of endogenous LC3 following TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , alone or with BafA1 , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV NPC1 patientfibroblasts . Graphs show densitometry of LC3II bands normalized to actin . Statistics ( G , Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - Student ' s t - . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

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

analysis of endogenous LC3 following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts. Graphs show densitometry of LC3II bands normalized to actin. Statistics (G, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.


( H ) Immunoblot of endogenous LC3 ( LC3I - II ) following TPC2 - A1 - P ( 30 µM ) or DMSO treatment , under fed ( complete Neurobasal / B27 ) or starvation ( DMEM / F12 free ) conditions in CTR MLIV iPSC derived cortical neurons . Graphs show densitometry of LC3II bands normalized to actin . Statistics H , Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - Student ' s t - . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

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

(H) Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P (30 µM) or DMSO treatment, under fed (complete Neurobasal/B27) or starvation (DMEM/F12 free) conditions in CTR and MLIV iPSC derived cortical neurons. Graphs show densitometry of LC3II bands normalized to actin. Statistics H, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.


Immunoblot of endogenous SQSTM1 ( P62 ) upon TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV NPC1 fibroblasts . Statistics J ) : Shown are mean values ± SD . n = 3 lysates per condition pooled from 3 independent experiments ; two - Student ' s t - . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 .

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

Immunoblot analysis of endogenous SQSTM1 (P62) upon TPC2-A1-P or ML-SA1 (30 µM, each) treatment, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patient fibroblasts. Statistics J): Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.


( D ) A cDNA array was used to map TPC2 transcripts in human brain .

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(D) A cDNA array was used to map TPC2 transcripts in the human brain.


( A ) TPC2 - A1 - P was injected intravenously , mice sacrificed at indicated time . TPC2 - A1 - P was measured in plasma brain by LC - MS / MS . TPC2 - A1 - P was rapidly eliminated , being undetectable by 240 min .

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(A) TPC2-A1-P was injected intravenously, and mice sacrificed at the indicated time. TPC2-A1-P was measured in plasma and brain by LC-MS/MS. TPC2-A1-P was rapidly eliminated, being undetectable by 240 min.


( B ) Elimination rate constants were determined using a semi - log plot . A two - phase decay model could fit obtained data points in plasma , while a one - phase decay model fit data in brain .

O O O O O O O O O O O O O O O O O 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) Elimination rate constants were determined using a semi-log plot. A two-phase decay model could fit the obtained data points in plasma, while a one-phase decay model fit the data in brain.


( C ) Brain [ TPC2 - A1 - P ] was simulated for various injected doses . 20 mg / kg TPC2 - A1 - P was chosen to avoid off - target activity while providing a therapeutic dose for > 20 min .

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(C) Brain [TPC2-A1-P] was simulated for various injected doses. 20 mg/kg TPC2-A1-P was chosen to avoid off-target activity while providing a therapeutic dose for >20 min.


( A B ) Tau increased β - catenin acetylation ( Ace - β - cat ) measured by immunoprecipitation using anti - β - cat , or Western blotting using anti - β - cat , anti - acetylated lysine ( Ace - lys ) HT7 ( probes specifically human total Tau ) . HEK293 transiently transfected with wildtype Tau40 ( Tau ) or empty vector ( Vec ) ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec Data were analyzed by Student ' s t

O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Tau increased β-catenin acetylation (Ace-β-cat) measured by immunoprecipitation using anti-β-cat, or Western blotting using anti-β-cat, anti-acetylated lysine (Ace-lys) and HT7 (probes specifically human total Tau). HEK293 cells transiently transfected with wildtype Tau40 (Tau) or the empty vector (Vec) (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec Data were analyzed by Student's t test


( C D ) β - cat mutation at K49 ( K49R ) but not K19 ( K19R ) abolished Tau - induced acetylation . HEK293 were co - transfected with eGFP - β - cat ( WT , or K19R , or K49R , or K19R / K49R ) Tau or empty vector for 48 h , then immunoprecipitated using anti - GFP Western blotting using anti - β - cat , anti - Ace - lys HT7 ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT ( D ) ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat WT plus Tau ( D ) ; & & , P < 0 . 01 , & & & , P < 0 . 001 vs β - cat K19R plus Tau ( D ) ; absence of asterisk indicates that difference is not significant . Data were analyzed by one - way

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

(C and D) β-cat mutation at K49 (K49R) but not K19 (K19R) abolished Tau-induced acetylation. HEK293 cells were co-transfected with eGFP-β-cat (WT, or K19R, or K49R, or K19R/K49R) and Tau or the empty vector for 48 h, and then immunoprecipitated using anti-GFP and Western blotting using anti-β-cat, anti-Ace-lys and HT7 (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT (D) ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat WT plus Tau (D) ; &&, P<0.01, &&&, P<0.001 vs β-cat K19R plus Tau (D); the absence of asterisk indicates that the difference is not significant. Data were analyzed by one-way ANOVA


( E F ) increased β - cat ( K49 ) acetylation was detected in hippocampal extracts of human Tau transgenic mice ( Tau + ) compared with endogenous Tau knockout mice ( Tau - ) , measured by Western blotting using acetylation site - specific antibody ( Ace - β - cat - K49 ) ; arrowhead in panel E denoted nonspecific band identified by molecular . remarkably increased of phosphorylated Tau at Ser199 ( pS199 ) AT8 epitopes were detected in Tau + mice ( at least 3 mice per group were used ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs vs Tau - ( F ) Data were analyzed by Student ' s t

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

(E and F) The increased β-cat (K49) acetylation was detected in the hippocampal extracts of human Tau transgenic mice (Tau+) compared with the endogenous Tau knockout mice (Tau-), measured by Western blotting using acetylation site-specific antibody (Ace-β-cat-K49); arrowhead in panel E denoted nonspecific band identified by molecular weight. The remarkably increased levels of phosphorylated Tau at Ser199 (pS199) and AT8 epitopes were detected in Tau+ mice (at least 3 mice per group were used). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs vs Tau- (F) Data were analyzed by Student's t test


( G H ) K18 deletion of Tau ( Tau - k18 ( - ) ) attenuated Tau - induced β - cat acetylation . HEK293 were transfected with empty Vec , or Tau40 or Tau - k18 ( - ) for 48 h , ace - β - cat was measured by immunoprecipitation using anti - β - cat Western blotting using anti - ace - lys , anti - β - cat , Tau5 ( detecting total Tau ; n = 3 from three independent experiments ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau ( H ) Data were analyzed by one - way

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

(G and H) K18 deletion of Tau (Tau-k18(-)) attenuated Tau-induced β-cat acetylation. HEK293 cells were transfected with empty Vec, or Tau40 or Tau-k18(-) for 48 h, and the ace-β-cat was measured by immunoprecipitation using anti-β-cat and Western blotting using anti-ace-lys, anti-β-cat, and Tau5 (detecting total Tau; n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (H) Data were analyzed by one-way ANOVA


increased total Ace - β - cat ( K49 ) positively correlated with increased phosphorylated Tau pS199 AT8 as detected by Western blotting in hippocampal extracts of AD compared with - matched controls ( n = 10 each group , Pearson was used for correlation Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec vs Ctrl ( J ) Data were analyzed by Student ' s t

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

The increased total and Ace-β-cat (K49) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age-matched controls (n=10 each group, Pearson analysis was used for correlation analysis Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec vs Ctrl (J) Data were analyzed by Student's t test


increased total Ace - β - cat ( K49 ) positively correlated with increased phosphorylated Tau pS199 AT8 as detected by Western blotting in hippocampal extracts of AD compared with - matched controls ( n = 10 each group Pearson was used for correlation

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

The increased total and Ace-β-cat (K49) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age-matched controls (n=10 each group Pearson analysis was used for correlation analysis


( M N ) Expressing pseudo - phosphorylated Tau ( TauS199E ) further increased K49 - acetylation of β - catenin . N2a were transiently transfected with Vec , Tau40 or TauS199E for 48 h , of K49 - acetylated β - cat , total β - cat total Tau were detected by Western blotting ( n = 3 biological replicates each group ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat WT plus vs Tau ( N ) Data were analyzed by one - way

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

(M and N) Expressing pseudo-phosphorylated Tau (TauS199E) further increased the K49-acetylation of β-catenin. N2a cells were transiently transfected with Vec, Tau40 or TauS199E for 48 h, levels of K49-acetylated β-cat, total β-cat and total Tau were detected by Western blotting (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat WT plus vs Tau (N) Data were analyzed by one-way ANOVA


( O P ) Inhibiting acetyltransferases CBP / P300 by TPOP146 ( 134 nM ) or PCAF by L - 45 ( 126 nM ) for 24 h did not significantly decrease K49 - acetylated β - cat level in Tau - overexpressing N2a ( n = 3 biological replicates each group ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec Data were analyzed by one - way

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 and P) Inhibiting acetyltransferases CBP/P300 by TPOP146 (134 nM) or PCAF by L-45 (126 nM) for 24 h did not significantly decrease the K49-acetylated β-cat level in Tau-overexpressing N2a cells (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec Data were analyzed by one-way ANOVA


( A ) Tau directly acetylates β - cat in - tube . Both Tau40 β - cat were purified from E . coli using Ni - NTA resin ( n = 3 from three independent experiments ) .

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(A) Tau directly acetylates β-cat in test-tube. Both Tau40 and β-cat were purified from E. coli using Ni-NTA resin (n = 3 from three independent experiments).


( B C ) Mutation of Tau40 at acetyltransferase activity region abolishes its acetylation activity on β - cat . eGFP - Tau40 , eGFP - Tau - 2CA eGFP - Tau - k18 ( - ) were purified from HEK293 ( using anti - GFP beads ) GST - β - cat was from E . coli ( B ) , or both Tau40 β - cat were from E . coli ( C ) ( n = 3 from three independent experiments ) .

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

(B and C) Mutation of Tau40 at acetyltransferase activity region abolishes its acetylation activity on β-cat. eGFP-Tau40, eGFP-Tau-2CA and eGFP-Tau-k18(-) were purified from HEK293 cells (using anti-GFP beads) and GST-β-cat was from E. coli (B), or both Tau40 and β-cat were from E. coli (C) (n = 3 from three independent experiments).


( D E ) Mutation of β - cat K49 abolishes its acetylation by Tau . Both myc - Tau40 eGFP - β - cat were purified from HEK293 ( using anti - GFP or anti - Myc beads ) ( D ) , or both from E . coli ( E ) ( n = 3 from three independent experiments ) .

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

(D and E) Mutation of β-cat K49 abolishes its acetylation by Tau. Both myc-Tau40 and eGFP-β-cat were purified from HEK293 cells (using anti-GFP or anti-Myc beads) (D), or both from E. coli (E) (n = 3 from three independent experiments).


Kinetics of β - cat acetylation by Tau . Tau acetylates β - cat in - ( F ) its acetyltransferase activity domain - dependent manner . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) .

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Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in concentration- (F) and its acetyltransferase activity domain-dependent manner. Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).


Kinetics of β - cat acetylation by Tau . Tau acetylates β - cat in time - ( G ) its acetyltransferase activity domain - dependent manner . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) .

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

Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in time- (G) and its acetyltransferase activity domain-dependent manner. Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).


Kinetics of β - cat acetylation by Tau . Tau acetylates β - cat in pH - ( H ) , its acetyltransferase activity domain - dependent manner . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) .

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

Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in pH- (H), and its acetyltransferase activity domain-dependent manner. Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).


Kinetics of β - cat acetylation by Tau . affinity ( Km ) of Tau to β - cat was measured by initial rate of Tau as a function of β - cat concentrations ( I , J ) . Michaelis - Menten parameters were determined using Graphpad Prism 6 ( n = 3 from three independent experiments ) .

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

Kinetics of β-cat acetylation by Tau. The affinity (Km) of Tau to β-cat was measured by the initial rate of Tau as a function of the β-cat concentrations (I, J). Michaelis-Menten parameters were determined using Graphpad Prism 6 (n = 3 from three independent experiments).


( A B ) Expressing Tau inhibited β - cat ubiquitination in HEK293 , measured by immunoprecipitation using antibody against β - cat Western blotting using anti - ubiquitination ( anti - Ub ) anti - β - cat , respectively ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Vec ( B ) Data were analyzed by Student ' s t .

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

(A and B) Expressing Tau inhibited β-cat ubiquitination in HEK293 cells, measured by immunoprecipitation using antibody against β-cat and Western blotting using anti- ubiquitination (anti-Ub) and anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec (B) Data were analyzed by Student's t test.


( C D ) Overexpressing Tau inhibited β - cat ubiquitination in hippocampi of Tau transgenic mice , measured by immunoprecipitation using anti - Ub Western blotting using anti - β - cat , respectively ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau - ( D ) Data were analyzed by Student ' s t .

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

(C and D) Overexpressing Tau inhibited β-cat ubiquitination in hippocampi of Tau transgenic mice, measured by immunoprecipitation using anti-Ub and Western blotting using anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau- (D) Data were analyzed by Student's t test.


( E F ) Expressing Tau - k18 ( - ) attenuated Tau - induced β - cat elevation . HEK293 were transfected with Vec , or Tau - 40 , or Tau - k18 ( - ) for 24 h , then were treated with Chx for 12 h or 24 h , followed by Western blotting . β - cat level was normalized to β - actin ( n = 4 biological replicates each group ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau ( F ) Data were analyzed by Two - way repeated - measures

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

(E and F) Expressing Tau-k18(-) attenuated Tau-induced β-cat elevation. HEK293 cells were transfected with Vec, or Tau-40, or Tau-k18(-) for 24 h, and then were treated with Chx for 12 h or 24 h, followed by Western blotting. β-cat protein level was normalized to β-actin (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (F) Data were analyzed by Two-way repeated-measures ANOVA


( G H ) K49 acetylation of β - cat inhibited its ubiquitination . HEK293 were co - transfected with HA - ubiquitin GFP - β - cat or pseudo - acetylated K49 - β - catenin ( K49Q ) , then β - cat was immunoprecipitated by anti - GFP blotted by anti - Ub anti - β - cat ( n = 3 from three independent experiments ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT Data were analyzed by Student ' s t .

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

(G and H) K49 acetylation of β-cat inhibited its ubiquitination. HEK293 cells were co-transfected with HA-ubiquitin and GFP-β-cat or pseudo-acetylated K49-β-catenin (K49Q), and then β-cat was immunoprecipitated by anti-GFP and blotted by anti-Ub and anti-β-cat (n=3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Student's t test.


( I J ) K49 acetylation of β - cat inhibited its degradation . HEK293 were transfected with β - cat WT or K49Q for 24 h , then treated with cycloheximide ( Chx , 100 μg / ml ) for 12 h or 24 h ( n = 4 biological replicates each group ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT Data were analyzed by Two - way repeated - measures

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

(I and J) K49 acetylation of β-cat inhibited its degradation. HEK293 cells were transfected with β-cat WT or K49Q for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Two-way repeated-measures ANOVA


β - cat mutation at K49 ( K49R ) restored degradation of β - catenin . HEK293 were transfected with β - cat WT or co - transfected with β - cat WT Tau40 , or co - transfected with β - cat K49R Tau40 for 24 h , then treated with cycloheximide ( Chx , 100 μg / ml ) for 12 h or 24 h ( n = 4 biological replicates each group ) .

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

β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group).


β - cat mutation at K49 ( K49R ) restored degradation of β - catenin . HEK293 were transfected with β - cat WT or co - transfected with β - cat WT Tau40 , or co - transfected with β - cat K49R Tau40 for 24 h , then treated with cycloheximide ( Chx , 100 μg / ml ) for 12 h or 24 h ( n = 4 biological replicates each group ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs Tau + β - cat WT ( L ) Data were analyzed by Two - way repeated - measures

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

β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau+β-cat WT (L) Data were analyzed by Two-way repeated-measures ANOVA


( M N ) K49 acetylation of β - cat inhibited its phosphorylation at N - terminal domain . HEK293 were transfected with GFP - vec , or eGFP - β - cat ( WT ) or K49Q for 48 h , then treated with wortmannin ( WO , 1 μM ) plus GF109203X ( GFX , 1 μM ) to activate GSK - 3β for 1 h followed by Western blotting ( n = 3 biological replicates each group ) . Data information : Data are presented as mean ± ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 vs β - cat WT plus WO / GFX ( N ) . data in ( N ) was analyzed by one - way ANOVA .

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

(M and N) K49 acetylation of β-cat inhibited its phosphorylation at N-terminal domain. HEK293 cells were transfected with GFP-vec, or eGFP-β-cat (WT) or K49Q for 48 h, then treated with wortmannin (WO, 1 μM) plus GF109203X (GFX, 1 μM) to activate GSK-3β for 1 h followed by Western blotting (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT plus WO/GFX (N). data in (N) was analyzed by one-way ANOVA.


( A B ) Expressing Tau - k18 ( - ) abolished its effect on β - cat K49 - acetylation c - cas - 3 ( caspase - 3 ) . N2a were transfected with Vec , or Tau40 , or Tau - k18 ( - ) for 48 h , then treated with 0 . 5 μM staurosporine ( STP , an apoptotic inducer ) for 4 h , followed by Western blotting ( n = 3 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs vec ( B ) # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs Vec plus STP ( B ) ; $ $ $ , P < 0 . 001 vs Tau plus STP ( B ) Data were analyzed by one - way ANOVA .

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

(A and B) Expressing Tau-k18(-) abolished its effect on β-cat K49-acetylation and c-cas-3 (cleaved caspase-3). N2a cells were transfected with Vec, or Tau40, or Tau-k18(-) for 48 h, and then treated with 0.5 μM staurosporine (STP, an apoptotic inducer) for 4 h, followed by Western blotting (n=3 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vec (B) #, P<0.05, ##, P<0.01, ###, P<0.001 vs Vec plus STP (B) ; $$$, P<0.001 vs Tau plus STP (B) Data were analyzed by one-way ANOVA.


Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . ; N2a , transfected with myc - Vec , or myc - Tau40 , or myc - Tau - k18 ( - ) , or myc - Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by flow cytometry using Annexin - FITC ( C ) Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau plus STP Data were analyzed by one - way ANOVA .

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

Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. ; N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by flow cytometry using Annexin-FITC (C) Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP Data were analyzed by one-way ANOVA.


Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a , transfected with eGFP - Vec , or eGFP - Tau40 , or eGFP - Tau - k18 ( - ) , or Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by co - with c - cas - 3 ( red ) ( D In panel D ( middle row ) , Tau expression ( arrowheads ) was largely not co - localized with c - casp - 3 ( yellow arrowheads ) in same visual field ( n = 4 ~ 8 biological replicates each group ; scale bar , 10 μm ) .

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

Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) (D In panel D (middle row), the Tau expression (white arrowheads) was largely not co-localized with c-casp-3 (yellow arrowheads) in the same visual field (n=4~8 biological replicates each group; scale bar, 10 μm).


Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a , transfected with eGFP - Vec , or eGFP - Tau40 , or eGFP - Tau - k18 ( - ) , or Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by co - with c - cas - 3 ( red ) apoptotic rate ( E ) was presented as of yellow ( c - casp - 3 GFP co - stained ) to green ( total GFP - expressing ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau plus STP Data were analyzed by one - way ANOVA .

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

Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) The apoptotic rate (E) was presented as the ratio of yellow (c-casp-3 and GFP co-stained) to green (the total GFP-expressing cells) staining. Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP Data were analyzed by one-way ANOVA.


Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a , transfected with myc - Vec , or myc - Tau40 , or myc - Tau - k18 ( - ) , or myc - Tau - 2CA for 48 h , were then treated with 0 . 5 μM STP for 4 h followed by CCK8 ( F ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau plus STP ♧ ♧ ♧ , P < 0 . 001 vs vec Data were analyzed by one - way ANOVA .

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

Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by CCK8 assay (F). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.


( G ) Non - acetylation modifiable mutation of β - cat at K49 ( K49R ) , not K19 ( K19R ) , abolished anti - apoptotic effect of Tau . N2a were co - transfected with Tau40 β - cat WT , or K49R , or K19R for 48 h , then treated with 0 . 5 μM STP for 4 h . viability was measured by CCK8 ( n = 8 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec plus STP ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs vs β - cat + Vec plus STP ( G ) ; $ $ $ , P < 0 . 001 vs β - cat + Tau plus STP ( G ) ; & & & , P < 0 . 001 vs β - cat K49R + Tau plus STP ( G ) . ♧ ♧ ♧ , P < 0 . 001 vs vec Data were analyzed by one - way ANOVA .

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

(G) Non-acetylation modifiable mutation of β-cat at K49 (K49R), not K19 (K19R), abolished the anti-apoptotic effect of Tau. N2a cells were co-transfected with Tau40 and β-cat WT, or K49R, or K19R for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs vs β-cat + Vec plus STP (G) ; $$$, P<0.001 vs β-cat + Tau plus STP (G) ; &&&, P<0.001 vs β-cat K49R+ Tau plus STP (G). ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.


( H ) Expressing β - cat K49Q most significantly increased viability . N2a were transfected with β - cat WT or K49R or K49Q or empty Vec for 48 h , then treated with 0 . 5 μM STP for 4 h . viability was measured by CCK8 ( n = 8 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec plus STP ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat plus STP ( H ) , ; $ $ $ , P < 0 . 001 vs β - cat K49Q plus STP ( H ) ♧ ♧ ♧ , P < 0 . 001 vs vec Data were analyzed by one - way ANOVA .

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

(H) Expressing β-cat K49Q most significantly increased cell viability. N2a cells were transfected with β-cat WT or K49R or K49Q or the empty Vec for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat plus STP (H), ; $$$, P<0.001 vs β-cat K49Q plus STP (H) ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.


( I ) Overexpressing Ser199 - phosphorylation mimic Tau ( TauS199E ) further augmented viability . N2a were transfected with empty vec , Tau40 or TauS199E for 48h then treated with 0 . 5 μM STP for 4 h . viability was measured by CCK8 ( n = 8 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec plus STP ; # , P < 0 . 05 , # # , P < 0 . 01 , # # # , P < 0 . 001 vs vs Tau plus STP ( I ) Data were analyzed by one - way ANOVA .

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

(I) Overexpressing Ser199-phosphorylation mimic Tau (TauS199E) further augmented cell viability. N2a cells were transfected with empty vec, Tau40 or TauS199E for 48h and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs vs Tau plus STP (I) Data were analyzed by one-way ANOVA.


( A ) Co - expressing myc - Tau eGFP - β - catenin increased translocation of β - catenin , while co - expressing myc - Tau eGFP - β - cat K49R abolished translocation of β - catenin in HEK293 . nuclei were stained using DAPI ( n = 3 from three independent experiments , scale bar , 5 μm ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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

(A) Co-expressing myc-Tau and eGFP-β-catenin increased nuclear translocation of β-catenin, while co-expressing myc-Tau and eGFP-β-cat K49R abolished the nuclear translocation of β-catenin in HEK293 cells. The nuclei were stained using DAPI (n=3 from three independent experiments, scale bar, 5 μm).


Expressing β - cat K49Q increased its translocation . HEK293 were transfected with eGFP - β - cat WT or K49Q for 48 h , then β - cat level in different fractions was measured by Western

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

Expressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h, and then β-cat protein level in different fractions was measured by Western blotting


Expressing β - cat K49Q increased its translocation . HEK293 were transfected with eGFP - β - cat WT or K49Q for 48 h , then β - cat level in different fractions was measured by Western blotting Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs β - cat WT ( C ) Data were analyzed by Student ' s t

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

Expressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h, and then β-cat protein level in different fractions was measured by Western blotting Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs β-cat WT (C) Data were analyzed by Student's t test


Expressing β - cat K49Q increased its translocation . HEK293 were transfected with eGFP - β - cat WT or K49Q for 48 h immunofluorescent labeling using a scanning confocal microscope ( D ) . nuclei ( blue ) was stained by DAPI ( n = 3 from three independent experiments , scale bar , 2 μm ) .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

Expressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h and immunofluorescent labeling using a laser scanning confocal microscope (D). The nuclei (blue) was stained by DAPI (n=3 from three independent experiments, scale bar, 2 μm).


( E ) Expressing β - cat K49Q further increased its transcription activity measured by dual Topflash / Fopflash reporter luciferase after transient transfection of β - cat WT , or K49Q , or Vec in HEK293 for 48 h , luciferase activity was normalized by β - cat WT or K49Q level ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec ; # # , P < 0 . 01 , # # # , P < 0 . 001 vs β - cat WT Data were analyzed by one - way

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 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 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 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

(E) Expressing β-cat K49Q further increased its transcription activity measured by dual Topflash/Fopflash reporter luciferase assay after transient transfection of β-cat WT, or K49Q, or Vec in HEK293 cells for 48 h, and the luciferase activity was normalized by β-cat WT or K49Q protein level (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec ; ##, P<0.01, ###, P<0.001 vs β-cat WT Data were analyzed by one-way ANOVA


( F ) Expressing Tau - k18 ( - ) abolished Tau - induced upregulation of mRNA of survival genes ( bcl2 survivin ) dkk1 ( downstream of Wnt signaling ) in N2a measured by Q - PCR ( n = 6 ~ 9 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Tau ( F Data were analyzed by one - way

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

(F) Expressing Tau-k18(-) abolished Tau-induced upregulation of mRNA levels of survival genes (bcl2 and survivin) and dkk1 (downstream of Wnt signaling) in N2a cells measured by Q-PCR (n=6~9 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau (F Data were analyzed by one-way ANOVA


( G ) Expressing Tau - k18 ( - ) or Tau - 2CA abolished Tau - induced Wnt / β - catenin transcriptional activation measured by dual reporter luciferase activity in HEK293 ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs vs Tau Data were analyzed by one - way

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

(G) Expressing Tau-k18(-) or Tau-2CA abolished Tau-induced Wnt/β-catenin transcriptional activation measured by dual reporter luciferase activity assay in HEK293 cells (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vs Tau Data were analyzed by one-way ANOVA


( H ) Expressing phosphor - mimic TauS199E ( TauS199E ) further increased transcription activity of Wnt / TCF / LEF1 by Tau in HEK293 ( n = 7 biological replicates each group ) . Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Vec ; # # , P < 0 . 01 , # # # , P < 0 . 001 vs Tau ( H ) . Data were analyzed by , one - way

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

(H) Expressing phosphor-mimic TauS199E (TauS199E) further increased the transcription activity of Wnt/TCF/LEF1 by Tau in HEK293 cells (n=7 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec ; ##, P<0.01, ###, P<0.001 vs Tau (H). Data were analyzed by , one-way ANOVA


increased mRNA of bcl2 , survivin dkk1 in AD brains compared with - / sex - matched controls detected by Q - PCR ( n = 7 each group ) Data information : Data are expressed as mean ± , * , P < 0 . 05 ; * * , P < 0 . 01 , * * * , P < 0 . 001 vs Ctrl ( I ) Data were analyzed by Student ' s t

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

The increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group) Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Ctrl (I) Data were analyzed by Student's t test


increased mRNA of bcl2 , survivin dkk1 in AD brains compared with - / sex - matched controls detected by Q - PCR ( n = 7 each group ) , positive correlations with increased K49 - acetylated β - catenin ( see Fig . 1I , J ) analyzed by Pearson .

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

The increased mRNA levels of bcl2, survivin and dkk1 in the AD brains compared with the age-/sex-matched controls detected by Q-PCR (n=7 each group), and the positive correlations with the increased K49-acetylated β-catenin (see Fig. 1I, J) analyzed by Pearson analysis.


( H , I ) . Secretory vesicles of chromaffin were examined by transmission electron microscopy after 100 nM AngII stimulation , with or without pre - incubation with 400 nM Compound 7 . Scale bars : ( H , I ) , 150 nm . Red arrows indicate morphologically docked LDCVs green arrows stands for undocked LDCVs . PM represents plasma . ( J ) . Vesicle numbers according to different distances from plasma were calculated in presence or absence of 100 nM AngII or 400 nM Compound 7 . PM represents plasma . Data information : Data were analyzed using one - way ANOVA displayed as mean ± s . e . m . # , P < 0 . 05 ; # # , P < 0 . 01 ; # # # , P < 0 . 001 : pre - incubated with PTP - MEG2 inhibitors compared with control vehicles . N . S . means no significant difference . Data were from 6 independent experiments . # indicates P < 0 . 05 # # # indicates P < 0 . 001 compared with control group .

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

(H, I). Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation, with or without pre-incubation with 400 nM Compound 7. Scale bars: (H, I), 150 nm. Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs. PM represents plasma membrane. (J). Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7. PM represents plasma membrane. Data information: Data were analyzed using one-way ANOVA and displayed as the mean ± s.e.m. #, P<0.05; ##, P<0.01; ###, P<0.001: cells pre-incubated with PTP-MEG2 inhibitors compared with control vehicles. N.S. means no significant difference. Data were from 6 independent experiments. # indicates P<0.05 and ### indicates P<0.001 compared with control group.


( D ) . Phosphorylated NSF interacted with PTP - MEG2 . PC12 were transfected with FLAG - NSF . After stimulation of with 100nM AngII in presence or absence of 100μM H2O2 for 5 minutes ( H2O2 was exploited to increase overall tyrosine phosphorylation of ) , potential PTP - MEG2 substrate in lysates was pulled down with GST - PTP - MEG2 - D470A or GST control .

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

(D). Phosphorylated NSF interacted with PTP-MEG2. PC12 cells were transfected with FLAG-NSF. After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes (H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins), the potential PTP-MEG2 substrate in cell lysates was pulled down with GST-PTP-MEG2-D470A or GST control.


( B ) . Relative values of decreased phosphatase activities of different PTP - MEG2 mutants towards pNPP ( p - Nitrophenyl phosphate ) compared with wild - type PTP - MEG2 . Data information : * * * P < 0 . 001 : PTP - MEG2 mutants compared with control group . Data were obtained from 3 independent experiments . N . S . means no significant difference . data were analyzed using one - way ANOVA , displayed as mean ± s . e . m . In red column indicates that mutation sites have greater effects on enzyme activities toward phospho - NSF segement than pNPP . green column indicates that mutation sites only have significant effects on enzyme activity toward NSF phospho - segement but not pNPP . column stands for mutants that have similar effects on pNPP NSF phospho - segement .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(B). Relative values of the decreased phosphatase activities of different PTP-MEG2 mutants towards pNPP (p-Nitrophenyl phosphate) compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.


( C ) . Fold - change decreases in phosphatase activities of different PTP - MEG2 mutants towards NSF - pY83 phospho - segment compared with wild - type PTP - MEG2 . Data information : * * * P < 0 . 001 : PTP - MEG2 mutants compared with control group . Data were obtained from 3 independent experiments . N . S . means no significant difference . data were analyzed using one - way ANOVA , displayed as mean ± s . e . m . In red column indicates that mutation sites have greater effects on enzyme activities toward phospho - NSF segement than pNPP . green column indicates that mutation sites only have significant effects on enzyme activity toward NSF phospho - segement but not pNPP . column stands for mutants that have similar effects on pNPP NSF phospho - segement .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(C). Fold-change decreases in the phosphatase activities of different PTP-MEG2 mutants towards the NSF-pY83 phospho-segment compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.


( D ) . Statistical diagram of quantal in Fig . 5A Appendix Figure S6A - D . secretion amount of each group was standardized with respect to control group . Data information : Green color represents which are found to modulate foot probability on structure , red color means which does not regulate foot probability . , * indicates PTP - MEG2 mutants overexpression group compared with WT overexpression group . # indicates overexpression group compared with control group . * * , P < 0 . 01 ; * * * P < 0 . 001 # , P < 0 . 05 ; # # , P < 0 . 01 ; # # # P < 0 . 001 . N . S . means no significant difference . Data were from 6 independent experiments . data were analyzed using one - way ANOVA showed as mean ± s . e . m .

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

(D). Statistical diagram of the quantal size in Fig. 5A and Appendix Figure S6A-D. The secretion amount of each group was standardized with respect to the control group. Data information: Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. , * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.


( C ) . GST pull - down suggested that PACSIN1 , MUNC18 - 1 , VAMP7 , SNAP25 , DYNAMIN1 DYNAMIN2 directly interact with PTP - MEG2 . PC12 were transfected with plasmids of candidate substrates , including SYN1 , MUNC18 - 3 , PACSIN1 , SCAMP1 , MUNC18 - 1 , PPP3CA , STX17 , VAMP7 , SYT7 , SYT11 , SNAP25 , DYNAMIN1 , DYNAMIN2 stimulated with 100 nM AngII . tyrosine phosphorylation of these was verified by specific anti - pY antibodies ( Appendix Figure S8A - E ) . potential substrates of PTP - MEG2 in lysates were pulled down with a GST - PTP - MEG2 - D470A trapping mutant then detected by Western blot . arrow stands for Western blot band consistent with predicted molecular of potential PTP - MEG2 substrate .

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

(C). The GST pull-down assay suggested that PACSIN1, MUNC18-1, VAMP7, SNAP25, DYNAMIN1 and DYNAMIN2 directly interact with PTP-MEG2. PC12 cells were transfected with plasmids of candidate substrates, including SYN1, MUNC18-3, PACSIN1, SCAMP1, MUNC18-1, PPP3CA, STX17, VAMP7, SYT7, SYT11, SNAP25, DYNAMIN1, and DYNAMIN2 stimulated with 100 nM AngII. The tyrosine phosphorylation of these proteins was verified by specific anti-pY antibodies (Appendix Figure S8A-E). The potential substrates of PTP-MEG2 in cell lysates were pulled down with a GST-PTP-MEG2-D470A trapping mutant and then detected by Western blot. White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP-MEG2 substrate.


( D ) . Interactions of PTP - MEG2 - trapping mutants with MUNC18 - 1 - Y145 mutants DYNAMIN2 - Y125 mutants . PC12 were transfected with FLAG - MUNC18 - 1 - Y145 , FLAG - DYNAMIN2 - Y125 different mutations of FLAG - MUNC18 - 1 - Y145A , Y145H , Y145E or Y145F , FLAG - DYNAMIN2 - Y125A , Y125E , Y125F , 24 hours before stimulation with 100 nM AngII , respectively . lysates were then incubated with GST - beads - PTP - MEG2 - D470A for 2 hours with constant rotation . potential PTP - MEG2 substrates were pulled down by GST - beads , their were examined by FLAG antibody with Western blot .

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(D). Interactions of the PTP-MEG2-trapping mutants with the MUNC18-1-Y145 mutants and DYNAMIN2-Y125 mutants. PC12 cells were transfected with FLAG-MUNC18-1-Y145, FLAG-DYNAMIN2-Y125 and different mutations of the FLAG-MUNC18-1-Y145A, Y145H, Y145E or Y145F, FLAG-DYNAMIN2-Y125A, Y125E, Y125F, 24 hours before stimulation with 100 nM AngII, respectively. The cell lysates were then incubated with GST-beads-PTP-MEG2-D470A complex for 2 hours with constant rotation. The potential PTP-MEG2 substrates were pulled down by GST-beads, and their levels were examined by the FLAG antibody with Western blot.


( H ) . MUNC18 - 1 - Y145 mutations decreased interaction between MUNC18 - 1 SYNTAXIN1 . PC12 were transfected with plasmid encoding SYNTAXIN - 1 . in lysates were pulled down with purified GST - beads - MUNC18 - 1 - Y145 GST - MUNC18 - 1 - Y145H / E / F , detected with SYNTAXIN1 antibody . right histogram shows quantified . Data information * indicates MUNC18 - 1 , mutants overexpression group compared with WT overexpression group . # indicates MUNC18 - 1 , overexpression group compared with control group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 N . S . means no significant difference . Data were from 3 ( H ) independent experiments . data were analyzed using one - way ANOVA , showed as mean ± s . e . m .

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(H). The MUNC18-1-Y145 mutations decreased the interaction between MUNC18-1 and SYNTAXIN1. PC12 cells were transfected with plasmid encoding SYNTAXIN-1. The proteins in cell lysates were pulled down with purified GST-beads-MUNC18-1-Y145 complex and the GST-MUNC18-1-Y145H/E/F, and detected with SYNTAXIN1 antibody. The right histogram shows the quantified protein levels. Data information * indicates MUNC18-1, mutants overexpression group compared with the WT overexpression group. # indicates MUNC18-1, overexpression group compared with the control group. *, P<0.05; **, P<0.01; *** P<0.001 N.S. means no significant difference. Data were from 3 (H) independent experiments. All the data were analyzed using one-way ANOVA, and showed as the mean ± s.e.m.


( E ) . Relative phosphatase activities of different PTP - MEG2 mutants towards MUNC18 - 1 - pY145 phospho - segment compared with wild - type PTP - MEG2 . ( F ) . Relative phosphatase activities of different PTP - MEG2 mutants towards DYNAMIN 2 - pY125 phospho - segment compared with wild - type PTP - MEG2 . Data information : ( E , F ) Residues are coloured according to Figure 8B . ( E , F ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 : PTP - MEG2 mutants compared with control . N . S . means no significant difference . Data were obtained from 3 independent experiments . * * , P < 0 . 01 ; * * * P < 0 . 001 # # P < 0 . 01 ; # # # P < 0 . 001 . N . S . means no significant difference . Data were obtained from 10 in each group of 6 independent experiments . . data were analyzed using one - way ANOVA showed as mean ± s . e . m .

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(E). Relative phosphatase activities of different PTP-MEG2 mutants towards the MUNC18-1-pY145 phospho-segment compared with wild-type PTP-MEG2. (F). Relative phosphatase activities of different PTP-MEG2 mutants towards the DYNAMIN 2-pY125 phospho-segment compared with wild-type PTP-MEG2.Data information: (E, F) Residues are coloured according to Figure 8B. (E, F), * P<0.05, ** P<0.01, *** P<0.001: PTP-MEG2 mutants compared with the control. N.S. means no significant difference. Data were obtained from 3 independent experiments. **, P<0.01; *** P<0.001 and ## P<0.01; ### P<0.001. N.S. means no significant difference. Data were obtained from 10 cells in each group of 6 independent experiments. . All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm pellets collected at indicated time points after STm infection were lysed , fractionated by SDS - PAGE immunoblotted for caspase - 1 ( E , F ) ( E Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) p20 ( casp . - 1 p20 ) bands ( E ) ( F , of band intensities for caspase - 1 p20 normalized to pro - caspase - 1 actin ( E ) from three independent experiments are shown as fold change ( F Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - Student ' s t - .

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WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 (E, F) (E Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20) bands (E) (F, Quantification of band intensities for caspase-1 p20 normalized to pro-caspase-1 and actin (E) from three independent experiments are shown as fold change (F Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.