Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown ( TMX1 kds ; two clones ) in immunodeficient mice . ( I ) Tumor growth in the first 19 days after injection ; ( J ) the corresponding tumor volume quantified after 19 days and ( K ) after 45 days .

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Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown (TMX1 kds; two clones) in immunodeficient mice. (I) Tumor growth in the first 19 days after injection; (J) the corresponding tumor volume quantified after 19 days and (K) after 45 days.


Kaplan - Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients , separated in groups with high or low expression of any of the three genes of interest ( GOI ) , TMX1 , TMX3 and NFAT1 .

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Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with high or low expression of any of the three genes of interest (GOI), TMX1, TMX3 and NFAT1.


mRNA expression levels of NFAT1 , TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients . In ( B ) , expression levels are based on the FPKM values of the TCGA samples ( center line : median ; box : 25 % and 75 % percentile ; whiskers : 1 . 5 times interquartile range ) .

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mRNA expression levels of NFAT1, TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients. In (B), expression levels are based on the FPKM values of the TCGA samples (center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range).


Kaplan - Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients , separated in groups with ( C ) BRAF WT and ( D ) BRAF V600E and with high or low expression of NFAT1 .

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Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with (C) BRAF WT and (D) BRAF V600E and with high or low expression of NFAT1.


( 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

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

(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

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

(C 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

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

(E 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

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

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


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

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


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

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


( M 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

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

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


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

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


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

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


( B 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 ) .

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(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 ) .

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(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 ) .

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

Kinetics of β-cat acetylation by Tau. Tau acetylates β-cat in 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 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 ) .

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

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


Kinetics of β - cat acetylation by Tau . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( A 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 .

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

(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 .

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

(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

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

(E 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 .

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

(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

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


β - cat mutation at K49 ( K49R ) restored 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 ) .

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

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


β - cat mutation at K49 ( K49R ) restored 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

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

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

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


( A 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 .

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

(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 .

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

Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. ; N2a cells, transfected with 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 ) .

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

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


Expressing Tau - k18 ( - ) or Tau - 2CA abolished its anti - apoptotic function . N2a 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 .

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

Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) 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 .

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

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


( G ) Non - acetylation modifiable mutation of β - cat at K49 ( K49R ) , not K19 ( K19R ) , abolished 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 .

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

(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 .

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

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


( I ) Overexpressing Ser199 - phosphorylation mimic Tau ( TauS199E ) further augmented 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( A ) Co - expressing myc - Tau 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 ) .

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

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


Expressing β - cat K49Q increased its 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

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

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


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

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

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


Expressing β - cat K49Q increased its 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 ) .

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

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


( E ) Expressing β - cat K49Q further increased its transcription activity measured by dual Topflash / Fopflash reporter luciferase 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

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

(E) 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

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

(F) 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

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

(G) 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

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

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


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

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

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


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

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

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


Clustered heatmap of the whole chemical genomics screen ; each subsection belongs to an individual strain ' s KO library . Grey cells indicate missing values .

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

Clustered heatmap of the whole chemical genomics screen; each subsection belongs to an individual strain's KO library. Grey cells indicate missing values.


S - score scatterplots for each pairwise strain comparison , highlighting conserved phenotypes ( black points ) , significant changes ( red points ) and gene - condition relationships for which no call can reliably be made ( grey points ) . " r " , pearson ' s r value .

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S-score scatterplots for each pairwise strain comparison, highlighting conserved phenotypes (black points), significant changes (red points) and gene-condition relationships for which no call can reliably be made (grey points). "r", pearson's r value.


Fraction of deletion phenotypes in each strain conserved with other stains in pairwise , three - way and four - way comparisons . Error bars represent standard deviation for all pairwise and three - way comparisons . Only a four - way comparison is possible for each strain so no error bars are represented in these cases .

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Fraction of deletion phenotypes in each strain conserved with other stains in pairwise, three-way and four-way comparisons. Error bars represent standard deviation for all pairwise and three-way comparisons. Only a four-way comparison is possible for each strain so no error bars are represented in these cases.


Gene exclusiveness : a measure of each gene ' s propensity to change its chemical genomics profile across strains . The top 10 genes ' names are reported .

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Gene exclusiveness: a measure of each gene's propensity to change its chemical genomics profile across strains. The top 10 genes' names are reported.


Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons .

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Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons.


Wild type fitness of each strain relative to S288C across the same conditions as in panel A ; each dot represents a specific replicate where colony sizes were measured .

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Wild type fitness of each strain relative to S288C across the same conditions as in panel A; each dot represents a specific replicate where colony sizes were measured.


Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C ; each dot represents a strain - condition replicate as in panel B . The conditions maltose , glycerol , and NaCl are highlighted .

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Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted.


Changes in gene deletion phenotypes for growth on osmotic stress conditions . The top heatmap contains genes belonging to the HOG pathway , while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS . Significant growth phenotypes are marked with " - " .

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Changes in gene deletion phenotypes for growth on osmotic stress conditions. The top heatmap contains genes belonging to the HOG pathway, while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS. Significant growth phenotypes are marked with "-".


Changes in deletion phenotypes for growth on glycerol and maltose . The top heatmap contains the MAL genes , while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains . Significant growth phenotypes are marked with " - " .

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Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".


S - score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening . B ) Number of variants significantly associated ( p - value < 1E - 6 ) with phenotypic variation in each growth condition . C ) Number of associated variants that overlap ( i . e . are in a 3kbp window ) with a conditionally essential gene in the same condition , in any of the four KO libraries .

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S-score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening. B) Number of variants significantly associated (p-value < 1E-6) with phenotypic variation in each growth condition. C) Number of associated variants that overlap (i.e. are in a 3kbp window) with a conditionally essential gene in the same condition, in any of the four KO libraries.


Manhattan plots showing examples of overlaps between associated variants and the KO screening . The top plot shows the associations between variants and growth in the natural isolates as a function of the - log10 of the association p - value , while the four bottom plots show the strength of the KO phenotypes across the four yeast strains , as a function of the - log10 of the corrected S - score p - value . Sections shaded in grey indicate the overlap between associations and KO data . Position in the yeast chromosome is reported in kilobase units .

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Manhattan plots showing examples of overlaps between associated variants and the KO screening. The top plot shows the associations between variants and growth in the natural isolates as a function of the -log10 of the association p-value, while the four bottom plots show the strength of the KO phenotypes across the four yeast strains, as a function of the -log10 of the corrected S-score p-value. Sections shaded in grey indicate the overlap between associations and KO data. Position in the yeast chromosome is reported in kilobase units.


( A and B ) Endogenous FYCO1 binds to GST - LC3B . GST - LC3B bound to glutathione - Sepharose beads was incubated with HeLa cell lysate , and copurified proteins were identified by Coomassie blue staining and mass spectrometry ( A ) or immunoblotting with anti - FYCO1 antibody ( B ) .

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(A and B) Endogenous FYCO1 binds to GST-LC3B. GST-LC3B bound to glutathione-Sepharose beads was incubated with HeLa cell lysate, and copurified proteins were identified by Coomassie blue staining and mass spectrometry (A) or immunoblotting with anti-FYCO1 antibody (B).


( C ) GST - LC3B but not GST - p62 or GST alone binds to FYCO1 in a GST pull - down assay . GST , GST - LC3B , or GST - p62 bound to glutathione - Sepharose beads was incubated with [ 35S ] methionine - labeled myc - FYCO1 . Formed protein complexes were isolated and visualized by autoradiography ( top ) or Coomassie blue staining ( bottom ) .

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(C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione-Sepharose beads was incubated with [35S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom).


( D ) GFP - LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate . HeLa cells expressing GFP or GFP - LC3B were lysed and processed as in B . IP , immunoprecipitation ; MM , molecular mass ; WB , Western blot .

(D) GFP-LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate. HeLa cells expressing GFP or GFP-LC3B were lysed and processed as in B. IP, immunoprecipitation; MM, molecular mass; WB, Western blot.


( B ) The region of FYCO1 between aa 1 , 276 and 1 , 294 is essential and sufficient for the interaction with LC3B . GST or GST - LC3B were incubated with [ S35 ] methionine - labeled deletion mutants of FYCO1 and processed as in Fig . 1 C .

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(B) The region of FYCO1 between aa 1,276 and 1,294 is essential and sufficient for the interaction with LC3B. GST or GST-LC3B were incubated with [S35]methionine-labeled deletion mutants of FYCO1 and processed as in Fig. 1 C.


( C ) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells . HeLa cells expressing the indicated constructs were imaged by confocal microscopy . Insets show an enlarged field of interest .

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(C) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells. HeLa cells expressing the indicated constructs were imaged by confocal microscopy. Insets show an enlarged field of interest.


( D ) Both N - and C - terminal domains of LC3B are needed for efficient interaction with FYCO1 . GST , GST - LC3B , or its deletion mutants were incubated with [ S35 ] methionine - labeled myc - FYCO1 and processed as in Fig . 1 C . ARG , autoradiography ; CB , Coomassie blue .

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(D) Both N- and C-terminal domains of LC3B are needed for efficient interaction with FYCO1. GST, GST-LC3B, or its deletion mutants were incubated with [S35]methionine-labeled myc-FYCO1 and processed as in Fig. 1 C. ARG, autoradiography; CB, Coomassie blue.


( E ) The interaction between FYCO1 and LC3B is direct . GST or GST - LC3B was incubated with recombinant MBP - FYCO1863 - 1 , 478 . Protein complexes were isolated and visualized by immunoblotting with anti - GST or anti - MBP antibodies . WB , Western blot . Bars : ( C ) 10 µm ; ( C , insets ) 1 µm .

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(E) The interaction between FYCO1 and LC3B is direct. GST or GST-LC3B was incubated with recombinant MBP-FYCO1863-1,478. Protein complexes were isolated and visualized by immunoblotting with anti-GST or anti-MBP antibodies. WB, Western blot. Bars: (C) 10 µm; (C, insets) 1 µm.


( A ) Endogenous FYCO1 localizes to the cytosolic punctuated structures . HeLa cells cultured in complete growth medium ( left ) or in HBSS for 3 h ( right ) were fixed and stained with anti - FYCO1 antibody ( green ) and Draq5 ( blue ) .

(A) Endogenous FYCO1 localizes to the cytosolic punctuated structures. HeLa cells cultured in complete growth medium (left) or in HBSS for 3 h (right) were fixed and stained with anti-FYCO1 antibody (green) and Draq5 (blue).


( B ) Overexpressed mCherry - FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells .

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(B) Overexpressed mCherry-FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells.


( C ) FYCO1 partially colocalizes with LC3B , LAMP1 , ATG5 , ATG16L , p62 , and HuntingtinQ64 and does not colocalize with EEA1 . HeLa cells or mouse embryonic fibroblasts ( for ATG16L staining ) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection . For p62 staining , HeLa cells were incubated with 0 . 2 µM BafA1 for 12 h before fixation .

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(C) FYCO1 partially colocalizes with LC3B, LAMP1, ATG5, ATG16L, p62, and HuntingtinQ64 and does not colocalize with EEA1. HeLa cells or mouse embryonic fibroblasts (for ATG16L staining) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection. For p62 staining, HeLa cells were incubated with 0.2 µM BafA1 for 12 h before fixation.


( D ) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures . HeLa cells cotransfected with GFP - FYCO1 , mCherry - LC3B , and myc - p62 were imaged by confocal microscopy 48 h after transfection . ( E and F ) FYCO1 - decorated structures are heterogenous in their luminal content .

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(D) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures. HeLa cells cotransfected with GFP-FYCO1, mCherry-LC3B, and myc-p62 were imaged by confocal microscopy 48 h after transfection. (E and F) FYCO1-decorated structures are heterogenous in their luminal content.


( E ) HeLa cells transfected with CFP - FYCO1 and mCherry - YFP - p62 were imaged 48 h after transfection .

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(E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection.


( F ) HeLa cells transfected with GFP - FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h in normal growth medium to stain late endocytic compartments ( left ) . Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647 - dextran ( 10 , 000 D ; right ) .

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(F) HeLa cells transfected with GFP-FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h in normal growth medium to stain late endocytic compartments (left). Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647-dextran (10,000 D; right).


( A ) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection . Insets show an enlarged field of interest .

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(A) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection. Insets show an enlarged field of interest.


( B - D ) Immunoelectron micrographs of HeLa cells transfected with GFP - FYCO1 and stained with anti - GFP polyclonal antibody ( 10 - nm gold particle ) .

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(B-D) Immunoelectron micrographs of HeLa cells transfected with GFP-FYCO1 and stained with anti-GFP polyclonal antibody (10-nm gold particle).


( B ) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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(B) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles. HeLa cells were imaged 24 h after transfection with the indicated constructs.


( C ) FYCO1 can self - interact in a CC - dependent manner . Full - length GFP - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc - tagged FYCO1 in rabbit reticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - myc antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation .

(C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.


( D ) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein - lipid overlay assay . PIP Strips were incubated with 1 - µg / ml solutions of MBP - FYVEFYCO1 , MBP - 2xFYVEFYCO1 , or MBP - FYCO1863 - 1 , 233 for 1 h , and bound proteins were detected by immunostaining with anti - MBP antibody . LPA , lysophosphatic acid ; LPC , lysophosphocholine ; PI , phosphatidylinositol ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine - 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

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(D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein-lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVEFYCO1, MBP-2xFYVEFYCO1, or MBP-FYCO1863-1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.


( E ) The construct containing a tandem fusion of two FYVE domains from FYCO1 ( 2xFYVEFYCO1 ) localizes to the nuclear envelope and ER in HeLa cells . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A and E ) Insets show an enlarged field of interest .

(E) The construct containing a tandem fusion of two FYVE domains from FYCO1 (2xFYVEFYCO1) localizes to the nuclear envelope and ER in HeLa cells. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A and E) Insets show an enlarged field of interest.


( F ) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs ( left ) and PXp40phox ( right ) . HeLa cells were imaged 24 h after transfection with the indicated constructs . Bars , 10 µm .

(F) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs (left) and PXp40phox (right). HeLa cells were imaged 24 h after transfection with the indicated constructs. Bars, 10 µm.


( A ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles . HeLa cells transiently transfected with FYCO1990 - 1 , 233 were imaged 24 h after transfection .

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(A) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles. HeLa cells transiently transfected with FYCO1990-1,233 were imaged 24 h after transfection.


( B ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 that can efficiently interact with the full - length FYCO1 . Full - length myc - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP - tagged FYCO1 in rabbitreticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - SDS - PAGE antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation .

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(B) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbitreticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-SDS-PAGE antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.


( C ) FYCO1990 - 1 , 233 strongly colocalizes with Rab7 , LysoTracker red , and Alexa Fluor 647 - dextran ( 10 , 000 D ) but only weakly with PXp40phox . HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h were imaged 24 h after transfection .

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(C) FYCO1990-1,233 strongly colocalizes with Rab7, LysoTracker red, and Alexa Fluor 647-dextran (10,000 D) but only weakly with PXp40phox. HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h were imaged 24 h after transfection.


( D ) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 ( FYCO1990 - 1 , 233 + 1 , 156 - 1 , 233 ) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A , C , and D ) Insets show an enlarged field of interest . Bars , 10 µm .

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(D) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 (FYCO1990-1,233+1,156-1,233) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A, C, and D) Insets show an enlarged field of interest. Bars, 10 µm.


( A ) Rab7Q67L recruits FYCO11 , 038 - 1 , 233 and FYCO11 , 091 - 1 , 233 but not FYCO11 , 156 - 1 , 233 to the cytosolic vesicles . HeLa cells transfected with GFP - FYCO11 , 038 - 1 , 233 ( top ) , GFP - FYCO11 , 091 - 1 , 233 ( bottom ) , or GFP - FYCO11 , 156 - 1 , 233 ( middle right ) with or without mCherry - Rab7Q67L were imaged 24 h after transfection . The graph shows the relative number of cells ± SD with GFP - FYCO11 , 038 - 1 , 233 recruited to intracellular membranes without and with cotransfected mCherry - Rab7Q67L .

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(A) Rab7Q67L recruits FYCO11,038-1,233 and FYCO11,091-1,233 but not FYCO11,156-1,233 to the cytosolic vesicles. HeLa cells transfected with GFP-FYCO11,038-1,233 (top), GFP-FYCO11,091-1,233 (bottom), or GFP-FYCO11,156-1,233 (middle right) with or without mCherry-Rab7Q67L were imaged 24 h after transfection. The graph shows the relative number of cells ± SD with GFP-FYCO11,038-1,233 recruited to intracellular membranes without and with cotransfected mCherry-Rab7Q67L.


( B ) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain ( FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 ) but not a 2xFYVE alone to the perinuclear vesicles . HeLa cells transfected with GFP - FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 or GFP - FYCO11 , 156 - 1 , 233 + 1 , 156 - 1 , 233 with or without mCherry - Rab7Q67L were imaged 24 h after transfection .

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(B) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain (FYCO11,091-1,233+1,156-1,233) but not a 2xFYVE alone to the perinuclear vesicles. HeLa cells transfected with GFP-FYCO11,091-1,233+1,156-1,233 or GFP-FYCO11,156-1,233+1,156-1,233 with or without mCherry-Rab7Q67L were imaged 24 h after transfection.


( C ) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate . HEK293 cells stably transfected with 3xFlag - FYCO1 were cotransfected with GTP - locked mutants of Rab5 , - 7 , - 11 , and - 24 and lysed 24 h after transfection , and protein complexes were immunoprecipitated with anti - Flag M2 agarose affinity gel . Bound proteins were eluted with Flag peptide , resolved by SDS - PAGE , and visualized by immunoblotting with anti - GFP and anti - Flag antibody . IP , immunoprecipitation ; WB , Western blot .

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(C) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate. HEK293 cells stably transfected with 3xFlag-FYCO1 were cotransfected with GTP-locked mutants of Rab5, -7, -11, and -24 and lysed 24 h after transfection, and protein complexes were immunoprecipitated with anti-Flag M2 agarose affinity gel. Bound proteins were eluted with Flag peptide, resolved by SDS-PAGE, and visualized by immunoblotting with anti-GFP and anti-Flag antibody. IP, immunoprecipitation; WB, Western blot.


( D ) Leucine - 1151 and tryptophan - 1152 are important for the recruitment of FYCO1 to Rab7 - containing vesicles . HeLa cells transfected with GFP - FYCO1990 - 1 , 233L1151A / W1152A with or without mCherry - Rab7Q67L were imaged 24 h after transfection . ( B and D ) Insets show an enlarged field of interest .

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(D) Leucine-1151 and tryptophan-1152 are important for the recruitment of FYCO1 to Rab7-containing vesicles. HeLa cells transfected with GFP-FYCO1990-1,233L1151A/W1152A with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (B and D) Insets show an enlarged field of interest.


( E ) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles . ( right ) HeLa cells cotransfected with GFP - RILP together with mCherry - FYCO1 and stained with Draq5 were imaged 24 h after transfection . ( left ) Relative number of HeLa cells cotransfected with mCherry - FYCO1 and the indicated constructs , in which FYCO1 is recruited to the intracellular vesicles . Error bars represent SD . Bars , 10 µm .

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(E) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles. (right) HeLa cells cotransfected with GFP-RILP together with mCherry-FYCO1 and stained with Draq5 were imaged 24 h after transfection. (left) Relative number of HeLa cells cotransfected with mCherry-FYCO1 and the indicated constructs, in which FYCO1 is recruited to the intracellular vesicles. Error bars represent SD. Bars, 10 µm.


( A ) Overexpressed FYCO1 is enriched in the cell periphery , close to the MT plus end . HeLa cells were imaged 48 h after transfection with mCherry - FYCO1 and GFP - tubulin .

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(A) Overexpressed FYCO1 is enriched in the cell periphery, close to the MT plus end. HeLa cells were imaged 48 h after transfection with mCherry-FYCO1 and GFP-tubulin.


( B ) Peripheral enrichment of FYCO1 is dependent on intact MTs . HeLa cells were transfected with GFP - FYCO1 and cultured in normal medium ( left ) or treated with 5 µM colcemid ( middle ) or 5 µM latrunculin A ( right ) for 2 h .

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(B) Peripheral enrichment of FYCO1 is dependent on intact MTs. HeLa cells were transfected with GFP-FYCO1 and cultured in normal medium (left) or treated with 5 µM colcemid (middle) or 5 µM latrunculin A (right) for 2 h.


( C ) FYCO1 - decorated vesicles move along the MT tracks . HeLa cells transfected with mCherry - FYCO1 and GFP - tubulin were live imaged by confocal microscopy 48 h after transfection . Movements of FYCO1 - decorated structures were tracked with ImageJ MTrackJ plug - in ( National Institutes of Health ) and presented as 8 - min - long tracks in the overlay image .

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(C) FYCO1-decorated vesicles move along the MT tracks. HeLa cells transfected with mCherry-FYCO1 and GFP-tubulin were live imaged by confocal microscopy 48 h after transfection. Movements of FYCO1-decorated structures were tracked with ImageJ MTrackJ plug-in (National Institutes of Health) and presented as 8-min-long tracks in the overlay image.


( D ) Full - length FYCO1 but not the FYCO1 lacking aa 555 - 1 , 136 can redistribute Rab7 and ORP1L to the cell periphery . HeLa cells were transfected with the indicated constructs and stained with DRAQ5 .

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(D) Full-length FYCO1 but not the FYCO1 lacking aa 555-1,136 can redistribute Rab7 and ORP1L to the cell periphery. HeLa cells were transfected with the indicated constructs and stained with DRAQ5.


( E ) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1 - decorated structures . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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(E) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1-decorated structures. HeLa cells were imaged 24 h after transfection with the indicated constructs.


( A ) Western blot ( WB ) of cell lysates from HeLa cells stably expressing GFP - LC3 48 h after transient transfection with siRNA against FYCO1 ( siRNAs : A , Applied Biosystems ; D , Thermo Fisher Scientific ) . MM , molecular mass .

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(A) Western blot (WB) of cell lysates from HeLa cells stably expressing GFP-LC3 48 h after transient transfection with siRNA against FYCO1 (siRNAs: A, Applied Biosystems; D, Thermo Fisher Scientific). MM, molecular mass.


( B ) Representative images of HeLa cells with or without the perinuclear clusters of GFP - LC3 - positive vesicles ( right ) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 ( left ) . Error bars represent SDs based on three independent experiments .

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(B) Representative images of HeLa cells with or without the perinuclear clusters of GFP-LC3-positive vesicles (right) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 (left). Error bars represent SDs based on three independent experiments.


( C , D , and E ) The perinuclear clusters of GFP - LC3 - positive vesicles colocalize with mCherry - Rab7 but not with Golgi protein marker 58K or LysoTracker red . GFP - LC3 HeLa cells were transiently transfected with siRNA against FYCO1 ( Thermo Fisher Scientific ) 48 h before imaging or cotransfected with mCherry - Rab7 . Cells were either fixed and stained with anti - Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h . Insets show an enlarged field of interest . Bars , 10 µm .

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(C, D, and E) The perinuclear clusters of GFP-LC3-positive vesicles colocalize with mCherry-Rab7 but not with Golgi protein marker 58K or LysoTracker red. GFP-LC3 HeLa cells were transiently transfected with siRNA against FYCO1 (Thermo Fisher Scientific) 48 h before imaging or cotransfected with mCherry-Rab7. Cells were either fixed and stained with anti-Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h. Insets show an enlarged field of interest. Bars, 10 µm.


A , B . Representative images of H & amp ; amp ; E - ( A ) and PAS - ( B ) stained colon sections from wild - type and Angptl2 - / - mice . Scale bar = 100μm . C , D . Representative images of colon crypts as assessed by Ki - 67 ( C ) and pH3 ( D ) staining . Scale bar = 100 μm .

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A, B. Representative images of H&amp;E- (A) and PAS- (B) stained colon sections from wild-type and Angptl2-/- mice. Scale bar = 100μm.C, D. Representative images of colon crypts as assessed by Ki-67 (C) and pH3 (D) staining. Scale bar = 100 μm.


E . mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild - type ( n = 4 ) and Angptl2 - / - ( n = 4 ) mice based on qRT - PCR analysis .

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E. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild-type (n=4) and Angptl2-/- (n=4) mice based on qRT-PCR analysis.


F . Western blotting analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 served as an internal control . Numbers below panels represent normalized expression of proteins .

F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 served as an internal control. Numbers below panels represent normalized expression of proteins.


G . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice . Scale bar = 50 μm .

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G. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice. Scale bar = 50 μm.


A . Survival rates of wild - type ( n = 33 ) and Angptl2 - / - ( n = 33 ) mice supplied with drinking water containing 2 . 5 % DSS for 6 days and then untreated water for 6 more days , from two different experiments . * * * p < 0 . 001 , by log - rank test .

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A. Survival rates of wild-type (n=33) and Angptl2-/- (n=33) mice supplied with drinking water containing 2.5 % DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***p < 0.001, by log-rank test.


B . Representative images of colons from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . C . Colon length of wild - type ( n = 7 ) and Angptl2 - / - ( n = 6 ) mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) .

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B. Representative images of colons from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).C. Colon length of wild-type (n=7) and Angptl2-/- (n=6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).


D , E , F . Representative images of colon crypts as assessed by H & amp ; amp ; E ( D ) , Ki - 67 ( E ) , and pH3 ( F ) staining in wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . Scale bar = 100 μm . G , H . The number of regenerating crypts as quantified using Ki - 67 ( G ) and pH3 ( H ) staining . Each data point represents the number of Ki - 67 - positive viable crypts or the number of pH3 - positive cells in a single field of view . Multiple areas in at least 6 mice per genotype were quantified . Numbers below panels represent the average .

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D, E, F. Representative images of colon crypts as assessed by H&amp;E (D), Ki-67 (E), and pH3 (F) staining in wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 100 μm.G, H. The number of regenerating crypts as quantified using Ki-67 (G) and pH3 (H) staining. Each data point represents the number of Ki-67-positive viable crypts or the number of pH3-positive cells in a single field of view. Multiple areas in at least 6 mice per genotype were quantified. Numbers below panels represent the average.


I . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . Scale bar = 50 μm .

I. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 50 μm.


J . mRNA levels of indicated genes in isolated crypts from wild - type ( n = 4 ) and Angptl2 - / - ( n = 4 ) mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) .

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J. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).


A . Weight loss of wild - type ( n = 4 ) and Angptl2 - / - ( n = 5 ) mice in days following 12 Gy irradiation .

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A. Weight loss of wild-type (n=4) and Angptl2-/- (n=5) mice in days following 12 Gy irradiation.


B . Small intestine and colon length from wild - type ( n = 4 ) and Angptl2 - / - ( n = 5 ) mice 5 days after 12 Gy irradiation .

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B. Small intestine and colon length from wild-type (n=4) and Angptl2-/- (n=5) mice 5 days after 12 Gy irradiation.


C , D . Representative images of crypts of small intestine as assessed by H & amp ; amp ; E ( C ) and Ki - 67 ( D ) staining in wild - type and Angptl2 - / - mice 5 days after 12 Gy irradiation . Scale bar = 100 μm . E . The number of regenerating crypts in small intestine as quantified by Ki - 67 ( D ) staining at days 3 and 5 . Each data point represents the number of Ki - 67 - positive viable crypts in a single field of view . Multiple areas in at least 3 mice per genotype were quantified . Numbers below panels represent average .

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C, D. Representative images of crypts of small intestine as assessed by H&amp;E (C) and Ki-67 (D) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.E. The number of regenerating crypts in small intestine as quantified by Ki-67 (D) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.


F , G . Representative images of colon crypts as assessed by H & amp ; amp ; E ( F ) or Ki - 67 ( G ) staining in wild - type and Angptl2 - / - mice 5 days after 12 Gy irradiation . Scale bar = 100 μm . H . The number of regenerating colon crypts as quantified by using Ki - 67 ( G ) staining at days 3 and 5 . Each data point represents the number of Ki - 67 - positive viable crypts in a single field of view . Multiple areas in at least 3 mice per genotype were quantified . Numbers below panels represent average .

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F, G. Representative images of colon crypts as assessed by H&amp;E (F) or Ki-67 (G) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.H. The number of regenerating colon crypts as quantified by using Ki-67 (G) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.


A . Angptl2 mRNA levels in small intestine ( proximal , mid and distal ) , cecum and colon ( proximal and distal ) of wild - type mice based on qRT - PCR analysis . n = 5 .

A. Angptl2 mRNA levels in small intestine (proximal, mid and distal), cecum and colon (proximal and distal) of wild-type mice based on qRT-PCR analysis. n=5.


B . ANGPTL2 IHC in colon from wild - type and Angptl2 - / - mice . The Angptl2 - / - colon image serves as a negative control . Scale bar = 50 μm .

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B. ANGPTL2 IHC in colon from wild-type and Angptl2-/- mice. The Angptl2-/- colon image serves as a negative control. Scale bar = 50 μm.


C . mRNA levels of indicated genes in wild - type ( n = 4 ) mice based on qRT - PCR analysis .

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C. mRNA levels of indicated genes in wild-type (n=4) mice based on qRT-PCR analysis.


D . Representative IF images of distal colon from wild - type mice as assessed with anti - ANGPTL2 ( red ) and anti - α - SMA ( green ) . Nuclei are counterstained with DAPI ( blue ) . Scale bar = 100 μm .

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D. Representative IF images of distal colon from wild-type mice as assessed with anti-ANGPTL2 (red) and anti-α-SMA (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.


A , B , C . mRNA levels of indicated genes in ISEMFs from wild - type ( n = 4 ) and Angptl2 - / - ( n = 3 ) mice based qRT - PCR analysis .

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A, B, C. mRNA levels of indicated genes in ISEMFs from wild-type (n=4) and Angptl2-/- (n=3) mice based qRT-PCR analysis.


D . Representative staining for P - Smad1 / 5 in crypts from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( DSS ) or untreated water for 12 days ( Untreated ) . Dashed lines indicate half of the crypt and arrowheads show P - Smad1 / 5 - positive cells in lower halves of crypts . Scale bar = 50 μm . E . The number of P - Smad1 / 5 - positive cells in the crypt base ( n = 60 ) from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( DSS ) or untreated water for 12 days ( Untreated ) . Multiple areas in 3 mice per genotype were quantified .

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D. Representative staining for P-Smad1/5 in crypts from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Dashed lines indicate half of the crypt and arrowheads show P-Smad1/5-positive cells in lower halves of crypts. Scale bar = 50 μm.E. The number of P-Smad1/5-positive cells in the crypt base (n=60) from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Multiple areas in 3 mice per genotype were quantified.


F . Western blotting analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 serves as an internal control . Numbers below panels represent normalized protein expression .

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F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 serves as an internal control. Numbers below panels represent normalized protein expression.


A . mRNA levels of indicated genes in colon and cultured ISEMFs from wild - type mice ( n = 4 ) based on qRT - PCR analysis . Inta5 , Integrin α5 ; Intb1 , Integrin β1 .

A. mRNA levels of indicated genes in colon and cultured ISEMFs from wild-type mice (n=4) based on qRT-PCR analysis. Inta5, Integrin α5; Intb1, Integrin β1.


B . Representative IHC of integrin α5 and β1 in colon from wild - type mouse . Scale bar = 50 μm .

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B. Representative IHC of integrin α5 and β1 in colon from wild-type mouse. Scale bar = 50 μm.


C , D . mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild - type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors . n = 3 . U0126 , ERK inhibitor ; LY294002 , PI3K inhibitor ; BAY11 - 7085 , NF - κB inhibitor .

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C, D. mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild-type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors. n=3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11-7085, NF-κB inhibitor.


A . Colon organoid cultures treated with vehicle or rANGPTL2 protein . Scale bar = 200 μm . B . Organoid growth efficiency and size following treatment with vehicle or rANGPTL2 . rA2 , recombinant ANGPTL2 . n = 4 . Data from vehicle / Wnt ( + ) was set at 1 .

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A. Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm.B. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n=4. Data from vehicle/Wnt (+) was set at 1.


D . Colon organoid cultures in presence of ISEMFs with or without Noggin ( Nog ) . Scale bar = 50

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D. Colon organoid cultures in presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm


E . Organoid growth efficiency and size in presence of ISEMFs with or without Noggin ( Nog ) . n = 4 . Data from Wild - type organoids / Wild - type ISEMFs / Nog ( + ) was set at 1 .

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E. Organoid growth efficiency and size in presence of ISEMFs with or without Noggin (Nog). n=4. Data from Wild-type organoids/Wild-type ISEMFs/Nog (+) was set at 1.


( A ) ELISA of supernatant IL - 1β for unprimed THP - 1 cells ( UT ) or LPS - primed ( 500 ng / ml , 3 hrs ) THP - 1 cells then infected with ZIKV ( MOI = 1 ) for 36 hrs . Uninfected cells serves as mock control .

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(A) ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 hrs) THP-1 cells then infected with ZIKV (MOI=1) for 36 hrs. Uninfected cells serves as mock control.


( B ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells , left untreated or primed by LPS ( 500 ng / ml , 3 hrs ) , after ZIKV infection ( MOI = 1 ) for 36 hrs .

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(B) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated or primed by LPS (500 ng/ml, 3 hrs), after ZIKV infection (MOI=1) for 36 hrs.


( C ) ELISA of IL - 1β in the supernatants of wild type ( WT ) , NLRP3 knockout ( KO ) # 1 or AIM2 KO # 1 THP - 1 cells left untreated ( UT ) , or pretreated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) stimulation , or stimulated with poly ( dA : dT ) ( 1mg / ml , 6 hrs ) , or infected with ZIKV ( MOI = 1 , 36 hrs ) .

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(C) ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1 or AIM2 KO#1 THP-1 cells left untreated (UT), or pretreated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) stimulation, or stimulated with poly (dA:dT) (1mg/ml, 6 hrs), or infected with ZIKV (MOI=1, 36 hrs).


( D ) ELISA of IL - 1β in the supernatants of two clones of NLRP3 KO THP - 1 cells with or without ZIKV infection ( MOI = 1 ) at the indicated time points .

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(D) ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI=1) at the indicated time points.


( E ) ELISA of IL - 1β in the supernatants of PBMCs from two donors transfected with control ( ctrl ) siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

(E) ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for 36 hrs.


( F ) ELISA of IL - 1β in the supernatant of WT , NLRP3 KO or AIM2 KO THP - 1 cells with ZIKV ( MR766 or GZ01 ) ( MOI = 1 ) or DENV - 2 ( MOI = 1 ) infection for 36 hrs .

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(F) ELISA of IL-1β in the supernatant of WT, NLRP3 KO or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI=1) or DENV-2 (MOI=1) infection for 36 hrs.


( G ) ELISA of IL - 1β in the supernatants of THP - 1 cells pre - infected with ZIKV for 24 hrs or mock control . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) treatment for 6 hrs .

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(G) ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 hrs or mock control. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) treatment for 6 hrs.


( H ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells pre - infected with ZIKV ( MOI = 1 ) or mock control for 24 hrs . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) stimulation for 6 hrs .

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(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.