b ) Box plot showing distribution of log2 ( FC ) of or selected miR - 29a targets in Data ref : Mazziotti et al , 2017 dataset of - regulated genes in visual cortex . Notches represent confidence intervals for median . Whiskers indicate 95th 5th percentile , box marks 25th 75th percentile . Significance vs . detected genes was assessed by Wilcoxon ' s ( miR - 29a targets p = 4 . 9x10 - 14 , top 200 miR - 29a targets p = 3 . 7x10 - 38 ) .

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b) Box plot showing the distribution of log2(FC) of all or selected miR-29a targets in the Data ref: Mazziotti et al, 2017 dataset of age-regulated genes in the visual cortex. Notches represent confidence intervals for the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes was assessed by Wilcoxon's test (miR-29a targets p=4.9x10-14, top 200 miR-29a targets p=3.7x10-38).


( d , e ) Mature miR - 29a ( n = 3 / 4 mice for each ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns not significant ) Dnmt3a expression level ( normalized to P10 ) at different ages measured by qPCR ( n = 3 / 4 mice for each ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns not significant ) . One way ANOVA p - values are reported . Error bars represent ± . f ) Scatter plot showing correlation of miR - 29a Dnmt3a in visual cortex display a strong negative correlation ( Pearson correlation : r = - 0 . 817 , p < 0 . 001 ) . Each circle represents data from a single .

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(d,e) Mature miR-29a (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test ***p<0.001, ****p<0.0001, ns not significant) and Dnmt3a expression level (normalized to P10) at different ages measured by qPCR (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test: *p<0.05, **p<0.01, ****p<0.0001, ns not significant). One way ANOVA p-values are reported. Error bars represent ±SEM. f) Scatter plot showing correlation of miR-29a and Dnmt3a levels in the visual cortex display a strong negative correlation (Pearson correlation: r=-0.817, p<0.001). Each circle represents data from a single animal.


g ) Representative examples of in situ hybridization on visual cortex ( left P25 , right P120 ) using a LNA probe against miR - 29a . Scale bar 180 µm . Cortical layers are delimited by roman numerals

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g) Representative examples of in situ hybridization on visual cortex (left P25, right P120) using a LNA probe against miR-29a. Scale bar 180 µm. Cortical layers are delimited by roman numerals and brackets


h ) Example of co - for PV ( green ) , miR - 29a ( red ) marker ( blue ) in a P120 mouse . triangles represent PV + / miR - 29a + . Scale bar 20 µm .

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h) Example of co-staining for PV (green), miR-29a (red) and nuclear marker (blue) in a P120 mouse. White triangles represent PV+/miR-29a+ cells. Scale bar 20 µm.


Mature miR - 29a expression ( miR29 ) in visually deprived mice : a ) normal rearing ( NR , N = 5 ) vs rearing ( DRb , N = 5 ) ; t - , p = 0 . 19 , b ) NR ( N = 4 ) vs rearing from P10 ( DRp10 , N = 5 ; t - , p = 0 . 70 . In both experiments , was performed at

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Mature miR-29a expression (miR29) in visually deprived mice: a) normal rearing (NR, N=5) vs dark rearing (DRb, N=5); t-test, p=0.19, b) NR (N=4) vs dark rearing from P10 (DRp10, N=5; t-test, p=0.70. In both experiments, analysis was performed at P25


Mature miR - 29a expression ( miR29 ) in visually deprived mice : c ) one week period of rearing beginning at P30 ( DRw ) followed by a 4 hour exposure to ( DRw + 4h ) vs control ( ctr , N = 4 for each group ; one - way ANOVA , p = 0 . 68 )

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Mature miR-29a expression (miR29) in visually deprived mice: c) one week period of dark rearing beginning at P30 (DRw) followed by a 4 hour exposure to light (DRw+4h light) vs control animals (ctr, N=4 for each group; one-way ANOVA, p=0.68)


Mature miR - 29a expression ( miR29 ) in visually deprived mice : d ) days of MD beginning at P25 , cortex vs cortex to deprived eye , N = 5 ; paired t , p = 0 . 52 ) . Error bars represent ± , symbols represent cases .

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Mature miR-29a expression (miR29) in visually deprived mice: d) days of MD beginning at P25, contralateral cortex vs ipsilateral cortex to deprived eye, N=5; paired t test, p=0.52). Error bars represent ±SEM, symbols represent individual cases.


b ) MiR - 29a Dnmt3a expression in cortex treated with scrambled or miR - 29a mimic ( N = 3 mice ) . Data were normalized to untreated cortex of same . Mir - 29a : scr vs mim29a , t - , p = 0 . 005 ; Dnmt3a scr vs mim29a t - , p < 0 . 0001 .

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b) MiR-29a and Dnmt3a expression in the cortex treated with scrambled or miR-29a mimic (N=3 mice). Data were normalized to the untreated cortex of the same animal. Mir-29a: scr vs mim29a, t-test, p=0.005; Dnmt3a scr vs mim29a t-test, p<0.0001.


c ) IOS . Ocular dominance index ( ODI ) of deprived treated with scrambled ( scr , N = 6 black lines ) or miR - 29a mimic ( mimic29 , N = 8 orange lines ) , or left untreated ( MD no treat , N = 5 grey lines ) before after 3 days of MD . Dashed lines represent single , continuous lines group average . two - way ANOVA RM , treatment X time , p = 0 . 028 ; post hoc Sidak ' s comparison , post - MD mim29a vs scr p < 0 . 05 , mim29a vs MD no treat p < 0 . 01 ; pre - MD scr vs post - MD scr p < 0 . 001 ; pre - MD MD no treat vs post - MD MD no treat p < 0 . 01 ; other comparisons not significant ) .

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c) IOS analysis. Ocular dominance index (ODI) of deprived animals treated with scrambled (scr, N=6 black lines) or miR-29a mimic (mimic29, N=8 orange lines), or left untreated (MD no treat, N=5 grey lines) before and after 3 days of MD. Dashed lines represent single animals, continuous lines the group average. two-way ANOVA RM, treatment X time, p= 0.028; post hoc Sidak's comparison, post-MD mim29a vs scr p<0.05, mim29a vs MD no treat p<0.01; pre-MD scr vs post-MD scr p<0.001; pre-MD MD no treat vs post-MD MD no treat p<0.01; other comparisons not significant).


d ) effects of MD on expression of Npas IV Bdnf exIV is reported as between expression level of deprived nondeprived cortex for treated with scrambled ( scr + MD ) miR - 29a mimic ( mim29a + MD ) . Each symbol represents result of a single mouse . ( MD + scr vs MD + mim29a ; N = 4 ; t - , Npas4 : p = 0 . 038 ; Bdnf exIV : p = 0 . 049 ) .

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d) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and miR-29a mimic (mim29a+MD). Each symbol represents the result of a single mouse. (MD+scr vs MD+mim29a; N=4; t-test, Npas4: p=0.038; Bdnf exIV: p=0.049).


b ) Effects of amiR - 29a on expression of miR - 29a Dnmt3a . Fold change values with respect to untreated cortex are reported ( ± ) for mice treated with amiR - 29a or scr . scr vs amiR - 29a ; N = 3 t - , miR - 29a * * p = 0 . 0010 ; Dnmt3a * * * * p = 0 . 0019 .

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b) Effects of amiR-29a on the expression of miR-29a and Dnmt3a. Fold change values with respect to the contralateral untreated cortex are reported (±SEM) for mice treated with amiR-29a or scr. scr vs amiR-29a; N=3 t-test, miR-29a **p=0.0010; Dnmt3a ****p=0.0019.


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

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

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

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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 expression changes induced by miR - 29a antagonization in adult mice at transcript - ( log2FC , x - axis ) - level ( log2FC , y - axis ) . Spearman coefficient correlation , r = 0 . 11 ; p = 0 . 006 , Fisher showing significant enrichment in I quadrant p = 0 . 03 . 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 . between eye responses ( C / I ± ) in treated with saline not deprived ( sal no MD ) , with scrambled LNA MD ( scr MD ) , with amiR - 29a LNA MD ( amiR - 29a MD ) . N = 6 / 7 ; one - way ANOVA , p = 0 . 0003 ; post hoc Tukey ' s : sal noMD vs scr + MD , ns ; sal noMD vs amiR - 29a + MD , p = 0 . 0002 ; scr + MD vs amiR - 29a + MD , p = 0 . 0169 .

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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 . ODI ± of deprived treated with scr ( N = 5 , black lines ) amiR - 29a LNA ( N = 6 , blue lines ) . Dashed lines connect pre - post - MD ODI values of each . Solid lines represent group average . Two - way ANOVA RM , treatment X time , p = 0 . 0074 ; post hoc Sidak ' s , 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 ( ± ) in response to eye ( contra ) 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 ; 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 IOS response to visual stimulation of eye ( contra ) pre - post - MD ( thick black ) . Grey delimited by thin black lines envelops traces from scr ( 95 % confidence interval ) ; blue envelop traces from amiR - 29a ( 95 % confidence interval ) . e ) Same as in d ) for eye ( ipsi ) pre - post - MD responses .

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

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

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

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

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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 of C4S in adult amiR - 29a treated mice ( N = 5 ) as compared to scrambled ( N = 4 ) . t , 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 : lanes corresponding to treated ( treat scr or treat amiR - 29a ) untreated ( untreat scr or untreat amiR - 29a ) cortex ( ctx ) of two amiR - 29a treated two scr treated mice are reported . Right : of data shows ArsB upregulation by amiR - 29a . results are expressed as fold change normalized to untreated cortex . Each symbol represents result of one . N = 4 , scr vs amiR - 29a treated cortex , unpaired t , p = 0 .

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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 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 . Box plots consist of median ( central ) , 25th 75th percentiles ( box ) highest / lowest value within 1 . 5 * interquartile range of box ( whiskers ) . Data information : n = 3 - 4 biological replicates per 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 fractionations of young aged liver samples probed with indicated antibodies .

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


E ) Heatmaps average profiles of Pol II its S2 S5 phosphorylated forms in young aged . 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 average profiles of SPT4 ChIP - seq signal at TSSs of tNET genes in young aged . 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 infected with lentiCRISPR v2 . 0 library where expression of GFP indicates latency reactivation . Target genes were enriched through two - round sorting . Continuously cultured C11 latent infected with lentiCRISPRv2 . 0 served as control . Blue dots encircled represent GFP - positive for flow cytometry .

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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 sorted C11 by PCR after lentiCRISPR v2 . 0 library infection .

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


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

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 abundance of target genes in sorted C11 . 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 top candidate genes in C11 . C11 were infected by lentiCRISPR v2 . 0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg / ml puromycin . of GFP - positive was measured by flow cytometry to determine 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.


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 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


effect of candidate genes on HIV latency was further verified in ACH2 ( C ) models of HIV latency . p24 in ACH2 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 were measured by Western blot after knock out in C11 by LvPEBP1 - sg1 . Mock C11 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 ) Western blot ( right panel ) in latently infected C11 parental Jurkat .

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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 . immunoprecipitation was performed in C11 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 Raf1 / ERK / IκB IKK / IκB / NF - κB signaling pathways . of indicated in total lysates ( C ) were analysed by Western blot in C11 - PEBP1 - KO mock C11 .

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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 Raf1 / ERK / IκB IKK / IκB / NF - κB signaling pathways . of NF - κB / p65 in nucleus ( D ) were analysed by Western blot in C11 - PEBP1 - KO mock C11 .

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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 recruitment into HIV LTR was analyzed by ChIP - qPCR with specific primers targeting HIV LTR after normalization to 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 . impact of PEBP1 on activation of HIV LTR was explored by luciferase reporter in 293T . 293T 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 .

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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 . expression of PEBP1 was analysed by Western blot in TZM - bl 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 were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from plasma whose viral load is 129 copies / ml . 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 were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from plasma whose viral load is 129 copies / ml . of p24 in supernatants ( C ) .

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

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 . expression level of PEBP1 was detected by Western blot using whole lysate of C11 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 CD4 + T . CD4 + T from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B , HIV - 1 was isolated from blood supernatants of receiving ART with a viral load of 129 copies / ml . Seventy - two hours post - treatment with 10 µM EGCG or DHA , 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 . induction of PEBP1 by EGCG or DHA suppressed HIV replication in CD4 + T . CD4 + T from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B E , HIV - 1 was isolated from blood supernatants of receiving ART with a viral load of 129 copies / ml . supernatants from HIV - 1 - infected CD4 + T 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 . CD4 + T were isolated from HIV - 1 positive on antiretroviral therapy ( n = 5 , P1 - P5 ) . CD4 + T were treated with α - CD3 / CD28 alone or α - CD3 / CD28 10 µM EGCG . - associated was extracted 7 days post - treatment . 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 CD4 + T . CD4 + T were treated with 10 μM EGCG . expression of PEBP1 in 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 was induced by EGCG in CD4 + T , which was determined by Western blot of whole lysate from CD4 + T .

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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 entry of NF - κB / p65 . This was measured by Western blot of of CD4 + T 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 CD4 + T 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 , mRNA expression of PEBP1 was measured by qPCR .

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


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

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 were treated with IFN - α , IFN - β IFN - γ for 24 h . expression 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 CD4 + T model of latency . 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 isolation of axons within a corticostriatal network live - imaging of axonal BDNF - mCherry transduced into mouse 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 of anterograde , retrograde , mean velocity , 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 - ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 Data are presented as means ± .

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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 ) of anterograde , retrograde , mean velocity , 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 absence of phosphorylation , or by an aspartic acid ( HTTS421D / S421D or HTTSD ) , mimicking constitutive phosphorylation ( 3 independent experiments ; one - way ANOVA with Tukey ' s multiple comparisons ) . Data information : , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . Data are presented as means ± .

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

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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 of anterograde , retrograde , mean velocity , 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 ) viability in WT , HTTSA , or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT ( WT siCtl : n = 3 ; WT siMecp2 : n = 3 ; HTTSD siMecp2 : n = 3 ; HTTSA siMecp2 : n = 2 ) . No differences were observed between different groups ( 3 independent experiments ; one - way ANOVA Kruskal - Wallis , 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 means ± .

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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 : between striatal cortical BDNF determined by quantifying level of BDNF at cortex striatum of 55 day - old WT ( n = 8 ) , Mecp2 KO ( n = 6 ) , Mecp2 KO / HTTSD ( mimicking absence of phosphorylation ) ( n = 4 ) Mecp2 KO / HTTSA mice ( mimicking constitutive phosphorylation ) ( n = 5 ) . Since striatal BDNF depends only on BDNF transport from cortex , this reflects BDNF transport through corticostriatal pathway ( Mann Whitney ) . Right : Quantitative of phospho TrkB level in striatum of KO WT mice KO HTTSD mice by immunoblotting . relative expression of phospho TrkB were normalized against GAPDH are presented as . ( n = 6 mice per group ) ( Mann Whitney ) Middle : Quantitative of PSD - 95 level in striatum of Mecp2 KO / HTT WT mice KO / HTTSD mice by immunoblotting . relative expression of PSD - 95 were normalized against GAPDH are presented as . ( n = 6 mice per group ) ( Mann Whitney ) Data information : Data are presented as means ± . * 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 behavior of Mecp2 KO mice at 35 , 45 , 55 days of assessed their survival . Right : Mecp2 KO / HTTSD mice ( n = 30 ) WT mice ( n = 17 ) had a significantly longer lifespan than KO ( n = 22 ) or KO / HTTSA mice ( n = 21 ) ( Kaplan - Meier survival ) . Data information : Data are presented as means ± . * 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 ) of 10 WT , 24 KO , 32 KO / HTTSD , 24 KO / HTTSA mice at P30 P50 ( one - way ANOVA with Tukey ' s comparison ) . Data information : Data are presented as means ± . * 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 , 9 KO / HTTSA mice at P35 P55 . ( Kruskal - Wallis with Dunn ' s comparison ) . Data information : Data are presented as means ± . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 accelerating rotarod continued to outperform Mecp2 KO ( n = 16 ) Mecp2 KO / HTTSA ( n = 13 ) at P55 ( one - way ANOVA with Fisher ' s LSD ) . Data information : Data are presented as means ± . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 onset of spontaneous locomotor activity of 17 WT , 20 KO , 19 KO / HTTSD , 17 KO / HTTSA mice at P55 . ( Kruskal - Wallis with Dunn ' s comparison ) . Data information : Data are presented as means ± . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of DIV 5 cortical neurons transfected with siMecp2 or siControl ( siCtl ) treated with 1 µM FK506 or vehicle for 1 h , of pS421 HTT ( n = 9 per group ) . Data are presented as means ± , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney .

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

(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 ) treated with 1 µM FK506 or vehicle for 1 h . Scale bar = 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 ) of anterograde , retrograde , mean velocity , linear flow rate number of BDNF - mCherry - containing vesicles from 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 mean ± 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 O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 analyzed brain extracts for endogenous HTT phosphorylation by western blotting , 2 h after administration , using an anti - phospho - HTT - S421 specific antibody . D7F7 antibody recognizes total HTT . relative level of phospho HTT was normalized on total HTT level are presented as ( KO FK506 n = 4 , KO Vehicle n = 4 ) . Data are presented as means ± , * p < 0 . 05 , Mann Whitney . )

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 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

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

O O O O O 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-CONTROLLED_VAR 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

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

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

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

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

O O O O O 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 miRNA seed region . A ) Cartoon representation of Ago2 - miRNA crystal structure . Ago2 is colored gray except for helix - 7 , which is yellow . miRNA guide colored red . C ) Close - up view of unpaired shows helix - 7 breaks nucleobase stacking in miRNA seed region by intercalating between g6 g7 .

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

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 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

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 bound ( 0 . 1 nM ) versus Ago2 - miR122 for wild type ( WT ) , helix - 7 double point mutant ( MI - AA ) , Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± standard deviation ( SD ) are plotted . Top panel shows target paired to seed region of guide ( miRNA - 122 ) .

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 ( 0 . 1 nM ) from Ago2 - miR122 ( 1 nM ) was monitored in presence of unlabeled target ( 100 nM ) . Fraction of target bound to Ago2 - miR122 is plotted as a function of time for WT 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 time traces ( with a resolution of 100 ms for wt , 300 ms for MI - AA Ηhelix - 7 ) show docking dissociation of wild type ( top ) , MI - AA ( middle ) , Ηhelix - 7 ( bottom ) Ago2 - miRNA at single spots in 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 B-MEASURED_VAR 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 6 , with wild type Ago2 ; histograms were fit with single exponential decay . first column of data was not included in to avoid potential artifacts arising from limit of time resolution . bin is 0 . 5 sec for N = 4 1 sec for N = 5 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 dependence of dwell time on N for wild type Ago2 . dashed red indicates observation time limit ( 300 s ) , which is constrained by photobleaching . For bar plots ( E , G I ) error bars are 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 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 MI - AA helix - 7 mutant . dwell time histogram for N = 6 fit with double exponential decay ( green ; R2 = 0 . 98 ) ; 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

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 bar plots ( E , G I ) error bars are 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

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 ∆ helix - 7 mutant . dwell time histograms for N = 5 N = 6 were fit with double exponential decays ( green ; R2 = 0 . 97 for N = 5 R2 = 0 . 96 for N = 6 ) ; dwell time for N = 5 N = 6 with single exponential fit was 9 . 1 ± 0 . 91 sec ( R2 = 0 . 86 ) 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

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 bar plots ( E , G I ) error bars are 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

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 g7 . A - C ) Plots of target ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 ( wild type Ηhelix - 7 ) for targets with an increasing number of G : U wobble pairs in seed 3 ' end . D ) Plot of target ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O 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 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 ( 0 . 1 nM ) in presence of increasing concentrations of unlabeled a competitor ( top panel ) . Fraction target bound to Ago2 is plotted as a function of off - target competitor . B ) Fraction of target bound to Ago2 in presence of increasing concentrations of unlabeled total . For A - B , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ; bound free RNAs were then separated using a filter - binding apparatus at various times . Representative time course for target ( 0 . 1 nM ) binding to wild type , MI - AA , Δhelix - 7 Ago2 ( 1 nM ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 . value is 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 ( ) MI - AA ( gray ) Ago2 . Helix - 7 colored yellow ; ordered region of 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 seed region in 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 seed region in wild type ( left ) 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 . ( A - C ) Human colon carcinoma HCT 116 were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . same experiment was performed in presence of bafilomycin A1 ( BafA1 ) , which inhibits fusion between lysosomes autophagosomes , to evaluate autophagic flux . ( A ) Representative . ( B ) Quantitative data .

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

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 . ( A - C ) Human colon carcinoma HCT 116 were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . same experiment was performed in presence of bafilomycin A1 ( BafA1 ) , which inhibits fusion between lysosomes autophagosomes , to evaluate autophagic flux . ( A ) Representative . ( B ) Quantitative data . ( C ) Representative immunoblots of HCT 116 transfected either with an unrelated siRNA or with a SIRT1 - specific siRNA showing LC3 lipidation after treatment with 100 - µM spermidine in presence or absence of bafilomycin A1 .

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

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


lifespan - extending 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 . EGFP - Atg8p localization ( bottom ) was visualized by microscopy . Yeast undergoing autophagy ( in which EGFP - Atg8p exhibits a prominent vacuolar localization ) are indicated by arrows . Yeast 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 vacuolar degradation of EGFP - Atg8p fusion , thereby representing autophagic flux . Notice that both WT Δsir2 yeast show similar free EGFP 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 .

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


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


life - extending autophagy - inducing effects of spermidine in C . elegans are not mediated by Sir2 . ( A ) microscopy of C . elegans transgenic expressing a full - plgg - 1DsRed : : LGG - 1 fusion indicative of autophagic activity . Two representative pictures of wild - type ( WT ) sir - 2 . 1 untreated ( Co , control ) or treated with 0 . 2 - mM spermidine ( Spd ) supplementation of food are shown . ( B ) of autophagic activity through measurement of DsRed : : LGG - 1 pixel from of WT shown in A . Data represent means ± ( n = 3 ) with ≥ 25 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 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 without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 01 ) . P - values were calculated using log - rank as described in Materials 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 phosphoproteome status after resveratrol / or spermidine treatment . ( A - C ) Human colon carcinoma HCT 116 were treated for 2 h with vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , 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 for effects on 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 , acetyl - CoA carboxylase a [ ACACA ] ) or affected by resveratrol or / spermidine treatment ( PTKB , AKT1 , MAPK8 , 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 were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , then treated with either vehicle or indicated dose of resveratrol or spermidine , alone or in combination , for 2 h . Quantitative data . Bars depict percentages ( means ± SD ; n = 3 ; * , P < 0 . 05 ) of showing accumulation of GFP - LC3 in ( 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 were cultured for 2 wk in three different SILAC media containing different arginine lysine isotopes . were treated with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) for 2 h , fractioned into , , mitochondrial extracts , processed for acetyl lysine peptide enrichment , analyzed by MS . ( A ) Hierarchical clustering of drug - specific organellar distributions of acetylated sites quantified in at least one fraction . Fold changes are calculated relative to untreated . 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 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 MotifX algorithm ( Schwartz Gygi , 2005 ) . Among sites that were hyperacetylated in response to both agents , K ( F / Y ) motif is significantly enriched when tested against whole proteome ( P < 0 . 00001 ) . ( C ) When testing against largest acetylation site dataset from Choudhary et al . ( 2009 ) ( acetylation background dataset [ ABD ] ) , 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 .

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

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 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 , KP motif is significantly enriched when tested against ABD ( P < 0 . 001 ) . ( C D ) When tested against ABD ( C ) or whole proteome ( D ) , 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 ( de ) acetylation reactions . ( A B ) HCT 116 were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , enucleated to obtain cytoplasts . 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 of cytoplasts indicative of autophagic activity . Arrows indicate GFP - LC3 - transfected cytoplasts , whereas insets show nonenucleated transfected . ( B ) Quantitative data . Bars show percentages of or cytoplasts showing accumulation of GFP - LC3 in ( 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 were cotransfected with a plasmid for 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 localization signal fused to GFP ( WT GFP - SIRT1 mtNLS GFP - SIRT1 , respectively ) for 12 or 24 h , fixed , analyzed by microscopy . ( C ) Representative 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.


( D ) Quantitative data . Bars depict percentages of exhibiting accumulation of RFP - LC3 in ( RFP - LC3vac ) . ( B D ) Means ± ; n = 3 ; * , P < 0 . 05 .

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(D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05.


( E ) Representative immunoblots for 24 - h endogenous LC3 lipidation . asterisk stands for a nonspecific band . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.