Figure 5Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( C - D ) Western blot analysis of cathepsin B ( CtsB ) in CTR and MCOLN1IVS3 - 2A > G neurons treated with TPC2 - A1 - P ( 30 µM ) or DMSO . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; two - tailed Student ' s t - test ( C ) . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 . Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker ( LyTr ) . Endolysosomal expansion was observed in MCOLN1IVS3 - 2A > G , CLN3D416G and CLN3ΔEx4 - 7 neurons , which was ameliorated by TPC2 - A1 - P ( 30 µM ) treatment . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 . Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( H - I ) Electron microscopy analysis of neuronal rosettes ( neuronal progenitor cells , NPC ) treated with DMSO or TPC2 - A1 - P . TPC2 - A1 - P treatment significantly decreased the number of inclusion bodies ( black arrow heads ) in MCOLN1IVS3 - 2A > G . CLN3D416G and CLN3ΔEx4 - 7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies . However , CLN3ΔEx4 - 7 NPC showed significantly more mitochondria with aberrant cristae numbers ( white arrow heads ) , a phenotype which was rescued by TPC2 - A1 - P ( 30 µM ) treatment . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

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

Figure 5Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (C-D) Western blot analysis of cathepsin B (CtsB) in CTR and MCOLN1IVS3-2A>G neurons treated with TPC2-A1-P (30 µM) or DMSO. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); two-tailed Student's t-test (C). **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker (LyTr). Endolysosomal expansion was observed in MCOLN1IVS3-2A>G, CLN3D416G and CLN3ΔEx4-7 neurons, which was ameliorated by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (H-I) Electron microscopy analysis of neuronal rosettes (neuronal progenitor cells, NPC) treated with DMSO or TPC2-A1-P. TPC2-A1-P treatment significantly decreased the number of inclusion bodies (black arrow heads) in MCOLN1IVS3-2A>G. CLN3D416G and CLN3ΔEx4-7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies. However, CLN3ΔEx4-7 NPC showed significantly more mitochondria with aberrant cristae numbers (white arrow heads), a phenotype which was rescued by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Figure 6 ( A ) Confocal images of plasma membrane ( PM ) LAMP1 immunofluorescence in CTR fibroblasts . LAMP1 on the PM is expressed as fold - change relative to DMSO - treated cells . ( B ) Statistical analysis of lysosomal exocytosis data as shown in A . ( C ) Lysosomal exocytosis in CTR , MLIV , NPC1 and JNCL human fibroblasts . PM - localized LAMP1 was measured by flow cytometry , expressed as % of CTR DMSO - treated cells . Ionomycin in A - C ( 4 µM ; 10 min treatment ) was used as positive control . TPC2 - A1 - P and ML - SA1 ( 30 µM , each ; 90 min treatment in A - C ) . Statistics ( B - C ) : Shown are mean values ± SEM . n > 3 for each tested condition ( in B each dot represents an imaged frame containing several cells , in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1 * 104 events ) ; two - way ANOVA , post hoc Dunnett ' s ( B ) or Tukey ' s ( C ) multiple comparisons test , * p - value < 0 . 05 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 . Immunoblot analysis of endogenous LC3 ( LC3I - II ) 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 . 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 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 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 .

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

Figure 6(A) Confocal images of plasma membrane (PM) LAMP1 immunofluorescence in CTR fibroblasts. LAMP1 on the PM is expressed as fold-change relative to DMSO-treated cells. (B) Statistical analysis of lysosomal exocytosis data as shown in A. (C) Lysosomal exocytosis in CTR, MLIV, NPC1 and JNCL human fibroblasts. PM-localized LAMP1 was measured by flow cytometry, expressed as % of CTR DMSO-treated cells. Ionomycin in A-C (4 µM; 10 min treatment) was used as positive control. TPC2-A1-P and ML-SA1 (30 µM, each; 90 min treatment in A-C). Statistics (B-C): Shown are mean values ± SEM. n > 3 for each tested condition (in B each dot represents an imaged frame containing several cells, in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1*104 events); two-way ANOVA, post hoc Dunnett's (B) or Tukey's (C) multiple comparisons test, *p-value < 0.05; ***p-value < 0.001; ****p-value < 0.0001.Immunoblot analysis of endogenous LC3 (LC3I-II) 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.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 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 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.


Figure 7 ( D ) A cDNA array was used to map TPC2 transcripts in the human brain .

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

Figure 7(D) A cDNA array was used to map TPC2 transcripts in the human brain.


Figure 8 ( 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 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 .

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

Figure 8(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 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.


Figure 1 ( 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 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 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 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 ANOVAThe 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 testThe 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 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 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

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

Figure 1(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 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 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 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 ANOVAThe 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 testThe 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 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 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


Figure 2 ( 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 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 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 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 ) 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 ) , 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 . 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 ) .

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

Figure 2(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 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 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 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) 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), 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. 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).


Figure 3 ( 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 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 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 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 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 ANOVAβ - 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 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 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 .

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

Figure 3(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 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 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 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 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 ANOVAβ-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 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 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.


Figure 4 ( 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 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 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 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 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 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 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 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 .

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

Figure 4(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 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 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 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 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 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 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 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.


Figure 5 ( 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 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 blottingExpressing 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 testExpressing 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 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 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 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 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 ANOVAThe 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 testThe 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 .

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

Figure 5(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 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 blottingExpressing 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 testExpressing 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 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 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 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 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 ANOVAThe 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 testThe 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.


Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 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 .

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

Figure 3(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 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.


Figure 5 ( 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 .

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Figure 5(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.


Figure 6 ( 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 .

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Figure 6(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.


Figure 7 ( 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 ) . 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 .

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Figure 7(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). 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.


Figure 8 ( 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 .

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Figure 8(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.


Figure 1A , B : Quantitative RT - PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes ( GV and MII ) , somatic tissues , ES cells ( A ) and preimplantation embryos ( B ) . n = 3 biological replicatesC : Cumulative numbers of pups per female showing fertility of WT and Cnot6l - / - female mice . n = 5 females for each genotypeD : Representative images results of oocytes collected from oviducts of WT and Cnot6l - / - mice at 16 h after hCG injection . scale bar , 100 μm . Arrows indicate polar body - 1 ( PB1 ) E : Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l - / - mice at 16 h after hCG injection . scale bar , 20 μm . Arrows indicate spindle polesF : Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l - / - female mice . The numbers of analyzed oocytes were indicated ( n ) G : Quantification of preimplantation embryos derived from WT and Cnot6l - / - females that develop to the indicated stages after hCG administration and mated with adult WT males . The numbers of analyzed embryos are indicated ( n ) H : Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l - / - female at indicated time points after hCG administration . Scale bar , 100 μm . Data information : Error bars , s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non -

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Figure 1A, B: Quantitative RT-PCR results showing relative expression levels of mouse Cnot6 and Cnot6l in oocytes (GV and MII), somatic tissues, ES cells (A) and preimplantation embryos (B). n = 3 biological replicatesC: Cumulative numbers of pups per female showing fertility of WT and Cnot6l-/- female mice. n = 5 females for each genotypeD: Representative images results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 100 μm. Arrows indicate polar body-1 (PB1)E: Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 20 μm. Arrows indicate spindle polesF: Rates of PB1 emission and normal spindle formation in oocytes ovulated by WT and Cnot6l-/- female mice. The numbers of analyzed oocytes were indicated (n)G: Quantification of preimplantation embryos derived from WT and Cnot6l-/- females that develop to the indicated stages after hCG administration and mated with adult WT males. The numbers of analyzed embryos are indicated (n)H: Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l-/- female at indicated time points after hCG administration. Scale bar, 100 μm. Data information: Error bars, s.e.m. **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif


Figure 2A : Rates of germinal vesicle breakdown ( GVBD ) and PB1 emission in oocytes cultured in vitro . Fully - grown GV oocytes were collected from PMSG - primed ( 44 h ) WT and Cnot6l - / - mice . PB1 : polar body - 1 . Error bars , s . e . m . The numbers of analyzed oocytes were indicated ( n ) B : Representative images of WT , Cnot6l - / - and Btg4 - / - oocytes showing PB1 emission at 16 h after culture . Arrows indicate PB1 . Scale bar , 100 μmC : Representative images of chromosome spreads made from WT and Cnot6l - / - oocytes after 16 h of in vitro maturation culture . Immunofluorescent staining of topoisomerase II ( TOP2 ) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres , respectively . Numbers of paired sister chromatids are indicated . Scale bar , 5 μmD : Percentage ( % ) of aneuploidy among in vitro cultured WT and Cnot6l - / - oocytes that have released PB1s . Error bars , s . e . m . * P < 0 . 05 ; * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were indicateE : Rates of GVBD , PB1 emission , and normal spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes cultured in vitro . Error bars , s . e . m . * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - significant . The numbers of analyzed oocytes were indicateF : Confocal microscopy results showing spindle assembly in WT , Cnot6l - / - and Btg4 - / - oocytes at metaphase I ( MI ) and metaphase II ( MII ) . Scale bar , 20 μmG : Pericentrin immunofluorescence showing MTOCs in cultured WT , Cnot6l - / - and Btg4 - / - oocytes at MI and MII stages . Spindle and DNA were labeled by microtubule nucleation factor ( TPX2 ) and DAPI , respectively . Scale bar , 20

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

Figure 2A: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)B: Representative images of WT, Cnot6l-/- and Btg4-/- oocytes showing PB1 emission at 16 h after culture. Arrows indicate PB1. Scale bar, 100 μmC: Representative images of chromosome spreads made from WT and Cnot6l-/- oocytes after 16 h of in vitro maturation culture. Immunofluorescent staining of topoisomerase II (TOP2) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres, respectively. Numbers of paired sister chromatids are indicated. Scale bar, 5 μmD: Percentage (%) of aneuploidy among in vitro cultured WT and Cnot6l-/- oocytes that have released PB1s. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicateE: Rates of GVBD, PB1 emission, and normal spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes cultured in vitro. Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicateF: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μmG: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm


Figure 3A : Live cell imaging results showing in vitro meiotic division of WT and Cnot6l - / - oocytes . Abbreviations : PM , prometaphase ; MI , metaphase I ; AI , anaphase I ; TI , anaphase I - metaphase II transition ; MII , metaphase II ; PB1 : polar body - 1 . Hours after released from GV arrest were indicated . Scale bar , 20 μmB : Data represent the mean and standard deviations of mCherry - Securin fluorescence intensity in WT and Cnot6l - / - oocytes at each time point . Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest . The numbers of analyzed oocytes were indicated ( n ) C : SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l - / - oocytes at 16 h after culture . Centromeres and DNA were labeled by CREST and DAPI , respectively . Scale bar , 5 μmD : BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l - / - oocytes at 9 . 5 h after culture . More than 8 oocytes were observed for each genotype with similar results . Scale bar , 5 μmE : PB1 emission rates in WT and Cnot6l - / - oocytes cultured with or without MPS1 inhibitor reversin ( 5 μm ) . Reversin is added at 9 hours after culture . Error bars , s . e . m . The numbers of analyzed oocytes were indicated ( n )

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

Figure 3A: Live cell imaging results showing in vitro meiotic division of WT and Cnot6l-/- oocytes. Abbreviations: PM, prometaphase; MI, metaphase I; AI, anaphase I; TI, anaphase I-metaphase II transition; MII, metaphase II; PB1: polar body-1. Hours after released from GV arrest were indicated. Scale bar, 20 μmB: Data represent the mean and standard deviations of mCherry-Securin fluorescence intensity in WT and Cnot6l-/- oocytes at each time point. Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest. The numbers of analyzed oocytes were indicated (n)C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μmD: BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l-/- oocytes at 9.5 h after culture. More than 8 oocytes were observed for each genotype with similar results. Scale bar, 5 μmE: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)


Figure 4A : Relative mRNA copy number dynamics in WT and Cnot6l - / - samples at the indicated stages . Error bars indicate values of the two biological replicatesB : Box plot showing gene expression levels of WT , Cnot6l - / - and Btg4 - / - oocytes at the MII stage . Genes were divided into 10 bins according to their expression levels in the WT MII oocytes . The box indicates upper and lower quantiles , the thick line in the box indicate the median , and the whiskers represent 2 . 5th and 97 . 5th percentiles . * * * P < 0 . 001 by two - tailed Student " s t test . n . s . : non - signifC : qRT - PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l - / - mice ( Cnot6l - / - / WT ) . Error bars , s . e . m . * * * P < 0 . 001 by two - tailed Student " s t test . n = 3 biological repliD : Scatter plot comparing transcripts between WT and Cnot6l - / - oocytes ( at GV , MI and MII stages ) and zygotes derived from these oocytes . Transcripts decreased or increased more than five folds in Cnot6l - / - oocyte samples were high - lighted with blue or red , respectivelyE : Heatmap showing transcripts that were degraded during meiotic maturation ( GV / Zygote > 10 ) in WT oocytes . The definition of cluster I - III is described in the textF : Relative mRNA copy number dynamics of the three gene clusters in WT , Cnot6l - / - and Btg4 - / -

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

Figure 4A: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicatesB: Box plot showing gene expression levels of WT, Cnot6l-/- and Btg4-/- oocytes at the MII stage. Genes were divided into 10 bins according to their expression levels in the WT MII oocytes. The box indicates upper and lower quantiles, the thick line in the box indicate the median, and the whiskers represent 2.5th and 97.5th percentiles. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signifC: qRT-PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l-/- mice (Cnot6l-/-/WT). Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n = 3 biological repliD: Scatter plot comparing transcripts between WT and Cnot6l-/- oocytes (at GV, MI and MII stages) and zygotes derived from these oocytes. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectivelyE: Heatmap showing transcripts that were degraded during meiotic maturation (GV/Zygote > 10) in WT oocytes. The definition of cluster I-III is described in the textF: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples


Figure 6Western blot results showing the levels of CPEB1 in WT and Cnot6l - / - oocytes at the indicated time points after meiotic resumption . Total proteins from 50 ( A oocytes are loaded in each lane . DDB1 ( A is blotted as a loading control . Experiments were performed three times with reproducible results ; a representative result is shownWestern blot results showing the levels of CPEB1 in WT and Cnot6l - / - oocytes at the indicated time points after meiotic resumption . Total proteins fro 100 ( B ) oocytes are loaded in each lane α - tubulin ( B ) is blotted as a loading control . Experiments were performed three times with reproducible results ; a representative result is shownImmunofluorescenc of L - homopropargylglycine ( HPG ) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l - / - mice . Scale bar , 20 μm . Error bars , standard deviations ( n = 6 oocytes for each genotype ) . * * P < 0 . 01 , * * * P < 0 . 001quantification ( D ) of L - homopropargylglycine ( HPG ) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l - / - mice . Scale bar , 20 μm . Error bars , standard deviations ( n = 6 oocytes for each genotype ) . * * P < 0 . 01 , * * * P < 0 . 001E : Relative mRNA copy numbers of polysome - bound transcripts in WT and Cnot6l - / - oocytes at indicated stages . Error bars indicate values of the replicatesF : Scatter plot comparing polysome - bound transcripts between WT and Cnot6l - / - oocytes at GV , MI and MII stages , respectively . Transcripts decreased or increased more than five folds in Cnot6l - / - oocyte samples were high - lighted with blue or red , respectivelyI : Quantitative RT - PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l - / - oocytes at GV , MI and MII stages . Error bars , standard deviations ( n = 3 biologica repeats )

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

Figure 6Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins from 50 (A oocytes are loaded in each lane. DDB1 (A is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shownWestern blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins fro 100 (B) oocytes are loaded in each lane α-tubulin (B) is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shownImmunofluorescenc of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001quantification (D) of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001E: Relative mRNA copy numbers of polysome-bound transcripts in WT and Cnot6l-/- oocytes at indicated stages. Error bars indicate values of the replicatesF: Scatter plot comparing polysome-bound transcripts between WT and Cnot6l-/- oocytes at GV, MI and MII stages, respectively. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectivelyI: Quantitative RT-PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l-/- oocytes at GV, MI and MII stages. Error bars, standard deviations (n = 3 biologica repeats)


Figure 7A : Presence of putative AREs ( AUUUA ) in transcripts of WT oocyte . Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change ( MII / GV ) in WT oocytesB : Presence of putative AREs in oocyte transcripts stabilized by Cnot6l - / - knockout . Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change ( Cnot6l - / - / WT ) at MII stageC : Co - IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7 . HeLa cells were co - transfected with plasmids expressing HA - ZFP36L2 and FLAG - CNOT6L / 7 for 48 h before immunoprecipitation . Experiments were performed three times with reproducible results ; a representative result is shownD : RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts , with or without the presence of ZFP36L2 . HeLa cells were co - transfected with plasmids expressing FLAG - ZFP36L2 and HA - CNOT6L for 48 h before immunoprecipitation using an anti - HA antibody . mRNAs recovered from the immunoprecipitates were subjected to qRT - PCR . Error bars , standard deviations ( n = 3 biological repeats ) . * * * P < 0 . 001 by two - tailed Student " s

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

Figure 7A: Presence of putative AREs (AUUUA) in transcripts of WT oocyte. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (MII/GV) in WT oocytesB: Presence of putative AREs in oocyte transcripts stabilized by Cnot6l-/- knockout. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (Cnot6l-/-/WT) at MII stageC: Co-IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7. HeLa cells were co-transfected with plasmids expressing HA-ZFP36L2 and FLAG-CNOT6L/7 for 48 h before immunoprecipitation. Experiments were performed three times with reproducible results; a representative result is shownD: RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts, with or without the presence of ZFP36L2. HeLa cells were co-transfected with plasmids expressing FLAG-ZFP36L2 and HA-CNOT6L for 48 h before immunoprecipitation using an anti-HA antibody. mRNAs recovered from the immunoprecipitates were subjected to qRT-PCR. Error bars, standard deviations (n = 3 biological repeats). ***P < 0.001 by two-tailed Student"s t