate the median , and the whiskers represent 2 . 5th and 97 . 5th percentiles . * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non -

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B: Box plot showing gene expression levels of WT, Cnot6l-/- and Btg4-/- oocytes at the MII stage. Genes were divided into 10 bins according to their expression levels in the WT MII oocytes. The box indicates upper and lower quantiles, the thick line in the box indicate the median, and the whiskers represent 2.5th and 97.5th percentiles. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif


C : qRT - PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l - / - mice ( Cnot6l - / - / WT ) . Error bars , s . e . m . * * * P < 0 . 001 by two - tailed Student " s t test . n = 3 biological

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C: qRT-PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l-/- mice (Cnot6l-/-/WT). Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n = 3 biological repli


D : Scatter plot comparing transcripts between WT and Cnot6l - / - oocytes ( at GV , MI and MII stages ) and zygotes derived from these oocytes . Transcripts decreased or increased more than five folds in Cnot6l - / - oocyte samples were high - lighted with blue or red ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Scatter plot comparing transcripts between WT and Cnot6l-/- oocytes (at GV, MI and MII stages) and zygotes derived from these oocytes. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively


E : Heatmap showing transcripts that were degraded during meiotic maturation ( GV / Zygote > 10 ) in WT oocytes . The definition of cluster I - III is described in the

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E: Heatmap showing transcripts that were degraded during meiotic maturation (GV/Zygote > 10) in WT oocytes. The definition of cluster I-III is described in the text


F : Relative mRNA copy number dynamics of the three gene clusters in WT , Cnot6l - / - and Btg4 - / -

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F: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples


Western blot results showing the levels of CPEB1 in WT and Cnot6l - / - oocytes at the indicated time points after meiotic resumption . Total proteins from 50 ( A oocytes are loaded in each lane . DDB1 ( A is blotted as a loading control . Experiments were performed three times with reproducible results ; a representative result is

Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins from 50 (A oocytes are loaded in each lane. DDB1 (A is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shown


Western blot results showing the levels of CPEB1 in WT and Cnot6l - / - oocytes at the indicated time points after meiotic resumption . Total proteins fro 100 ( B ) oocytes are loaded in each lane α - tubulin ( B ) is blotted as a loading control . Experiments were performed three times with reproducible results ; a representative result is

Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins fro 100 (B) oocytes are loaded in each lane α-tubulin (B) is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shown


Immunofluorescenc of L - homopropargylglycine ( HPG ) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l - / - mice . Scale bar , 20 μm . Error bars , standard deviations ( n = 6 oocytes for each genotype ) . * * P < 0 . 01 , * * * P < 0 .

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

Immunofluorescenc of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001


quantification ( D ) of L - homopropargylglycine ( HPG ) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l - / - mice . Scale bar , 20 μm . Error bars , standard deviations ( n = 6 oocytes for each genotype ) . * * P < 0 . 01 , * * * P < 0 .

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quantification (D) of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001


E : Relative mRNA copy numbers of polysome - bound transcripts in WT and Cnot6l - / - oocytes at indicated stages . Error bars indicate values of the

O O O O O 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: Relative mRNA copy numbers of polysome-bound transcripts in WT and Cnot6l-/- oocytes at indicated stages. Error bars indicate values of the replicates


F : Scatter plot comparing polysome - bound transcripts between WT and Cnot6l - / - oocytes at GV , MI and MII stages , respectively . Transcripts decreased or increased more than five folds in Cnot6l - / - oocyte samples were high - lighted with blue or red ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Scatter plot comparing polysome-bound transcripts between WT and Cnot6l-/- oocytes at GV, MI and MII stages, respectively. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively


I : Quantitative RT - PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l - / - oocytes at GV , MI and MII stages . Error bars , standard deviations ( n = 3 biologica repeats )

O O O O O O O O O O O O O O O O O O O O O O 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: Quantitative RT-PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l-/- oocytes at GV, MI and MII stages. Error bars, standard deviations (n = 3 biologica repeats)


A : Presence of putative AREs ( AUUUA ) in transcripts of WT oocyte . Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change ( MII / GV ) in WT

O O O O O O O O O O O O O O O O O O O O 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: Presence of putative AREs (AUUUA) in transcripts of WT oocyte. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (MII/GV) in WT oocytes


B : Presence of putative AREs in oocyte transcripts stabilized by Cnot6l - / - knockout . Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change ( Cnot6l - / - / WT ) at MII

O O O O O O O O O O O O O O O O O O O O O O O O O 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: Presence of putative AREs in oocyte transcripts stabilized by Cnot6l-/- knockout. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (Cnot6l-/-/WT) at MII stage


C : Co - IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7 . HeLa cells were co - transfected with plasmids expressing HA - ZFP36L2 and FLAG - CNOT6L / 7 for 48 h before immunoprecipitation . Experiments were performed three times with reproducible results ; a representative result is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7. HeLa cells were co-transfected with plasmids expressing HA-ZFP36L2 and FLAG-CNOT6L/7 for 48 h before immunoprecipitation. Experiments were performed three times with reproducible results; a representative result is shown


D : RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts , with or without the presence of ZFP36L2 . HeLa cells were co - transfected with plasmids expressing FLAG - ZFP36L2 and HA - CNOT6L for 48 h before immunoprecipitation using an anti - HA antibody . mRNAs recovered from the immunoprecipitates were subjected to qRT - PCR . Error bars , standard deviations ( n = 3 biological repeats ) . * * * P < 0 . 001 by two - tailed Student " s

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoprecipitation results showing interaction of CNOT6L with indicated transcripts, with or without the presence of ZFP36L2. HeLa cells were co-transfected with plasmids expressing FLAG-ZFP36L2 and HA-CNOT6L for 48 h before immunoprecipitation using an anti-HA antibody. mRNAs recovered from the immunoprecipitates were subjected to qRT-PCR. Error bars, standard deviations (n = 3 biological repeats). ***P < 0.001 by two-tailed Student"s t


fluorescence microscopy results ( B ) showing the translation activities of the Cnot6l 3 " - UTR ( WT and CPE mutated ) in GV - ( maintained by 2 μM milrinone ) or MII - ( released from milrinone ) arrested oocytes . GFP signal indicated translational activation of Cnot6l 3 " - UTR . An in vitro transcribed and polyadenylated mCherry mRNA was co - microinjected as a positive control . Scale

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fluorescence microscopy results (B) showing the translation activities of the Cnot6l 3"-UTR (WT and CPE mutated) in GV- (maintained by 2 μM milrinone) or MII- (released from milrinone) arrested oocytes. GFP signal indicated translational activation of Cnot6l 3"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive control. Scale b


GFP signal indicated translational activation of Cnot6l 3 " - UTR . An in vitro transcribed and polyadenylated mCherry mRNA was co - microinjected as a positive co Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte . Error bars , s . e . m . * P < 0 . 05 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

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

GFP signal indicated translational activation of Cnot6l 3"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive co Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


fluorescence microscopy results ( E ) showing the expression of GFP - fused Cnot6l 3 " - UTR in GV and MII oocytes with or without U0126 treatment ( 20 μM ) . Scale bar ,

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fluorescence microscopy results (E) showing the expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U0126 treatment (20 μM). Scale bar, 1


e expression of GFP - fused Cnot6l 3 " - UTR in GV and MII oocytes with or without U01 F : Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte The numbers of analyzed oocytes were indicated ( n )

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e expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U01 F: Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte The numbers of analyzed oocytes were indicated (n)


G : Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126 . Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L , CNOT7 and / or BTG4 and are released from milrinone at 12 h after microinjection . Then the oocytes were further culture for 24 h with or without adding U0126 to the medium . Error bars , s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were

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

G: Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126. Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L, CNOT7 and/or BTG4 and are released from milrinone at 12 h after microinjection. Then the oocytes were further culture for 24 h with or without adding U0126 to the medium. Error bars, s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate


A . Equal aliquots ( 2 mg ) of a Triton X - 100 solubilized lysate from rat brain were incubated with GST or GST - ROC . Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated 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

A. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


B . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb . Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated 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

B. Brain lysate as in (A) was incubated with GST or GST-nCLCb. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


C . Full - length GST - nCLCb ( 1 - 228 ) and various truncation mutants were purified from bacteria . Equal aliquots of purified protein were incubated with GST or GST - ROC . Specifically bound proteins were detected by Western blot with antibody against CLCs .

C. Full-length GST-nCLCb (1-228) and various truncation mutants were purified from bacteria. Equal aliquots of purified protein were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibody against CLCs.


D . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb proteins with boundaries as indicated . Specifically bound proteins were detected by Western blot with antibody against CLCs .

O O O O O O O O O O O O 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. Brain lysate as in (A) was incubated with GST or GST-nCLCb proteins with boundaries as indicated. Specifically bound proteins were detected by Western blot with antibody against CLCs.


F . Highly enriched triskelia were stripped from purified CCVs . The triskelia were incubated with GST or GST - ROC . Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins . For A - D / F , aliquots of the starting material ( SM ) equal to 10 % of that added to the beads , was analyzed in parallel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Highly enriched triskelia were stripped from purified CCVs. The triskelia were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. For A-D/F, aliquots of the starting material (SM) equal to 10% of that added to the beads, was analyzed in parallel.


PCR results of LRRK2 - WT from clone E1 ( 1 ) using primers that detect endogenous LRRK2 . Clone E1 is positive for 3xHA - LRRK2 ( 2 ) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2 . ( 3 ) Control unedited COS - 7 cells using the same primer combination as in ( 2 ) . ( 4 ) 1Kb marker .

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PCR results of LRRK2-WT from clone E1 (1) using primers that detect endogenous LRRK2. Clone E1 is positive for 3xHA-LRRK2 (2) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2. (3) Control unedited COS-7 cells using the same primer combination as in (2). (4) 1Kb marker.


COS - 7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488 - EGF , after which the cells were fixed and processed for immunofluorescence using HA antibody . Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels .

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COS-7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488-EGF, after which the cells were fixed and processed for immunofluorescence using HA antibody. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


COS - 7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies . Scale bar = 10 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

COS-7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies. Scale bar = 10 m.


COS - 7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies . Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels .

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COS-7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


Lysates from COS - 7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins . Band intensities of blots as in A are presented as % control siRNA . Two - tailed Student t - test , Mann - Whiteney post hoc test , * * * p < 0 . 001 , N = 3 .

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Lysates from COS-7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins.Band intensities of blots as in A are presented as % control siRNA. Two-tailed Student t-test, Mann-Whiteney post hoc test, ***p < 0.001, N = 3.


NP - 40 soluble lysates were prepared from COS - 7 cells transfected with siRNA as indicated and equal protein aliquots ( 1 . 5 mg ) were incubated with GST - PAK - CRIB domain . Specifically bound proteins were detected by Western blot with antibody recognizing Rac1 . An aliquot of the starting material ( SM ) equal to 5 % of that added to GST - PAK - CRIB was analyzed in parallel .

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NP-40 soluble lysates were prepared from COS-7 cells transfected with siRNA as indicated and equal protein aliquots (1.5 mg) were incubated with GST-PAK-CRIB domain. Specifically bound proteins were detected by Western blot with antibody recognizing Rac1. An aliquot of the starting material (SM) equal to 5% of that added to GST-PAK-CRIB was analyzed in parallel.


Band intensities of blots as in A were presented as % control siRNA . Bars represent mean sem . One - way ANOVA , Bonferroni ' s post hoc test , * * * p < 0 . 001 , N = 3 .

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Band intensities of blots as in A were presented as % control siRNA. Bars represent mean sem. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


COS - 7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin - 647 and antibody recognizing CLCs . Scale bar = 10 m .

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COS-7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin-647 and antibody recognizing CLCs. Scale bar = 10 m.


Perimeter and area of cells as in A were measured and a perimeter : area ratio was plotted as a % of the control siRNA treated cells . One - way ANOVA , Bonferroni ' s post hoc test , * * * p < 0 . 001 , N = 3 .

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Perimeter and area of cells as in A were measured and a perimeter:area ratio was plotted as a % of the control siRNA treated cells. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus ( at 7 days in vitro ) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa , CLCb , or both viruses in combination . The lysates were processed for Western blot with antibody that recognizes all forms of CLCs .

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Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus (at 7 days in vitro) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa, CLCb, or both viruses in combination. The lysates were processed for Western blot with antibody that recognizes all forms of CLCs.


Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre - synaptic protein synapsin and monoclonal antibody specific for the post - synaptic protein PSD95 . The lentivirus drives expression of GFP . The areas indicated by white boxes in the low power images ( squares ) are shown enlarged immediately below . The scale bars = 10 µm for the low mag and 2 . 5 µm for the high mag .

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Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre-synaptic protein synapsin and monoclonal antibody specific for the post-synaptic protein PSD95. The lentivirus drives expression of GFP. The areas indicated by white boxes in the low power images (squares) are shown enlarged immediately below. The scale bars = 10 µm for the low mag and 2.5 µm for the high mag.


Ai - x . Lateral view of the adult fly head . Scale bar = 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused ( arrows ) and disorganized but still separated ommatidia ( arrowheads ) . ii / iii . Rac1 overexpression with KD of dlrrk ( UAS - dlrrk - RNAi ( v22139 or v22140 ) / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused . iv / v . Rac1 overexpression with KD of Clc ( UAS - Clc - RNAi ( v22318 or v106632 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , results in smaller eyes with some ommatidia fused . vi . Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 ( irk3 ) , a non - relevant RNAi control ( UAS - irk3 - RNAi ( v101174 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , has no effect . vii / viii . KD of dlrrk with GMR - GAL4 / + ; UAS - dlrrk - RNAi ( v22139 or 22140 ) / + , respectively as control have no effect on eye morphology . ix / x . KD of Clc with GMR - GAL4 / UAS - Clc - RNAi ( v22318 or v106632 ) , respectively as control have no effect on eye morphology .

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Ai-x. Lateral view of the adult fly head. Scale bar = 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused (arrows) and disorganized but still separated ommatidia (arrowheads).ii/iii. Rac1 overexpression with KD of dlrrk (UAS-dlrrk-RNAi (v22139 or v22140)/longGMR-GAL4, UAS-Rac1W), results in smaller eyes where most ommatidia are fused.iv/v. Rac1 overexpression with KD of Clc (UAS-Clc-RNAi (v22318 or v106632)/+;longGMR-GAL4,UAS-Rac1W/+), results in smaller eyes with some ommatidia fused.vi. Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 (irk3), a non-relevant RNAi control (UAS-irk3-RNAi(v101174)/+; longGMR-GAL4,UAS-Rac1W/+), has no effect.vii/viii. KD of dlrrk with GMR-GAL4/+; UAS-dlrrk-RNAi(v22139 or 22140)/+, respectively as control have no effect on eye morphology.ix/x. KD of Clc with GMR-GAL4/UAS-Clc-RNAi (v22318 or v106632), respectively as control have no effect on eye morphology.


Bi - ii . Lateral view of the adult fly head . Scale bar : 100 μm . i . Rac1 overexpression with removing one copy of dlrrk ( dlrrke03680 / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused together . ii . Removing one copy of dlrrke03680 in GMR - GAL4 background , as control has no effect on eye morphology ( GMR - GAL4 / + ; dlrrke03680 / + ) .

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Bi-ii. Lateral view of the adult fly head. Scale bar: 100 μm.i. Rac1 overexpression with removing one copy of dlrrk (dlrrke03680/longGMR-GAL4,UAS-Rac1W), results in smaller eyes where most ommatidia are fused together.ii. Removing one copy of dlrrke03680 in GMR-GAL4 background, as control has no effect on eye morphology (GMR-GAL4/+;dlrrke03680/+).


Ci - viii . Lateral view of the adult fly head . Scale bar : 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia ( as explained in Ai ) . ii . Rac1 co - expression with dlrrk - WT using UAS - dlrrk - WT / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . iii . Rac1 co - expression with Clc using UAS - GFP - Clc / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . iv . Rac1 co - expression with dlrrk - WT - 3KD using UAS - dlrrk - 3KD / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . v . A wild - type eye of the longGMR - GAL4 promoter as a control . vi - viii . Overexpression of dlrrk - WT , dlrrk - 3KD , or Clc alone with GMR - GAL4 as control , have no effect on eye morphology .

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Ci-viii. Lateral view of the adult fly head. Scale bar: 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia (as explained in Ai).ii. Rac1 co-expression with dlrrk-WT using UAS-dlrrk-WT/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iii. Rac1 co-expression with Clc using UAS-GFP-Clc/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iv. Rac1 co-expression with dlrrk-WT-3KD using UAS-dlrrk-3KD/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.v. A wild-type eye of the longGMR-GAL4 promoter as a control.vi-viii. Overexpression of dlrrk-WT, dlrrk-3KD, or Clc alone with GMR-GAL4 as control, have no effect on eye morphology.


( A ) Cardiac GATA4 protein abundance analyzed by immunoblotting . GAPDH was the loading control . + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus . ( B ) Densitometric quantification of the immunoblot shown under ( A ) ; * * * p = 0 . 0003 for P1 vs . P7 and P1 vs . P21 ; p < 0 . 0001 for P1 vs . P60 . The number within bars indicates the number of mice analyzed in that particular group .

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(A) Cardiac GATA4 protein abundance analyzed by immunoblotting. GAPDH was the loading control. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus.(B) Densitometric quantification of the immunoblot shown under (A); ***p=0.0003 for P1 vs. P7 and P1 vs. P21; p<0.0001 for P1 vs. P60. The number within bars indicates the number of mice analyzed in that particular group.


( C ) Immunoblot of cardiac GATA4 abundance in control ( Con ) and cardiomyocyte specific Gata4 knock - out mice ( CM - G4 - KO ) at postnatal day ( P ) 1 . ( D ) Quantification of the immunoblot shown in ( C ) ; * p = 0 . 018 .

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(C) Immunoblot of cardiac GATA4 abundance in control (Con) and cardiomyocyte specific Gata4 knock-out mice (CM-G4-KO) at postnatal day (P) 1.(D) Quantification of the immunoblot shown in (C); *p=0.018.


( E ) Cardiac immunofluorescence staining and its quantification from Con and CM - G4 - KO mice at P1 . Scale bar : 25 µm ; * * * * p < 0 . 0001 .

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(E) Cardiac immunofluorescence staining and its quantification from Con and CM-G4-KO mice at P1. Scale bar: 25 µm; ****p<0.0001.


( F ) Transversal sections of mouse hearts 7 days after cryoinjury as indicated , stained with Sirius - Red . Scale bar : 500 µm . The quantification of the left ventricular ( LV ) scar area ( red , arrows ) 7 days after injury is shown on the right ; * * p = 0 . 0027 . ( G ) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury ; * p = 0 . 0261 . ( H ) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury ; * * * * p < 0 . 0001 .

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(F) Transversal sections of mouse hearts 7 days after cryoinjury as indicated, stained with Sirius-Red . Scale bar: 500 µm. The quantification of the left ventricular (LV) scar area (red, arrows) 7 days after injury is shown on the right; **p=0.0027.(G) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury; *p=0.0261.(H) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury; ****p<0.0001.


( I ) Echocardiography analysis of left ventricular systolic function ( measured as fractional area change ) in the indicated mice 7 days after cryoinjury ; * p = 0 . 033 between Con mice after sham or cryoinjury and p = 0 . 04 between Con and CM - G4 - KO mice after cryoinjury .

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(I) Echocardiography analysis of left ventricular systolic function (measured as fractional area change) in the indicated mice 7 days after cryoinjury; *p=0.033 between Con mice after sham or cryoinjury and p=0.04 between Con and CM-G4-KO mice after cryoinjury.


( A ) Representative pictures of TUNEL , WGA ( wheat germ agglutinin to mark cardiomyocyte membranes ) and DAPI immunofluorescence staining of mouse hearts as indicated . The white dashed line encircles the infarcted area ( arrows ) . Scale bar : 200µm . The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right ( A ´ ) . The number within bars indicates the number of mice analyzed in that particular group .

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(A) Representative pictures of TUNEL, WGA (wheat germ agglutinin to mark cardiomyocyte membranes) and DAPI immunofluorescence staining of mouse hearts as indicated. The white dashed line encircles the infarcted area (arrows). Scale bar: 200µm. The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right (A´). The number within bars indicates the number of mice analyzed in that particular group.


( B ) Representative merged and single channel pictures of cardiac CD31 , DAPI and WGA immunostaining in mouse hearts as indicated . The quantification of the myocardial capillary density as capillaries / cardiomyocyte ( CM ) ratio , 7 days ( d ) after cryoinjury or sham surgery is shown as bar graph ( B ´ ) . Scale bar : 20µm . * * p = 0 . 0039 .

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(B) Representative merged and single channel pictures of cardiac CD31, DAPI and WGA immunostaining in mouse hearts as indicated. The quantification of the myocardial capillary density as capillaries/cardiomyocyte (CM) ratio, 7 days (d) after cryoinjury or sham surgery is shown as bar graph (B´). Scale bar: 20µm. **p=0.0039.


( C ) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho - histone H3 ( pH3 ) , troponin T and DAPI in mice as indicated . Enlarged pH3 positive cardiomyocyte nuclei are shown separately . The arrows indicate pH3 positive cardiomyocyte nuclei . Scale bar : 25µm . The quantification of pH3 positive ( + ) cardiomyocytes per microscopic field is shown as bar graph ( C ´ ) ; * p = 0 . 046 .

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(C) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho-histone H3 (pH3), troponin T and DAPI in mice as indicated. Enlarged pH3 positive cardiomyocyte nuclei are shown separately. The arrows indicate pH3 positive cardiomyocyte nuclei. Scale bar: 25µm. The quantification of pH3 positive (+) cardiomyocytes per microscopic field is shown as bar graph (C´); *p=0.046.


( D ) Representative myocardial immunostaining for aurora B , DAPI and troponin T ( TropT ) . The arrow points towards a cardiomyocyte cell division , in which aurora B localizes at the midbody in cytokinesis . Scale bar : 25µm . The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph ( D ´ ) ; * p = 0 . 0104 .

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(D) Representative myocardial immunostaining for aurora B, DAPI and troponin T (TropT). The arrow points towards a cardiomyocyte cell division, in which aurora B localizes at the midbody in cytokinesis. Scale bar: 25µm. The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph (D´); *p=0.0104.


( E ) Determination of cardiac gene - expression by qPCR 7 days after sham or cryoinjury for the indicated genes ; * p = 0 . 039 for Il13 and p = 0 . 049 for Vegfa ; * * p = 0 . 0043 for Ccne1 , p = 0 . 0027 for Cdk4 , p = 0 . 0096 for Cenpa , p = 0 . 0015 for Zfp191 ; * * * p = 0 . 0009 for Ccna2 , p = 0 . 0002 for Cenpa .

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(E) Determination of cardiac gene-expression by qPCR 7 days after sham or cryoinjury for the indicated genes; *p=0.039 for Il13 and p=0.049 for Vegfa; **p=0.0043 for Ccne1, p=0.0027 for Cdk4, p=0.0096 for Cenpa, p=0.0015 for Zfp191; ***p=0.0009 for Ccna2, p=0.0002 for Cenpa.


( A ) Representative immunoblots for GATA4 and GAPDH ( as loading control ) of fetal cardiomyocytes treated with adenoviruses ( Ad ) expressing control shRNA ( shCon ) or shRNA against Gata4 ( shGATA4 ) for 48 hours .

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(A) Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 hours.


( B ) Number of cardiomyocytes ( CM ) in a defined low magnification ( 50x ) microscopic field before the adenoviral infection and 48h later . The number in the bars indicates the number of cell culture plates analyzed . * * * * p < 0 . 0001 .

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(B) Number of cardiomyocytes (CM) in a defined low magnification (50x) microscopic field before the adenoviral infection and 48h later. The number in the bars indicates the number of cell culture plates analyzed. ****p<0.0001.


( C ) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU , troponin I ( Trop I ) and DAPI . Scale bar : 50µm ; p = 0 . 0169 .

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(C) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU, troponin I (Trop I) and DAPI. Scale bar: 50µm; p=0.0169.


( D ) Representative pictures and quantification of cardiomyocytes positive for phospho - histone H3 ( pH3 ) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3 , troponin I and DAPI . Scale bar : 50µm ; * p = 0 . 0145 .

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(D) Representative pictures and quantification of cardiomyocytes positive for phospho-histone H3 (pH3) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3, troponin I and DAPI. Scale bar: 50µm; *p=0.0145.


( E ) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B ( Auro B ) , troponin I and DAPI . Scale bar : 50µm .

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(E) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B (Auro B), troponin I and DAPI. Scale bar: 50µm.


( F ) Representative pictures , single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3 , troponin I and DAPI . Scale bar : 100µm .

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(F) Representative pictures, single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3, troponin I and DAPI. Scale bar: 100µm.