A . Representative confocal images from EGFP - GSK3α FRAP experiments . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but for spines expressing EGFP - GSK3β . Scale bars = 1 µm .

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A. Representative confocal images from EGFP-GSK3α FRAP experiments. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but for spines expressing EGFP-GSK3β. Scale bars = 1 µm.


E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test . N = 12 , 11 and 11 spines for EGFP - GSK3α baseline , cLTD and washout groups and 20 , 11 and 9 spines for EGFP - GSK3β baseline , cLTD and washout groups , respectively .

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E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test. N = 12, 11 and 11 spines for EGFP-GSK3α baseline, cLTD and washout groups and 20, 11 and 9 spines for EGFP-GSK3β baseline, cLTD and washout groups, respectively.


F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α or EGFP - GSK3β in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α or EGFP-GSK3β in 'baseline', 'cLTD' or 'recovery' conditions. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test.


G . Representative western blots from whole extracts of organotypic slices incubated in ACSF ( untreated ; ' baseline ' ) , treated with 20 µM NMDA for 5 minutes ( ' cLTD ' ) or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ( ' washout ' ) . Blots were probed for pSer21 / 9 GSK3α / β , total GSK3α / β , pSer845 GluA1 , and total GluA1 . H . Quantification of experiments as those shown in G . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 4 independent experiments for all conditions . Asterisks ( * * ) denote P < 0 . 01 .

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G. Representative western blots from whole extracts of organotypic slices incubated in ACSF (untreated; 'baseline'), treated with 20 µM NMDA for 5 minutes ('cLTD') or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ('washout'). Blots were probed for pSer21/9 GSK3α/β, total GSK3α/β, pSer845 GluA1, and total GluA1. H. Quantification of experiments as those shown in G. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 4 independent experiments for all conditions. Asterisks (**) denote P < 0.01.


A . Representative confocal images from EGFP - GSK3α FRAP experiments in organotypic slices cultured from C57 wild - type mice . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in wild - type mouse slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but in slices cultured from C57 tauKO mice . Scale bars = 1 µm .

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A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout ') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm.


E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 13 , 11 and 12 spines for wt baseline , cLTD and recovery and 9 , 7 and 5 spines for tauKO baseline , cLTD and recovery groups , respectively .

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E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 13, 11 and 12 spines for wt baseline, cLTD and recovery and 9, 7 and 5 spines for tauKO baseline, cLTD and recovery groups, respectively.


F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α in wild - type or tauKO slices in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α in wild-type or tauKO slices in 'baseline', 'cLTD' or 'recovery' conditions. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - expressing neurons ( green traces ) in C57 wt or tauKO slices .

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B. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α-expressing neurons (green traces) in C57 wt or tauKO slices.


C - F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in C57 wt ( C ) or tauKO slices ( D ) , or at + 40 mV ( NMDAR - mediated EPSCs ) in wt ( E ) or tauKO slices ( F ) . Red circles represent the averages of all pairs in a given plot , and n denotes the number of cell pairs . In all cases , error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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C-F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in C57 wt (C) or tauKO slices (D), or at +40 mV (NMDAR-mediated EPSCs) in wt (E) or tauKO slices (F). Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs. In all cases, error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . CBA analysis of IL - 1β ( A ) in the culture supernatants .

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LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) in the culture supernatants.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . ELISA of IL - 18 ( B ) in the culture supernatants .

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LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. ELISA of IL-18 (B) in the culture supernatants.


CBA analysis of IL - 1β secretion from Abro1 + / + and Abro1 − / − BMDMs transduced with GFP - or ABRO1 - GFP - expressing lentiviruses prior to stimulation with LPS and ATP . Cell lysates were immunoblotted with anti - ABRO1 antibody .

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CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody.


ELISA analysis of IL - 1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium ( Con ) , LPS or LPS plus ATP . Cell lysates were immunoblotted with anti - ABRO1 and anti - GAPDH antibodies .

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ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies.


Abro1 + / + and Abro1 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with nigericin , silica , MSU , MDP , Alum , poly ( dA : dT ) , flagellin or LTx ( Anthrax Lethal Factor ) . CBA analysis of IL - 1β secretion in the culture supernatants .

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Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with nigericin, silica, MSU, MDP, Alum, poly (dA: dT), flagellin or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants.


Representative immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Abro1 + / + and Abro1 − / − BMDMs treated with ATP , nigericin , MSU , flagellin or poly ( dA : dT ) .

Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin or poly (dA: dT).


Flow cytometric analysis of caspase - 1 activity in LPS - primed Abro1 + / + and Abro1 − / − BMDMs stimulated with nigericin for indicated times .

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Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times.


Immunostaining of endogenous ASC specks in LPS - primed Abro1 + / + and Abro1 − / − BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . Scale bars , 10 μm . Quantification of ASC specks from ( H ) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei . At least 100 cells from each treatment condition were quantified . UD , undetectable .

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Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly (dA:dT). Scale bars, 10 μm. Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti - ASC antibody .

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LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody.


WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( A ) . n = 6 for PBS groups and n = 12 for MSU groups .

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WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). n = 6 for PBS groups and n = 12 for MSU groups.


WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . Flow cytometric analysis of peritoneal cell exudates ( B ) . n = 6 for PBS groups and n = 12 for MSU groups .

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WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) . Flow cytometric analysis of caspase - 1 activity in peritoneal exudate monocytes and neutrophils . n = 12 per group .

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Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β in the peritoneal lavage fluid ( D ) n = 6 for PBS groups and n = 12 for Alum groups .

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Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) n = 6 for PBS groups and n = 12 for Alum groups.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( E ) . n = 6 for PBS groups and n = 12 for Alum groups .

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Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups.


Survival of Abro1 + / + and Abro1 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

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Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Abro1 + / + and Abro1 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 , ASC or pro - casapase - 1 ( Pro - Casp - 1 ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 or NLRP3 deletion mutants as indicated in ( F ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 and Flag - tagged ABRO1 or ABRO1 deletion mutants as indicated in ( H ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - tagged NLRP3 ( WT ) , NLRP3 ( S194D ) , or NLRP3 ( S194A ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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 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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-tagged NLRP3 (WT), NLRP3 (S194D), or NLRP3 (S194A). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT BMDMs were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194A ) were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

O 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 O O O O O O B-CONTROLLED_VAR 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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194D ) were treated with or without 100 nM SP600125 for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT and Nlrp3S194A / S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 , Flag - ABRO1 and HA - ubiquitin ( HA - Ub ) , HA - Ub K48 only or K63 only . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding HA - Ub , Flag - ABRO1 , and NLRP3 truncations as indicated . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of ubiquitination of NLRP3 truncations ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP ( left ) or nigericin ( right ) . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were left unstimulated or stimulated with LPS or LPS plus ATP . Immunoblot analysis of NLRP3 and NLRP3 ( A350V ) ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 ( A350V ) and HA - ubiquitin ( HA - Ub ) . Immunoblot analysis of NLRP3 ( A350V ) ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

O O 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 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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were stimulated with or without 100ng / ml LPS for 1 h . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 , and HA - ubiquitin ( HA - Ub ) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

O 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 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 B-CONTROLLED_VAR 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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


LPS - primed WT and Nlrp3S194A / S194A BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


WT BMDMs were stimulated with LPS for 1 h . Immunoblot analysis of NLRP3 , ABRO1 , and BRCC3 from the cell lysates immunoprecipitated with control IgG , anti - ABRO1 , anti - BRCC3 or anti - NLRP3 antibody .

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

WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Brcc3 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of ABRO1 . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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

Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


Abro1 + / + and Abro1 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Abro1 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of BRCC3 . Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti - BRCC3 antibody .

Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.


A constant amount of Myc - NLRP3 plasmid together with an increasing amount of Myc - ABRO1 plasmid were co - transfected in HEK - 293T cells . Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti - BRCC3 antibody .

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

A constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Brcc3 + / + and Brcc3 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with ATP , nigericin , MSU , Alum , poly ( dA : dT ) or flagellin . CBA analysis of IL - 1β secretion in the culture supernatants .

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

Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.


Immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs treated with ATP , nigericin , MSU , flagellin , and poly ( dA : dT ) .

Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).


Immunostaining of endogenous ASC specks in LPS - primed Brcc3 + / + and Brcc3 - / - BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . ASC speck positive cells were counted and analyzed as described in Fig . 1I . UD , undetectable .

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

Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.


LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs were left unstimulated or stimulated with nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Survival of Brcc3 + / + and Brcc3 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

O O B-CONTROLLED_VAR 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

Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Brcc3 + / + and Brcc3 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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

ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( G )

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

Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G)


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( H ) . n = 6 for PBS groups and n = 12 for MSU groups .

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

Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.


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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 and

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

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 - 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 .

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

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 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

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

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 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 )

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

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 , contralateral cortex vs ipsilateral cortex to deprived eye , N = 5 ; paired t test , p = 0 . 52 ) . Error bars represent ± SEM , symbols represent individual cases .

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

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 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 .

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 ) .

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

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 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 .

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

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 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 .

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

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 .

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

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 .

O O O O O O O O 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 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

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 .

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

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 .

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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 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 .

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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 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 .

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

(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 .

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

(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 .

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

(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.