Figure 3 ( A ) Growth curves of MCF10A cells exposed to oxidative stress ( 200μM H2O2 ) relative to control cells ( n = 3 ) . Two - way ANOVA was used to test for significance . ( B ) Northern blot of MCF10A cells expressing a control short - hairpin RNA or a hairpin targeting tRNATyrGUA . ( C ) Western blot of MCF10A expressing a control short - hairpin RNA or a hairpin targeting the tyrosine - tRNA synthetase , YARS ( red arrow ) . HSC70 was used as a loading control . ( D ) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin ( n = 3 ) . Two - way ANOVA was used to test for significance . ( E ) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector ( n = 3 ) . A one - tailed Mann - Whitney test was used to test for significance at day 3 .

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Figure 3(A) Growth curves of MCF10A cells exposed to oxidative stress (200μM H2O2) relative to control cells (n=3). Two-way ANOVA was used to test for significance.(B) Northern blot of MCF10A cells expressing a control short-hairpin RNA or a hairpin targeting tRNATyrGUA.(C) Western blot of MCF10A expressing a control short-hairpin RNA or a hairpin targeting the tyrosine-tRNA synthetase, YARS (red arrow). HSC70 was used as a loading control.(D) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin (n=3). Two-way ANOVA was used to test for significance.(E) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector (n=3). A one-tailed Mann-Whitney test was used to test for significance at day 3.


Figure 4 ( A ) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2 - fold change of 0 . 5 or more . This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion . ( C ) Quantitative western blot validation depicting abundances of protein targets ( EPCAM , SCD , and USP3 ) identified from ( A ) . HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA . ( D ) Quantification of western blot analysis in ( C ) ( n = 4 ) . A one - tailed Mann - Whitney test was used to test for statistical significance . ( E ) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM , SCD , or USP3 . Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments . Two - way ANOVA was used to test for significance ( n = 3 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion.(C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA. (D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance. (E) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM, SCD, or USP3. Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments. Two-way ANOVA was used to test for significance (n=3).


Figure 5 ( A ) Quantitative western blot EPCAM , SCD , and USP3 in MCF10A cells 24 hours after treatment with H2O2 ( 200μM ) . HSC70 was used as a loading control . ( B ) Quantification of western results in ( A ) ( n = 9 ) . A one - tailed Mann - Whitney test was used to test for statistical significance . ( D ) Quantitative western blot for the Myc - tag and Luciferase ( top ) with normalized fluorescent intensities ( bottom ) are shown ( n = 3 ) . ( E ) Ribosome occupancy of 20 - 22nt RPFs in tRNATyrGUA - depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA - depleted cells using a Wilcoxon test .

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Figure 5(A) Quantitative western blot EPCAM, SCD, and USP3 in MCF10A cells 24 hours after treatment with H2O2 (200μM). HSC70 was used as a loading control. (B) Quantification of western results in (A) (n=9). A one-tailed Mann-Whitney test was used to test for statistical significance. (D) Quantitative western blot for the Myc-tag and Luciferase (top) with normalized fluorescent intensities (bottom) are shown (n=3).(E) Ribosome occupancy of 20-22nt RPFs in tRNATyrGUA-depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA-depleted cells using a Wilcoxon test.


Figure 6 ( A - B ) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A ( A ) ( n = 4 ) and in HBEC30 ( B ) ( n = 6 ) with mean ± s . e . m shown for each cell line . ( C ) Volcano plot of mass spectrometry results from a synthetic 5 ' - biotinylated tRFTyrGUA co - precipitation experiment with cell lysate . Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples . ( D ) Western blot validation of mass spectrometry results for three of the top hits in ( A ) , showing co - precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence ( Scr ) .

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Figure 6(A-B) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A (A) (n=4) and in HBEC30 (B) (n=6) with mean ± s.e.m shown for each cell line.(C) Volcano plot of mass spectrometry results from a synthetic 5'-biotinylated tRFTyrGUA co-precipitation experiment with cell lysate. Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples.(D) Western blot validation of mass spectrometry results for three of the top hits in (A), showing co-precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence (Scr).


Figure 7 ( B ) IGV plots from an hnRNPA1 HITS - CLIP ( Huelga et al . , 2012 ) reveals interactions with tRFTyrGUA . ( C ) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion . SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA . ( D ) Similar to the IGV plots shown in ( C ) , but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion . ( E ) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3 ' UTR hnRNPA1 CLIP binding ( Huelga et al . , 2012 ) . Transfection of tRFTyrGUA led to a significant right - shift in the expression levels of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was measured using the Kolmogorov - Smirnov test . ( F ) Cumulative distribution as in ( E ) . Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left - shift in mRNA stability of 3 ' UTR bound hnRNPA1 transcripts . Statistical significance was assessed using the Kolmogorov - Smirnov test .

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Figure 7(B) IGV plots from an hnRNPA1 HITS-CLIP (Huelga et al., 2012) reveals interactions with tRFTyrGUA.(C) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion. SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA. (D) Similar to the IGV plots shown in (C), but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion. (E) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3' UTR hnRNPA1 CLIP binding (Huelga et al., 2012). Transfection of tRFTyrGUA led to a significant right-shift in the expression levels of 3' UTR bound hnRNPA1 transcripts. Statistical significance was measured using the Kolmogorov-Smirnov test. (F) Cumulative distribution as in (E). Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left-shift in mRNA stability of 3'UTR bound hnRNPA1 transcripts. Statistical significance was assessed using the Kolmogorov-Smirnov test.


Figure 1 ( A ) Expression levels of NAT4 analyzed by qRT - PCR using total RNA extracted from a wild - type ( BY4741 ) and Pste5 - NAT4 strain grown in 2 % ( NCR ) , or 0 . 1 % glucose ( CR ) . NAT4 levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM ( Standard Error of the Mean ) of 3 independent experiments . * * p ≤ 0 . 01 ; calculated by unpaired two - tailed Student ' s t - test . ( B ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown in the same conditions as in ( A ) . Chromatin was immunoprecipitated using an antibody against N - acH4 and analyzed by qRT - PCR using primers for the different rDNA regions . Enrichment was normalized to Histone H4 levels . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 compared values to wild - type NCR conditions ; arrowhead p ≤ 0 . 05 compared values to wild - type CR conditions ( C ) Replicative lifespan ( RLS ) for BY4741 wild - type and nat4Δ strains . 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 . ( D ) Mean RLS for wild - type and nat4 ∆ strains using different genetic backgrounds . Values outside of parenthesis indicate mean lifespan , while values in parenthesis indicate the number of cells examined . ( E ) Mean RLS for BY4741 wild - type and nat4Δ strains under NCR and CR conditions . ( F ) Mean RLS for BY4741 wild - type and Pste5 - NAT4 strains under NCR and CR conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student's t-test.(B) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown in the same conditions as in (A). Chromatin was immunoprecipitated using an antibody against N-acH4 and analyzed by qRT-PCR using primers for the different rDNA regions. Enrichment was normalized to Histone H4 levels. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01 compared values to wild-type NCR conditions; arrowhead p ≤ 0.05 compared values to wild-type CR conditions(C) Replicative lifespan (RLS) for BY4741 wild-type and nat4Δ strains. 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.(D) Mean RLS for wild-type and nat4∆ strains using different genetic backgrounds. Values outside of parenthesis indicate mean lifespan, while values in parenthesis indicate the number of cells examined.(E) Mean RLS for BY4741 wild-type and nat4Δ strains under NCR and CR conditions.(F) Mean RLS for BY4741 wild-type and Pste5-NAT4 strains under NCR and CR conditions.


Figure 2 ( 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 . ( D ) Polysome profiling analysis performed in BY4741 wild - type and nat4Δ strains . The plots are representative of two independent experiments .

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Figure 2(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.(D) Polysome profiling analysis performed in BY4741 wild-type and nat4Δ strains. The plots are representative of two independent experiments.


Figure 3 ( 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 .

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Figure 3(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.


Figure 4 ( 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 . ( 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 .

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Figure 4(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.(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.


Figure 5 ( 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 .

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Figure 5(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.


Figure 6 ( 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 . ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(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.(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.(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.


Figure 1 ( 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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Figure 1(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.


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( D - G ) Weight of tibialis anterior ( D ) , gastrocnemius ( E ) , quadricep ( F ) and soleus ( G ) muscle normalized to initial body weight .

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Figure 3(D-G) Weight of tibialis anterior (D), gastrocnemius (E), quadricep (F) and soleus (G) muscle normalized to initial body weight.


Figure 4 ( 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 .

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Figure 4(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.


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

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


Figure 6Glycolytic index of basal metabolism .

Figure 6Glycolytic index of basal metabolism.


Figure 7Western blot analysis for ETC protein complex subunits .

Figure 7Western blot analysis for ETC protein complex subunits.


Figure 8Male 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 .

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Figure 8Male 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.


Figure 1B ) 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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Figure 1B) 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.


Figure 2A ) 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 ) . 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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Figure 2A) 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).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).


Figure 3 ( 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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Figure 3(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).


Figure 4A ) 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 . 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 ) 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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Figure 4A) 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.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)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.


Figure 5A ) 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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Figure 5A) 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.


Figure 6B , 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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Figure 6B,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.


Figure 7A ) 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7A) 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).


Figure 8B , 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8B, 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.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.


Figure 1A , 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 replicatesC : Cumulative numbers of pups per female showing fertility of WT and Cnot6l - / - female mice . n = 5 females for each genotypeD : 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 polesF : 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 ) 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 -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A, 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 replicatesC: Cumulative numbers of pups per female showing fertility of WT and Cnot6l-/- female mice. n = 5 females for each genotypeD: 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 polesF: 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)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


Figure 2A : 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 μmC : 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 μmD : 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 indicateE : 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 indicateF : Confocal microscopy results showing spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes at metaphase I ( MI ) and metaphase II ( MII ) . Scale bar , 20 μmG : 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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 2A: 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 μmC: 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 μmD: 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 indicateE: 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 indicateF: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μmG: 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


Figure 3A : 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 μmB : 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 μmD : 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 μmE : 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 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A: 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 μmB: 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 μmD: 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 μmE: 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)


Figure 4A : Relative mRNA copy number dynamics in WT and Cnot6l - / - samples at the indicated stages . Error bars indicate values of the two biological replicatesB : 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 - signifC : 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 repliD : 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 , respectivelyE : 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 textF : Relative mRNA copy number dynamics of the three gene clusters in WT , Cnot6l - / - and Btg4 - / -

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicatesB: 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-signifC: 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 repliD: 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, respectivelyE: 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 textF: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples


Figure 6Western 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 shownWestern 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 shownImmunofluorescenc 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 . 001quantification ( 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 . 001E : Relative mRNA copy numbers of polysome - bound transcripts in WT and Cnot6l - / - oocytes at indicated stages . Error bars indicate values of the replicatesF : 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 , respectivelyI : 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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 6Western 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 shownWestern 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 shownImmunofluorescenc 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 < 0.001quantification (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 < 0.001E: Relative mRNA copy numbers of polysome-bound transcripts in WT and Cnot6l-/- oocytes at indicated stages. Error bars indicate values of the replicatesF: 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, respectivelyI: 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)


Figure 7A : 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 oocytesB : 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 stageC : 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 shownD : 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

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Figure 7A: 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 oocytesB: 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 stageC: 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 shownD: 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


Figure 8fluorescence 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 bGFP 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 indicatefluorescence 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 , 1e 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8fluorescence 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 bGFP 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 indicatefluorescence 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, 1e 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 indicate


Figure 1A . 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 . 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 . 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 .

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Figure 1A. 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.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.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.


Figure 2PCR 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O 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 2PCR 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.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.


Figure 3Lysates 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Lysates 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.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.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.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.