c ) Distribution of log2 ( FC ) for the significant cases in transcriptomics ( N = 4 scr treated and N = 3 amiR - 29a treated biological replicates ) and proteomics ( N = 4 biological replicates per group ) datasets of animals treated with amiR - 29a versus scr animals . The black line represents the average . ( d , e ) Box plot showing distribution of log2 ( FC ) of miR29a targets as d ) transcripts ( RNA ) and e ) proteins ( PROT ) in transcriptomic and proteomic datasets of adult animals treated with amiR - 29a . Notches correspond to confidence intervals of the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance vs . all detected genes and proteins , respectively , assessed by Wilcoxon ' s test ( p - values shown in figure ) .

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

c) Distribution of log2(FC) for the significant cases in transcriptomics (N=4 scr treated and N=3 amiR-29a treated biological replicates) and proteomics (N=4 biological replicates per group) datasets of animals treated with amiR-29a versus scr animals. The black line represents the average. (d,e) Box plot showing distribution of log2(FC) of miR29a targets as d) transcripts (RNA) and e) proteins (PROT) in transcriptomic and proteomic datasets of adult animals treated with amiR-29a. Notches correspond to confidence intervals of the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes and proteins, respectively, assessed by Wilcoxon's test (p-values shown in figure).


f ) Box plots reporting distribution of log2 ( FC ) of age down - regulated miR29a targets after amiR - 29a treatment ( N = 4 scr treated and N = 3 amiR - 29a treated biological replicates ) . Notches correspond to confidence intervals of the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance was assessed with Wilcoxon ' s test ( p - values shown in figure ) .

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

f) Box plots reporting distribution of log2(FC) of age down-regulated miR29a targets after amiR-29a treatment (N=4 scr treated and N=3 amiR-29a treated biological replicates). Notches correspond to confidence intervals of the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance was assessed with Wilcoxon's test (p-values shown in figure).


h ) Scatter plot correlating the expression changes of genes significantly regulated by age ( log2FC , x - axis ) and miR - 29a antagonization by amiR29a in adult mice ( log2FC , y - axis ) . ( Spearman r = - 0 . 22 p = 1 . 4x10 - 8 ; Fisher test showing significant enrichment in the IV quadrant p = 2 . 2x10 - 16 ) . The number of genes for each quadrant is reported in figure .

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

h) Scatter plot correlating the expression changes of genes significantly regulated by age (log2FC, x-axis) and miR-29a antagonization by amiR29a in adult mice (log2FC, y-axis). (Spearman r=-0.22 p=1.4x10-8; Fisher test showing significant enrichment in the IV quadrant p=2.2x10-16). The number of genes for each quadrant is reported in figure.


i ) Scatter plot correlating the expression changes induced by miR - 29a antagonization in adult mice at the transcript - ( log2FC , x - axis ) and protein - level ( log2FC , y - axis ) . Spearman coefficient correlation , r = 0 . 11 ; p = 0 . 006 , Fisher test showing significant enrichment in the I quadrant p = 0 . 03 . The number of genes for each quadrant is reported in figure .

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i) Scatter plot correlating the expression changes induced by miR-29a antagonization in adult mice at the transcript- (log2FC, x-axis) and protein-level (log2FC, y-axis). Spearman coefficient correlation, r=0.11; p=0.006, Fisher test showing significant enrichment in the I quadrant p=0.03. The number of genes for each quadrant is reported in figure.


a ) VEP analysis . Ratio between contralateral and ipsilateral eye responses ( C / I ratio ± SEM ) in animals treated with saline and not deprived ( sal no MD ) , with scrambled LNA and MD ( scr MD ) , with amiR - 29a LNA and MD ( amiR - 29a MD ) . N = 6 / 7 ; one - way ANOVA , p = 0 . 0003 ; post hoc Tukey ' s test : sal noMD vs scr + MD , ns ; sal noMD vs amiR - 29a + MD , p = 0 . 0002 ; scr + MD vs amiR - 29a + MD , p = 0 . 0169 .

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

a) VEP analysis. Ratio between contralateral and ipsilateral eye responses (C/I ratio ± SEM) in animals treated with saline and not deprived (sal no MD), with scrambled LNA and MD (scr MD), with amiR-29a LNA and MD (amiR-29a MD). N=6/7; one-way ANOVA, p=0.0003; post hoc Tukey's test: sal noMD vs scr+MD, ns; sal noMD vs amiR-29a+MD, p=0.0002; scr+MD vs amiR-29a+MD, p=0.0169.


b ) IOS analysis . ODI ± SEM of deprived animals treated with scr ( N = 5 , black lines ) and amiR - 29a LNA ( N = 6 , blue lines ) . Dashed lines connect pre - and post - MD ODI values of each animal . Solid lines represent the group average . Two - way ANOVA RM , treatment X time , p = 0 . 0074 ; post hoc Sidak ' s test , pre - MD scr vs post - MD scr , ns ; pre - MD vs post - MD amiR - 29a , p = 0 . 0005 .

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b) IOS analysis. ODI ± SEM of deprived animals treated with scr (N=5, black lines) and amiR-29a LNA (N=6, blue lines). Dashed lines connect pre- and post-MD ODI values of each animal. Solid lines represent the group average. Two-way ANOVA RM, treatment X time, p= 0.0074; post hoc Sidak's test, pre-MD scr vs post-MD scr, ns; pre-MD vs post-MD amiR-29a, p=0.0005.


c ) IOS amplitude ( ± SEM ) in response to contralateral eye ( contra ) and ipsilateral eye ( ipsi ) stimulation . Other conventions as in b ) . amiR - 29a ( N = 6 ) vs scr ( N = 5 ) . Contra eye : two - way ANOVA RM , time X treatment interaction p = 0 . 034 ; post hoc Sidak ' s test ; pre - MD vs post - MD scr , ns ; pre - MD vs post - MD amiR - 29a , p = 0 . 0008 . Ipsi eye : two - way ANOVA RM , time X treatment interaction , ns .

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c) IOS amplitude (± SEM) in response to contralateral eye (contra) and ipsilateral eye (ipsi) stimulation. Other conventions as in b). amiR-29a (N=6) vs scr (N=5). Contra eye: two-way ANOVA RM, time X treatment interaction p=0.034; post hoc Sidak's test; pre-MD vs post-MD scr, ns; pre-MD vs post-MD amiR-29a, p=0.0008. Ipsi eye: two-way ANOVA RM, time X treatment interaction, ns.


d ) Average time course of the IOS response to visual stimulation of the contralateral eye ( contra ) pre - and post - MD ( thick black line ) . Grey area delimited by thin black lines envelops traces from scr animals ( 95 % confidence interval ) ; blue area envelop traces from amiR - 29a animals ( 95 % confidence interval ) . e ) Same as in d ) for ipsilateral eye ( ipsi ) pre - and post - MD responses .

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d) Average time course of the IOS response to visual stimulation of the contralateral eye (contra) pre- and post-MD (thick black line). Grey area delimited by thin black lines envelops traces from scr animals (95% confidence interval); blue area envelop traces from amiR-29a animals (95% confidence interval). e) Same as in d) for ipsilateral eye (ipsi) pre- and post-MD responses.


f ) The effects of MD on Npas IV and Bdnf exIV expression is reported as the ratio between expression levels of deprived and nondeprived cortex for animals treated with scrambled ( scr + MD ) and amiR - 29a ( amiR - 29a + MD ) . Each symbol represent the result of one animal . Error bars represent ± SEM . scr vs amiR - 29a ; t - test ; N = 7 - 9 , Npas4 : p = 0 . 035 ; N = 4 - 5 ; Bdnf exIV : p = 0 . 026 .

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

f) The effects of MD on Npas IV and Bdnf exIV expression is reported as the ratio between expression levels of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and amiR-29a (amiR-29a+MD). Each symbol represent the result of one animal. Error bars represent ±SEM. scr vs amiR-29a; t-test; N=7-9, Npas4: p=0.035; N=4-5; Bdnf exIV: p=0.026.


g ) Scatter plot correlating the changes in protein abundance induced by MD in young mice ( MD CP , x - axis ) with the effects observed in the adult deprived mice treated with amiR - 29a ( MD + LNA _ AD , y - axis ) . Proteins present in both data sets and showing an absolute expression fold change ( FC > 0 . 1 ) are represented . Green symbols correspond to proteins with concordant direction of regulation ; red symbols correspond to discordant regulation . Pearson correlation : r = 0 . 19 , p = 0 . 019 . The number of genes in each quadrant is reported in figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot correlating the changes in protein abundance induced by MD in young mice (MD CP, x-axis) with the effects observed in the adult deprived mice treated with amiR-29a (MD+LNA_AD, y-axis). Proteins present in both data sets and showing an absolute expression fold change (FC>0.1) are represented. Green symbols correspond to proteins with concordant direction of regulation; red symbols correspond to discordant regulation. Pearson correlation: r=0.19, p=0.019. The number of genes in each quadrant is reported in figure.


a ) Images of WFA - labeled PNNs of scrambled treated visual cortex ( left ) and miR - 29a mimic ( right ) in young mice . Scale bar 60 µm . ( b , c ) Quantification of PNN density ( scr vs mim29a , N = 3 mice per group ; t - test , p = 0 . 20 ) and intensity for each animal ( scr vs mim29a , N = 3 mice per group ; t - test , p = 0 . 014 ) . d ) Cumulative frequency of PNNs staining intensity in scrambled and mimic treated young mice scr ( N = 1438 cells vs mim29a N = 1749 cells , Kolmogorov - Smirnov test , p < 0 . 0001 ) . e ) PNN distribution in classes of intensity in mice treated with scrambled and mimic 29a .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of WFA-labeled PNNs of scrambled treated visual cortex (left) and miR-29a mimic (right) in young mice. Scale bar 60 µm. (b,c) Quantification of PNN density (scr vs mim29a, N=3 mice per group; t-test, p=0.20) and intensity for each animal (scr vs mim29a, N=3 mice per group; t-test, p=0.014). d) Cumulative frequency of PNNs staining intensity in scrambled and mimic treated young mice scr (N=1438 cells vs mim29a N=1749 cells, Kolmogorov-Smirnov test, p<0.0001). e) PNN distribution in classes of intensity in mice treated with scrambled and mimic 29a.


f ) Images of WFA - labeled PNNs of scrambled treated visual cortex ( left ) and amiR - 29a LNA ( right ) in adult mice . Scale bar 60 µm . ( g , h ) Quantification of PNN density ( scr vs amiR - 29a , N = 3 mice per group ; t - test , p = 0 . 03 ) and intensity for each animal scr vs amiR - 29a , N = 3 mice per group ; t - test , p = 0 . 047 . i ) Cumulative frequency of PNNs intensity in scrambled and amiR - 29a treated adult mice . scr N = 1883 cells vs amiR - 29a N = 1937 cells , Kolmogorov - Smirnov test , p < 0 . 0001 . j ) PNN distribution in classes of intensity in mice treated with scrambled and amiR - 29a .

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

f) Images of WFA-labeled PNNs of scrambled treated visual cortex (left) and amiR-29a LNA (right) in adult mice. Scale bar 60 µm. (g,h) Quantification of PNN density (scr vs amiR-29a, N=3 mice per group; t-test, p=0.03) and intensity for each animal scr vs amiR-29a, N=3 mice per group; t-test, p=0.047. i) Cumulative frequency of PNNs intensity in scrambled and amiR-29a treated adult mice. scr N=1883 cells vs amiR-29a N=1937 cells, Kolmogorov-Smirnov test, p< 0.0001. j) PNN distribution in classes of intensity in mice treated with scrambled and amiR-29a.


k ) Biochemical quantification of C4S in adult amiR - 29a treated mice ( N = 5 ) as compared to scrambled animals ( N = 4 ) . t test , p = 0 . 0008 .

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k) Biochemical quantification of C4S in adult amiR-29a treated mice (N=5) as compared to scrambled animals (N=4). t test, p=0.0008.


l ) Example of Arylsulfatase B ( ArsB ) western blot . Left : The lanes corresponding to the treated ( treat scr or treat amiR - 29a ) and untreated ( untreat scr or untreat amiR - 29a ) cortex ( ctx ) of two amiR - 29a treated and two scr treated mice are reported . Right : quantification of the data shows ArsB upregulation by amiR - 29a . The results are expressed as fold change normalized to untreated cortex . Each symbol represents the result of one animal . N = 4 , scr vs amiR - 29a treated cortex , unpaired t test , p = 0 .

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

l) Example of Arylsulfatase B (ArsB) western blot. Left: The lanes corresponding to the treated (treat scr or treat amiR-29a) and untreated (untreat scr or untreat amiR-29a) cortex (ctx) of two amiR-29a treated and two scr treated mice are reported. Right: quantification of the data shows ArsB upregulation by amiR-29a. The results are expressed as fold change normalized to untreated cortex. Each symbol represents the result of one animal. N=4, scr vs amiR-29a treated cortex, unpaired t test, p=0.043


C ) Violin and boxplots of Pol II density at enhancer regions ( n = 8 , 144 ) as a means of quantifying eRNA production . p - values were calculated using a two - sided Wilcoxon rank - sum test . Box plots consist of the median ( central line ) , the 25th and 75th percentiles ( box ) and the highest / lowest value within 1 . 5 * interquartile range of the box ( whiskers ) . Data information : n = 3 - 4 biological replicates per age group for tNET -

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C) Violin and boxplots of Pol II density at enhancer regions (n = 8,144) as a means of quantifying eRNA production. p-values were calculated using a two-sided Wilcoxon rank-sum test. Box plots consist of the median (central line), the 25th and 75th percentiles (box) and the highest/lowest value within 1.5 * interquartile range of the box (whiskers). Data information: n=3-4 biological replicates per age group for tNET-seq


D ) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies .

D) Western Blots of cellular fractionations of young and aged liver samples probed with the indicated antibodies.


E ) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals . Data information : n = 2 for ChIP -

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E) Heatmaps and average intensity profiles of RNA Pol II and its S2 and S5 phosphorylated forms in young and aged animals. Data information: n=2 for ChIP-seq


F ) Heatmaps and average intensity profiles of SPT4 ChIP - seq signal at TSSs of tNET genes in young and aged animals . Data information : n = 2 for ChIP -

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F) Heatmaps and average intensity profiles of SPT4 ChIP-seq signal at TSSs of tNET genes in young and aged animals. Data information: n=2 for ChIP-seq


B . Flow cytometry of cells infected with the lentiCRISPR v2 . 0 library where the expression of GFP indicates latency reactivation . Target genes were enriched through two - round sorting . Continuously cultured C11 latent cells infected with the lentiCRISPRv2 . 0 served as control . Blue dots and the encircled area represent the GFP - positive cells for flow cytometry analysis .

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B. Flow cytometry of cells infected with the lentiCRISPR v2.0 library where the expression of GFP indicates latency reactivation. Target genes were enriched through two-round sorting. Continuously cultured C11 latent cells infected with the lentiCRISPRv2.0 served as control. Blue dots and the encircled area represent the GFP-positive cells for flow cytometry analysis.


C . Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2 . 0 library infection .

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C. Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2.0 library infection.


D . Before next generation sequencing , the GFP expression in two - round sorted C11 cells was confirmed by fluorescence microscopy . Scale bar , 100 μm .

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D. Before next generation sequencing, the GFP expression in two-round sorted C11 cells was confirmed by fluorescence microscopy. Scale bar, 100 μm.


E . Fold change ( Log2 ) of the abundance of target genes in sorted C11 cells . Enriched sgRNA genes are highlighted .

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E. Fold change (Log2) of the abundance of target genes in sorted C11 cells. Enriched sgRNA genes are highlighted.


A . Validation of the top candidate genes in the C11 cell line . C11 cells were infected by lentiCRISPR v2 . 0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg / ml puromycin . The percentage of GFP - positive cells was measured by flow cytometry to determine the level of HIV - 1 reactivation .

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A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation.


The effect of candidate genes on HIV latency was further verified in J - Lat 10 . 6 ( B ) models of HIV latency . GFP expression in J - Lat 10 . 6 cells was analyzed by flow

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The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) models of HIV latency. GFP expression in J-Lat 10.6 cells was analyzed by flow cytometry


The effect of candidate genes on HIV latency was further verified in ACH2 ( C ) models of HIV latency . p24 in ACH2 cells was analyzed by ELISA , respectively .

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The effect of candidate genes on HIV latency was further verified in ACH2 (C) models of HIV latency. p24 in ACH2 cells was analyzed by ELISA, respectively.


E . PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1 - sg1 . Mock C11 cells served as control .

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E. PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1-sg1. Mock C11 cells served as control.


A . PEBP1 expression was measured by qPCR ( left panel ) and Western blot ( right panel ) in the latently infected C11 cells and the parental Jurkat cells .

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A. PEBP1 expression was measured by qPCR (left panel) and Western blot (right panel) in the latently infected C11 cells and the parental Jurkat cells.


B . The immunoprecipitation assay was performed in C11 cell lysates with anti - PEBP1 antibody followed by Western blot with anti - Raf1 or anti - IKK antibodies .

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B. The immunoprecipitation assay was performed in C11 cell lysates with anti-PEBP1 antibody followed by Western blot with anti-Raf1 or anti-IKK antibodies.


Effect of PEBP1 on the Raf1 / ERK / IκB and IKK / IκB / NF - κB signaling pathways . The levels of indicated proteins in total protein lysates ( C ) were analysed by Western blot in C11 - PEBP1 - KO cells and mock C11 cells .

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Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. The levels of indicated proteins in total protein lysates (C) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.


Effect of PEBP1 on the Raf1 / ERK / IκB and IKK / IκB / NF - κB signaling pathways . the levels of NF - κB / p65 protein in nucleus ( D ) were analysed by Western blot in C11 - PEBP1 - KO cells and mock C11 cells .

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Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. the levels of NF-κB/p65 protein in nucleus (D) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.


E . NF - κB / p65 protein recruitment into HIV LTR was analyzed by ChIP - qPCR with specific primers targeting the HIV LTR after normalization to the input .

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E. NF-κB/p65 protein recruitment into HIV LTR was analyzed by ChIP-qPCR with specific primers targeting the HIV LTR after normalization to the input.


F . The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells . 293T cells were co - transfected with PEBP1 - sg1 alone or PEBP1 - sg1 with HIV - 1 LTR - empty plasmids , HIV - 1 wild type LTR - luciferase plasmids , HIV - 1 LTR - Δsp1 - luciferase , HIV - 1 LTR - ΔNF - κB - luciferase , HIV - 1 LTR - ΔAp1 - luciferase or HIV - 1 LTR - ΔYY1 - luciferase . Transcription of HIV - 1 was determined by luciferase reporter assay .

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F. The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells. 293T cells were co-transfected with PEBP1-sg1 alone or PEBP1-sg1 with HIV-1 LTR-empty plasmids, HIV-1 wild type LTR-luciferase plasmids, HIV-1 LTR-Δsp1-luciferase, HIV-1 LTR-ΔNF-κB-luciferase, HIV-1 LTR-ΔAp1-luciferase or HIV-1 LTR-ΔYY1-luciferase. Transcription of HIV-1 was determined by luciferase reporter assay.


A . The expression of PEBP1 was analysed by Western blot in TZM - bl cells infected with mock , Lv - PCDH - empty or Lv - PCDH - PEBP1 plasmids .

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A. The expression of PEBP1 was analysed by Western blot in TZM-bl cells infected with mock, Lv-PCDH-empty or Lv-PCDH-PEBP1 plasmids.


Overexpression of PEBP1 suppressed HIV - 1 replication . TZM - bl cells were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies / ml . The transcription of HIV - 1 was evaluated 72 h post - infection by luciferase

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Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV-1 was evaluated 72 h post-infection by luciferase activity


Overexpression of PEBP1 suppressed HIV - 1 replication . TZM - bl cells were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies / ml . levels of p24 in the supernatants ( C ) .

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Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. levels of p24 in the supernatants (C).


D . The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h .

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D. The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h.


E . Induction of PEBP1 by EGCG or DHA in HIV - 1 infected primary CD4 + T cells . Primary CD4 + T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B , HIV - 1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies / ml . Seventy - two hours post - treatment with 10 µM EGCG or DHA , the expression of PEBP1 was detected by qPCR .

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E. Induction of PEBP1 by EGCG or DHA in HIV-1 infected primary CD4+ T cells. Primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy-two hours post-treatment with 10 µM EGCG or DHA, the expression of PEBP1 was detected by qPCR.


F . The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4 + T cells . The primary CD4 + T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B and E , HIV - 1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies / ml . The supernatants from HIV - 1 - infected CD4 + T cell were collected 1 , 2 , 3 or 4 days post - infection . Replication of HIV - 1 was analyzed by p24 ELISA .

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F. The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4+ T cells. The primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B and E, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV-1-infected CD4+ T cell were collected 1, 2, 3 or 4 days post-infection. Replication of HIV-1 was analyzed by p24 ELISA.


A . EGCG inhibited α - CD3 / CD28 reactivation of latent HIV - 1 . Primary CD4 + T cells were isolated from HIV - 1 positive patients on antiretroviral therapy ( n = 5 , P1 - P5 ) . Primary CD4 + T cells were treated with α - CD3 / CD28 alone or α - CD3 / CD28 and 10 µM EGCG . Cell - associated RNA was extracted 7 days post - treatment . The transcription of HIV - 1 was determined by real - time qPCR .

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A. EGCG inhibited α-CD3/CD28 reactivation of latent HIV-1. Primary CD4+ T cells were isolated from HIV-1 positive patients on antiretroviral therapy (n=5, P1-P5). Primary CD4+ T cells were treated with α-CD3/CD28 alone or α-CD3/CD28 and 10 µM EGCG. Cell-associated RNA was extracted 7 days post-treatment. The transcription of HIV-1 was determined by real-time qPCR.


B . PEBP1 mRNA was induced by EGCG in patient primary CD4 + T cells . Primary CD4 + T cells were treated with 10 μM EGCG . The expression of PEBP1 in the cells was measured by qPCR .

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B. PEBP1 mRNA was induced by EGCG in patient primary CD4+ T cells. Primary CD4+ T cells were treated with 10 μM EGCG. The expression of PEBP1 in the cells was measured by qPCR.


C . PEBP1 protein was induced by EGCG in patient primary CD4 + T cells , which was determined by Western blot of whole cell lysate from primary CD4 + T cells .

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C. PEBP1 protein was induced by EGCG in patient primary CD4+ T cells, which was determined by Western blot of whole cell lysate from primary CD4+ T cells.


D . EGCG - induced PEBP1 suppressed nuclear entry of NF - κB / p65 . This was measured by Western blot of nuclear protein of primary CD4 + T cells treated with α - CD3 / CD28 or α - CD3 / CD28 plus 10 μM EGCG .

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D. EGCG-induced PEBP1 suppressed nuclear entry of NF-κB/p65. This was measured by Western blot of nuclear protein of primary CD4+ T cells treated with α-CD3/CD28 or α-CD3/CD28 plus 10 μM EGCG.


B . Transcription of HIV - 1 in the primary CD4 + T cells was determined by nanoluc luciferase assays during HIV - 1 infection .

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B. Transcription of HIV-1 in the primary CD4+ T cells was determined by nanoluc luciferase assays during HIV-1 infection.


C . After infection with VSV - G pseudotyped HIV - 1 NL4 . 3 - nanoluc , the mRNA expression of PEBP1 was measured by qPCR .

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C. After infection with VSV-G pseudotyped HIV-1 NL4.3-nanoluc, the mRNA expression of PEBP1 was measured by qPCR.


Jurkat CD4 + T cells were treated with IFN - α , IFN - β and IFN - γ for 24 h . The mRNA expression levels of PEBP1 were measured by

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Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The mRNA expression levels of PEBP1 were measured by qPCR


Jurkat CD4 + T cells were treated with IFN - α , IFN - β and IFN - γ for 24 h . The protein expression levels of PEBP1 were measured by Western blot .

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Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The protein expression levels of PEBP1 were measured by Western blot.


F . PEBP1 knockout enhanced HIV - 1 transcription in the primary CD4 + T cell model of latency . The expression of HIV - 1 was measured by nanoluc after gene knockout where α - CD3 / CD28 stimulation served as a positive control .

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F. PEBP1 knockout enhanced HIV-1 transcription in the primary CD4+ T cell model of latency. The expression of HIV-1 was measured by nanoluc after gene knockout where α-CD3/CD28 stimulation served as a positive control.


( A ) Microchamber that allows the isolation of axons within a corticostriatal network and live - cell imaging of axonal BDNF - mCherry transduced into mouse primary cortical neurons . Data information : Scale bars = 20 µm .

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(A) Microchamber that allows the isolation of axons within a corticostriatal network and live-cell imaging of axonal BDNF-mCherry transduced into mouse primary cortical neurons. Data information: Scale bars = 20 µm.


( B ) Representative kymographs and quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry - containing vesicles in cortical neurons transfected with siMecp2 ( n = 753 vesicles / 81 axons ) or siControl ( siCtl ) ( n = 894 vesicles / 94 axons ) , inducing significant silencing of Mecp2 ( 3 independent experiments ; unpaired t - test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 Data are presented as the means ± SEM .

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(B) Representative kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 (n= 753 vesicles/ 81 axons) or siControl (siCtl) (n= 894 vesicles/ 94 axons), inducing significant silencing of Mecp2 (3 independent experiments; unpaired t-test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Data are presented as the means ± SEM.


( C ) Quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry trafficking into cortical neurons obtained from WT ( n = 753 vesicles / 81 axons ) , HTTSA ( n = 812 vesicles / 81 axons ) , or HTTSD ( n = 787 vesicles / 82 axons ) homozygous knock - in mice in which S421 of HTT was replaced by an alanine ( HTTS421A / S421A or HTTSA ) , mimicking the absence of phosphorylation , or by an aspartic acid ( HTTS421D / S421D or HTTSD ) , mimicking constitutive phosphorylation ( 3 independent experiments ; one - way ANOVA test with Tukey ' s multiple comparisons ) . Data information : , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . Data are presented as the means ± SEM .

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(C) Quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry trafficking into cortical neurons obtained from WT (n= 753 vesicles/ 81 axons), HTTSA (n= 812 vesicles/ 81 axons), or HTTSD (n= 787 vesicles/ 82 axons) homozygous knock-in mice in which S421 of HTT was replaced by an alanine (HTTS421A/S421A or HTTSA), mimicking the absence of phosphorylation, or by an aspartic acid (HTTS421D/S421D or HTTSD), mimicking constitutive phosphorylation (3 independent experiments; one-way ANOVA test with Tukey's multiple comparisons). Data information: , ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.


( D ) Cell viability in WT ( n = 3 ) , HTTSA ( n = 1 ) , or HTTSD ( n = 2 ) neurons measured by MTT assay . No differences were observed between the different groups ( 3 independent experiments ; one - way ANOVA Kruskal - Wallis test , p = 0 . 5701 Data information : ns = not significant . Data are presented as the means ± SEM .

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(D) Cell viability in WT (n=3), HTTSA (n=1), or HTTSD (n=2) neurons measured by MTT assay. No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701 Data information: ns = not significant. Data are presented as the means ± SEM.


( E ) Kymographs and quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry axonal trafficking into WT ( n = 894 vesicles / 94 axons ) , HTTSA ( n = 812 vesicles / 82 axons ) , or HTTSD ( n = 787 vesicles / 94 axons ) cortical neurons transfected with siCtl or siMecp2 , which significantly silenced Mecp2 ( 3 independent experiments ; one - way ANOVA with Tukey ' s multiple comparisons ) . ( F ) Cell viability in WT , HTTSA , or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay ( WT siCtl : n = 3 ; WT siMecp2 : n = 3 ; HTTSD siMecp2 : n = 3 ; HTTSA siMecp2 : n = 2 ) . No differences were observed between the different groups ( 3 independent experiments ; one - way ANOVA Kruskal - Wallis test , p = 0 . 5701 ) Data information : Scale bars = 20 µm . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . Data are presented as the means ± SEM .

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(E) Kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry axonal trafficking into WT (n= 894 vesicles/ 94 axons), HTTSA (n= 812 vesicles/ 82 axons), or HTTSD (n=787 vesicles/ 94 axons) cortical neurons transfected with siCtl or siMecp2, which significantly silenced Mecp2 (3 independent experiments; one-way ANOVA with Tukey's multiple comparisons). (F) Cell viability in WT, HTTSA, or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay (WT siCtl: n=3; WT siMecp2: n=3; HTTSD siMecp2: n=3; HTTSA siMecp2: n=2). No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701) Data information: Scale bars = 20 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.


( A ) Left : The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day - old WT ( n = 8 ) , Mecp2 KO ( n = 6 ) , Mecp2 KO / HTTSD ( mimicking the absence of phosphorylation ) ( n = 4 ) and Mecp2 KO / HTTSA mice ( mimicking constitutive phosphorylation ) ( n = 5 ) . Since striatal BDNF depends only on BDNF transport from the cortex , this ratio reflects BDNF transport through corticostriatal pathway ( Mann Whitney test ) . Right : Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting . The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio . ( n = 6 mice per group ) ( Mann Whitney test ) Middle : Quantitative analysis of PSD - 95 protein level in striatum of Mecp2 KO / HTT WT mice and KO / HTTSD mice by immunoblotting . The relative expression levels of PSD - 95 were normalized against GAPDH and are presented as the ratio . ( n = 6 mice per group ) ( Mann Whitney test ) Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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(A) Left: The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day-old WT (n=8), Mecp2 KO (n=6), Mecp2 KO/HTTSD (mimicking the absence of phosphorylation) (n=4) and Mecp2 KO/HTTSA mice (mimicking constitutive phosphorylation) (n=5). Since striatal BDNF depends only on BDNF transport from the cortex, this ratio reflects BDNF transport through corticostriatal pathway (Mann Whitney test). Right: Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting. The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Middle: Quantitative analysis of PSD-95 protein level in striatum of Mecp2 KO/HTT WT mice and KO/HTTSD mice by immunoblotting. The relative expression levels of PSD-95 were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( B ) Left : We investigated the behavior of Mecp2 KO mice at 35 , 45 , and 55 days of age and assessed their survival . Right : Mecp2 KO / HTTSD mice ( n = 30 ) and WT mice ( n = 17 ) had a significantly longer lifespan than KO ( n = 22 ) or KO / HTTSA mice ( n = 21 ) ( Kaplan - Meier survival test ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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(B) Left: We investigated the behavior of Mecp2 KO mice at 35, 45, and 55 days of age and assessed their survival. Right: Mecp2 KO/HTTSD mice (n = 30) and WT mice (n = 17) had a significantly longer lifespan than KO (n=22) or KO/HTTSA mice (n = 21) (Kaplan-Meier survival test). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( C ) Body weight of 10 WT , 24 KO , 32 KO / HTTSD , and 24 KO / HTTSA mice at P30 and P50 ( one - way ANOVA with Tukey ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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(C) Body weight of 10 WT, 24 KO, 32 KO/HTTSD, and 24 KO/HTTSA mice at P30 and P50 (one-way ANOVA with Tukey's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( D ) Frequency of apnea of 9 WT , 10 KO , 14 KO / HTTSD , and 9 KO / HTTSA mice at P35 and P55 . ( Kruskal - Wallis test with Dunn ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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(D) Frequency of apnea of 9 WT, 10 KO, 14 KO/HTTSD, and 9 KO/HTTSA mice at P35 and P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( E ) Mecp2 KO / HTTSD mice ( n = 19 ) performed as well as WT ( n = 17 ) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO ( n = 16 ) and Mecp2 KO / HTTSA ( n = 13 ) at P55 ( one - way ANOVA with Fisher ' s LSD test ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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(E) Mecp2 KO/HTTSD mice (n=19) performed as well as WT (n=17) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO (n=16) and Mecp2 KO/ HTTSA (n=13) at P55 (one-way ANOVA with Fisher's LSD test). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( F ) Time before the onset of spontaneous locomotor activity of 17 WT , 20 KO , 19 KO / HTTSD , and 17 KO / HTTSA mice at P55 . ( Kruskal - Wallis test with Dunn ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ns = not significant .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time before the onset of spontaneous locomotor activity of 17 WT, 20 KO, 19 KO/HTTSD, and 17 KO/HTTSA mice at P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ns = not significant.


( A ) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl ( siCtl ) and treated with 1 µM FK506 or vehicle for 1 h , and quantification of pS421 HTT ( n = 9 per group ) . Data are presented as means ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test .

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

(A) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h, and quantification of pS421 HTT (n=9 per group). Data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test.


( B ) Representative kymographs showing axonal trafficking of BDNF - mCherry - containing vesicles in cortical neurons transfected with siMecp2 or siControl ( siCtl ) and treated with 1 µM FK506 or vehicle for 1 h . Scale bar = 20 µm .

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

(B) Representative kymographs showing axonal trafficking of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h. Scale bar = 20 µm.


( C ) Quantification of anterograde , retrograde , mean velocity , linear flow rate and number of BDNF - mCherry - containing vesicles from the data in ( A ) ( siMecp2 + Vehicle : n = 801 vesicles / 95 axons ; siMecp2 + FK506 : n = 1020 vesicles / 116 axons ; siCtl + Vehicle : n = 780 vesicles / 83 axons ; siCtl + FK506 : n = 1029 vesicles / 102 axons ) . Data are presented as the mean ± SEM of at least three independent experiments ( one - way ANOVA with Tukey ' s comparison ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of anterograde, retrograde, mean velocity, linear flow rate and number of BDNF-mCherry-containing vesicles from the data in (A) (siMecp2+Vehicle: n=801 vesicles/95 axons; siMecp2+FK506: n=1020 vesicles/116 axons; siCtl+Vehicle: n=780 vesicles/83 axons; siCtl+FK506: n=1029 vesicles/102 axons). Data are presented as the mean ± SEM of at least three independent experiments (one-way ANOVA with Tukey's comparison). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( A ) Western blot of HTT S421 phosphorylation . We treated 30 - day - old Mecp2 KO mice intraperitoneally with FK506 ( 5 mg / kg ) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting , 2 h after administration , using an anti - phospho - HTT - S421 specific antibody . The D7F7 antibody recognizes total HTT . The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio ( KO FK506 n = 4 , KO Vehicle n = 4 ) . Data are presented as means ± SEM , * p < 0 . 05 , Mann Whitney test . )

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

(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.)


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( B ) FK506 - treated KO mice lived longer than vehicle - treated KO mice and FK506 - treated KO HTTSA mice ( Kaplan - Meier survival test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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 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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (B) FK506-treated KO mice lived longer than vehicle-treated KO mice and FK506-treated KO HTTSA mice (Kaplan-Meier survival test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( C ) Body weight of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice at P35 and P55 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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 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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( D ) Frequency of apnea of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice at P35 and P55 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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 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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (D) Frequency of apnea of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( E ) Motor coordination of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice on the accelerating rotarod test at P30 and P50 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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 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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (E) Motor coordination of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice on the accelerating rotarod test at P30 and P50 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( F ) Forelimb strength of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice assessed by the grip strength test at P40 and P60 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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 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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (F) Forelimb strength of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice assessed by the grip strength test at P40 and P60 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


Figure 1 . Helix - 7 dynamically interacts with the miRNA seed region . A ) Cartoon representation of the Ago2 - miRNA crystal structure . Ago2 is colored gray except for helix - 7 , which is yellow . miRNA guide colored red . C ) Close - up view of the unpaired complex shows helix - 7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7 .

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

Figure 1. Helix-7 dynamically interacts with the miRNA seed region. A) Cartoon representation of the Ago2-miRNA crystal structure. Ago2 is colored gray except for helix-7, which is yellow. miRNA guide colored red.C) Close-up view of the unpaired complex shows helix-7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7.


B ) Crystal structure of the Ago2 - miRNA - target RNA ternary complex . Target RNA is colored blue . D ) Close - up view of the Ago2 - miRNA - target complex shows helix - 7 docks into the minor groove of the guide : target duplex , directly contacting base pairs at positions g6 and g7 .

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

B) Crystal structure of the Ago2-miRNA-target RNA ternary complex. Target RNA is colored blue. D) Close-up view of the Ago2-miRNA-target complex shows helix-7 docks into the minor groove of the guide:target duplex, directly contacting base pairs at positions g6 and g7.


Figure 2 . Helix - 7 is not required for target recognition . A ) Plot of g2 - g8 target RNA bound ( 0 . 1 nM ) versus Ago2 - miR122 concentration for wild type ( WT ) , helix - 7 double point mutant ( MI - AA ) , and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± standard deviation ( SD ) are plotted . Top panel shows target RNA paired to seed region of the guide RNA ( miRNA - 122 ) .

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

Figure 2. Helix-7 is not required for target recognition. A) Plot of g2-g8 target RNA bound (0.1 nM) versus Ago2-miR122 concentration for wild type (WT), helix-7 double point mutant (MI-AA), and Ηhelix-7 Ago2. Average values from at least three independent experiments ± standard deviation (SD) are plotted. Top panel shows target RNA paired to seed region of the guide RNA (miRNA-122).


B ) Dissociation of a 32P - labeled target RNA ( 0 . 1 nM ) from the Ago2 - miR122 complex ( 1 nM ) was monitored in the presence of unlabeled target RNA ( 100 nM ) . Fraction of the target RNA bound to Ago2 - miR122 is plotted as a function of time for WT and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± SD were fit to single exponential decays .

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

B) Dissociation of a 32P-labeled target RNA (0.1 nM) from the Ago2-miR122 complex (1 nM) was monitored in the presence of unlabeled target RNA (100 nM). Fraction of the target RNA bound to Ago2-miR122 is plotted as a function of time for WT and Ηhelix-7 Ago2. Average values from at least three independent experiments ± SD were fit to single exponential decays.


C ) Representative fluorescence time traces ( with a resolution of 100 ms for wt , 300 ms for MI - AA and Ηhelix - 7 ) show docking and dissociation of wild type ( top ) , MI - AA ( middle ) , and Ηhelix - 7 ( bottom ) Ago2 - miRNA at single spots in the microfluidic chamber .

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

C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber.


D ) Dwell time histograms obtained for N = 4 , 5 and 6 , with wild type Ago2 ; histograms were fit with single exponential decay . The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution . The bin size is 0 . 5 sec for N = 4 and 1 sec for N = 5 and 6 .

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

D) Dwell time histograms obtained for N = 4, 5 and 6, with wild type Ago2; histograms were fit with single exponential decay. The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution. The bin size is 0.5 sec for N =4 and 1 sec for N =5 and 6.


E ) Bar plot showing the dependence of dwell time on N for wild type Ago2 . The dashed red line indicates the observation time limit ( 300 s ) , which is constrained by photobleaching . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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

E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching.For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


F ) Dwell time histogram for different N ' s with the MI - AA helix - 7 mutant . The dwell time histogram for N = 6 fit with double exponential decay ( green ; R2 = 0 . 98 ) ; The dwell time for N = 6 with single exponential fit was 15 . 2 ± 3 . 41 sec , R2 = 0 . 96 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dwell time histogram for different N's with the MI-AA helix-7 mutant. The dwell time histogram for N = 6 fit with double exponential decay (green; R2 = 0.98); The dwell time for N = 6 with single exponential fit was 15.2 ± 3.41 sec, R2 = 0.96).


G ) Bar plot for dwell times of MI - AA Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

O O O O O O O O O O O O O O O O O O O O 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) Bar plot for dwell times of MI-AA Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


H ) Dwell time histogram for different N ' s with the ∆ helix - 7 mutant . The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays ( green ; R2 = 0 . 97 for N = 5 and R2 = 0 . 96 for N = 6 ) ; The dwell time for N = 5 and N = 6 with single exponential fit was 9 . 1 ± 0 . 91 sec ( R2 = 0 . 86 ) and 22 . 3 ± 5 . 08 sec ( R2 = 0 . 90 ) respectively ) .

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

H) Dwell time histogram for different N's with the ∆helix-7 mutant. The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays (green; R2 = 0.97 for N = 5 and R2 = 0.96 for N = 6); The dwell time for N = 5 and N = 6 with single exponential fit was 9.1 ± 0.91 sec (R2 = 0.86) and 22.3 ± 5.08 sec (R2 = 0.90) respectively).


I ) Bar plot for dwell times of ∆ helix - 7 Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

O O O O O O O O O O O O O O O O O O O O O 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) Bar plot for dwell times of ∆helix-7 Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


Figure 4 . Helix - 7 discourages non - Watson Crick pairing at g6 and g7 . A - C ) Plots of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 ( wild type and Ηhelix - 7 ) concentration for targets with an increasing number of G : U wobble pairs in the seed 3 ' end . D ) Plot of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 concentration for a target with mismatches at positions g6 - g8 . For A - E , average values from at least three independent experiments ± SD are plotted .

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

Figure 4. Helix-7 discourages non-Watson Crick pairing at g6 and g7. A-C) Plots of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 (wild type and Ηhelix-7) concentration for targets with an increasing number of G:U wobble pairs in the seed 3' end. D) Plot of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 concentration for a target with mismatches at positions g6-g8. For A-E, average values from at least three independent experiments ± SD are plotted.


E ) Bar plots of the Kd of wild type ( left ) Ηhelix - 7 ( right ) Ago2 - miR122 for indicated target RNAs . For A - E , average values from at least three independent experiments ± SD are plotted .

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

E) Bar plots of the Kd of wild type (left) Ηhelix-7 (right) Ago2-miR122 for indicated target RNAs. For A-E, average values from at least three independent experiments ± SD are plotted.


Figure 5 . ΗHelix - 7 Ago2 is sensitive to inhibition by off - target RNAs . A ) Ago2 - miRNA122 ( 1 nM ) was incubated with a 32P - labeled , seed - matched target RNA ( 0 . 1 nM ) in the presence of increasing concentrations of unlabeled a competitor RNA ( top panel ) . Fraction target RNA bound to Ago2 is plotted as a function of off - target competitor RNA concentration . B ) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA . For A - B , average values from at least three independent experiments ± SD are plotted .

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

Figure 5. ΗHelix-7 Ago2 is sensitive to inhibition by off-target RNAs. A) Ago2-miRNA122 (1 nM) was incubated with a 32P-labeled, seed-matched target RNA (0.1 nM) in the presence of increasing concentrations of unlabeled a competitor RNA (top panel). Fraction target RNA bound to Ago2 is plotted as a function of off-target competitor RNA concentration. B) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA. For A-B, average values from at least three independent experiments ± SD are plotted.


Figure 6 . Helix - 7 mutants display reduced target - binding rates . A ) Ago2 - miR122 complexes were mixed with a 32P - labeled seed matched target RNA ; bound and free RNAs were then separated using a filter - binding apparatus at various times . Representative time course for target RNA ( 0 . 1 nM ) binding to wild type , MI - AA , and Δhelix - 7 Ago2 ( 1 nM ) . Average values from at least three independent experiments ± SD are plotted .

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Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted.


B ) Observed binding rates ( kon , obs ) plotted as a function of Ago2 - miR122 concentration . Average values ± SD are plotted .

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B) Observed binding rates (kon, obs) plotted as a function of Ago2-miR122 concentration. Average values ± SD are plotted.


C ) Arrival time distribution of single Ago2 - miRNA complexes binding immobilized targets in a microfluidic chamber . The value is the average of three independent measurements .

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C) Arrival time distribution of single Ago2-miRNA complexes binding immobilized targets in a microfluidic chamber. The value is the average of three independent measurements.


Figure 7 . Structure of a helix - 7 Ago2 mutant . A ) Superposition of wild type ( white ) and MI - AA ( gray ) Ago2 . Helix - 7 colored yellow ; ordered region of the guide RNAs colored in red .

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Figure 7. Structure of a helix-7 Ago2 mutant. A) Superposition of wild type (white) and MI-AA (gray) Ago2. Helix-7 colored yellow; ordered region of the guide RNAs colored in red.


B ) Close - up view shows helix - 7 tilts away from the seed region in the MI - AA mutant .

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B) Close-up view shows helix-7 tilts away from the seed region in the MI-AA mutant.


C ) Side - by - side comparison of the seed region in wild type ( left ) and MI - AA ( right ) Ago2 crystal structures .

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C) Side-by-side comparison of the seed region in wild type (left) and MI-AA (right) Ago2 crystal structures.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data . ( C ) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1 - specific siRNA showing LC3 lipidation after treatment with 100 - µM spermidine in the presence or absence of bafilomycin A1 .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1.


( D - F ) HCT 116 cells were left transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h , and treated with either vehicle ( Co ) , 100 - µM resveratrol , or 100 - µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h . ( D ) Representative images . ( E ) Quantitative data . ( B and E ) Bars depict the percentages of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ; means ± SEM ; n = 3 ; * , P < 0 . 05 ) . ( F ) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100 - µM spermidine in the presence or absence of EX527 . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D-F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


The lifespan - extending and autophagy - inducing effects of spermidine in yeast are not mediated by Sir2 . ( A - E ) EGFP - Atg8p was ectopically expressed in wild - type ( WT ) or Δsir2 S . cerevisiae undergoing chronological aging on small synthetic 2 % glucose media with or without ( Co , control ) supplementation of 4 - mM spermidine ( Spd ) . ( A ) Representative images . EGFP - Atg8p localization ( bottom ) was visualized by fluorescence microscopy . Yeast cells undergoing autophagy ( in which EGFP - Atg8p exhibits a prominent vacuolar localization ) are indicated by arrows . Yeast morphology was monitored by differential interference contrast ( DIC ; top ) .

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The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A-E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top).


( B ) Representative immunoblots against EGFP . Free EGFP indicates the vacuolar degradation of EGFP - Atg8p fusion , thereby representing the autophagic flux . Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine - mediated autophagy induction .

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(B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction.


( C ) Relative alkaline phosphatase ( ALP ) activity indicative of autophagy . n = 3 .

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(C) Relative alkaline phosphatase (ALP) activity indicative of autophagy. n = 3.


( E ) Quantification of reactive oxygen species . Bars indicate the percentages of cells exhibiting the reactive oxygen species - mediated conversion of dihydroethidine ( DHE ) into ethidium ( Eth ; n = 4 ) . Data represent means ± SEM ; * , P < 0 . 001 as compared with untreated cells of the same genotype . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase . RFU , relative fluorescence unit .

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(E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species-mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.


The life - extending and autophagy - inducing effects of spermidine in C . elegans are not mediated by Sir2 . ( A ) Fluorescence microscopy of C . elegans transgenic embryos expressing a full - length plgg - 1DsRed : : LGG - 1 fusion protein indicative of autophagic activity . Two representative pictures of wild - type ( WT ) and sir - 2 . 1 embryos untreated ( Co , control ) or treated with 0 . 2 - mM spermidine ( Spd ) supplementation of food are shown . ( B ) Quantification of autophagic activity through the measurement of DsRed : : LGG - 1 pixel intensity from images of WT animals shown in A . Data represent means ± SEM ( n = 3 ) with ≥ 25 images processed for each trial .

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The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial.


C ) Survival of WT C . elegans during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 005 ) . ( D ) Survival of sir - 2 . 1 C . elegans ( ok434 phenotype ) during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 01 ) . P - values were calculated using the log - rank test as described in Materials and methods .

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C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.


Convergent alterations in the phosphoproteome status after resveratrol and / or spermidine treatment . ( A - C ) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , and 100 - µM spermidine ( Spd ) , alone or in combination ( Resv + Spd ) . ( A ) Representative phosphoprotein arrays are shown .

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Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A-C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown.


( B ) Clustering analysis for the effects on protein kinase phosphorylation .

(B) Clustering analysis for the effects on protein kinase phosphorylation.


( C ) Representative immunoblots of selected kinases whose phosphorylation status was unaffected ( PRKAA1 , RPS6KB1 , and acetyl - CoA carboxylase a [ ACACA ] ) or affected by resveratrol or / and spermidine treatment ( PTKB , AKT1 , MAPK8 , and CDKN1B ) , validating phosphoprotein array data .

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(C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data.


( D ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle or the indicated dose of resveratrol or spermidine , alone or in combination , for 2 h . Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Convergent acetylproteome modification after resveratrol or spermidine treatment . ( A - C ) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes . Cells were treated with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) for 2 h , fractioned into cytoplasmic , nuclear , and mitochondrial extracts , processed for acetyl lysine peptide enrichment , and analyzed by MS . ( A ) Hierarchical clustering of drug - specific organellar distributions of all acetylated sites quantified in at least one fraction . Fold changes are calculated relative to untreated cells . Only sites regulated > 1 . 5 - fold were included in statistical analyses . ( B ) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way . n = 560 .

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Convergent acetylproteome modification after resveratrol or spermidine treatment. (A-C) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes. Cells were treated with 100-µM resveratrol (Resv) or spermidine (Spd) for 2 h, fractioned into cytoplasmic, nuclear, and mitochondrial extracts, processed for acetyl lysine peptide enrichment, and analyzed by MS. (A) Hierarchical clustering of drug-specific organellar distributions of all acetylated sites quantified in at least one fraction. Fold changes are calculated relative to untreated cells. Only sites regulated >1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560.


Significant motifs among sites undergoing acetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Consensus acetylation motifs identified upon resveratrol ( left ) or spermidine ( right ) treatment are depicted using the MotifX algorithm ( Schwartz and Gygi , 2005 ) . Among sites that were hyperacetylated in response to both agents , the K ( F / Y ) motif is significantly enriched when tested against the whole proteome ( P < 0 . 00001 ) . ( C ) When testing against the largest acetylation site dataset from Choudhary et al . ( 2009 ) ( acetylation background dataset [ ABD ] ) , the SxK motif is significant ( P < 0 . 0001 ) for sites whose acetylation increased upon spermidine treatment . No general consensus motifs were found for sites whose acetylation increased in response to both agents .

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Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P < 0.00001). (C) When testing against the largest acetylation site dataset from Choudhary et al. (2009) (acetylation background dataset [ABD]), the SxK motif is significant (P < 0.0001) for sites whose acetylation increased upon spermidine treatment. No general consensus motifs were found for sites whose acetylation increased in response to both agents.


Significant motifs among sites undergoing deacetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Among sites that were hypoacetylated in response to both agents , the KP motif is significantly enriched when tested against ABD ( P < 0 . 001 ) . ( C and D ) When tested against the ABD ( C ) or the whole proteome ( D ) , the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment ( P < 0 . 0001 ) . No general consensus motifs were found for sites whose acetylation decreased in response to both agents .

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Significant motifs among sites undergoing deacetylation. (A) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Among sites that were hypoacetylated in response to both agents, the KP motif is significantly enriched when tested against ABD (P < 0.001). (C and D) When tested against the ABD (C) or the whole proteome (D), the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment (P < 0.0001). No general consensus motifs were found for sites whose acetylation decreased in response to both agents.


Autophagy can be efficiently regulated by cytoplasmic ( de ) acetylation reactions . ( A and B ) HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and enucleated to obtain cytoplasts . The cytoplasts were treated with either vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , or 100 - µM spermidine ( Spd ) for 4 h . 1 - µM rapamycin ( Rapa ) was used as a positive control . ( A ) Representative images of cytoplasts indicative of autophagic activity . Arrows indicate GFP - LC3 - transfected cytoplasts , whereas insets show nonenucleated transfected cells . ( B ) Quantitative data . Bars show the percentages of cells or cytoplasts showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) .

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Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3-transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac).


( C - E ) HCT 116 cells were cotransfected with a plasmid for the expression of RFP - LC3 together with an empty vector ( pcDNA3 ) or plasmids encoding wild - type ( WT ) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP ( WT GFP - SIRT1 and mtNLS GFP - SIRT1 , respectively ) for 12 or 24 h , fixed , and analyzed by fluorescence microscopy . ( C ) Representative images indicative of 24 - h autophagic activity .

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(C-E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity.