293T - FcR - Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab7ADN ( E ) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


EM micrographs of double immuno - gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC , WT MEF and LMP2 KO MEF . Large gold particles ( 15 nm , black arrow head ) labels LMP2 and small gold particles labels LAMP1 ( 5 nm , white arrow head ) ( Scale bars = 500nm ) . The insets are the magnification of region of interest containing LAMP1 positive vacuole ( Scale bars = 100nm ) , marked by the black rectangle .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O 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-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O

EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF and LMP2 KO MEF. Large gold particles (15 nm, black arrow head) labels LMP2 and small gold particles labels LAMP1 (5 nm, white arrow head) (Scale bars = 500nm). The insets are the magnification of region of interest containing LAMP1 positive vacuole (Scale bars = 100nm), marked by the black rectangle.


The distribution of LMP2 positive signals ( n = 22 images ) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles .

O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR O

The distribution of LMP2 positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles.


Immuno - gold labeling with antibodies against constitutive proteasome subunits , α5 , β5 and 19 S2 ( 15 nm , black arrow head ) , co - labeled with an anti - LAMP1 antibody ( 5 nm , white arrow head ) in BMDC ( Scale bars = 500nm ) . Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets ( Scale bars = 100nm ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

Immuno-gold labeling with antibodies against constitutive proteasome subunits, α5, β5 and 19 S2 (15 nm, black arrow head), co-labeled with an anti-LAMP1 antibody (5 nm, white arrow head) in BMDC (Scale bars = 500nm). Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets (Scale bars = 100nm).


Immuno - electron microscopy images ( n = 23 ) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments ( D ) . These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle ( E ) .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SUBCELLULAR O O O O

Immuno-electron microscopy images (n = 23) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments (D). These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle (E).


BMDC transduced with genes encoding GFP , GFP - Rab5ACA and GFP - Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647 - OVA coated bead uptake . A single optical section of 10 optical sections of a representative cell are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by white rectangles , are magnified as inset ( Scale bars = 1μm )

B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O 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-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647-OVA coated bead uptake. A single optical section of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


BMDC transduced with genes encoding GFP , GFP - Rab5ACA and GFP - Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D - rendering of 10 optical sections of a representative cell are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by white rectangles , are magnified as inset ( Scale bars = 1μm )

B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D-rendering of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647 - OVA enclosed within a limiting membrane positive for LAMP1 . The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647 - OVA was calculated and data compiled from independent experiments ( n = 3 ) are plotted ( H ) . For each transduced cell type , images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point .

O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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 same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647-OVA enclosed within a limiting membrane positive for LAMP1. The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647-OVA was calculated and data compiled from independent experiments (n=3) are plotted (H).For each transduced cell type, images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point.


BMDC co - expressing LMP2 - RFP and GFP - Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . Images were acquired every 3min and images taken at 12min interval are presented ( I - K ) . The arrowheads in panel ( I and J ) mark the phagosome or vacuoles containing LMP2 , respectively .

B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O

BMDC co-expressing LMP2-RFP and GFP-Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively.


BMDC co - expressing LMP2 - RFP and GFP - Rab22ACA ( J , K ) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . Images were acquired every 3min and images taken at 12min interval are presented ( I - K ) . The arrowheads in panel ( I and J ) mark the phagosome or vacuoles containing LMP2 , respectively . The boxed region in panel ( J ) marks a phagosome that has been enlarged in panel ( K ) ( Bar 1μm ) .

B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

BMDC co-expressing LMP2-RFP and GFP-Rab22ACA (J, K) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively. The boxed region in panel (J) marks a phagosome that has been enlarged in panel (K) (Bar 1μm).


Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent - treated phagosomes treated or untreated with Epoxomicin . The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of

O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent-treated phagosomes treated or untreated with Epoxomicin. The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of Epoxomicin


Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647 - OVA . Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP , lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs . Post incubation latex beads were treated with detergent and analyzed by flow cytometry ( B ) . The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted ( n = 4 ) ( C ) .

O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647-OVA. Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP, lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs. Post incubation latex beads were treated with detergent and analyzed by flow cytometry (B). The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted (n=4) (C).


Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post - detergent extraction from BMDC expressing GFP or GFP - Ub ( D ) . The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted ( E ) .

B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O

Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post-detergent extraction from BMDC expressing GFP or GFP-Ub (D). The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted (E).


( A ) Colocalization of LacCer and LysoTracker ( LyTr ) in different CTR and LSD patient fibroblasts . Mander ´ s coefficients were calculated using the Fiji JACoP plugin . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s ( A , multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of LacCer and LysoTracker (LyTr) in different CTR and LSD patient fibroblasts. Mander´s coefficients were calculated using the Fiji JACoP plugin. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's (A, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( B - C ) Confocal images ( B ) and statistical analysis ( C ) showing colocalization of LacCer and LyTr in human CTR and MLIV fibroblasts , treated with TPC2 - A1 - P ( 30 µM , 16h ) . ( D - E ) Confocal images ( D ) and statistical analysis ( E ) showing colocalization of LacCer and LyTr in human CTR and NPC1 fibroblasts , treated with TPC2 - A1 - P ( 30 µM , 48h ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s C , E , multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O B-DISEASE B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O B-DISEASE B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Confocal images (B) and statistical analysis (C) showing colocalization of LacCer and LyTr in human CTR and MLIV fibroblasts, treated with TPC2-A1-P (30 µM, 16h). (D-E) Confocal images (D) and statistical analysis (E) showing colocalization of LacCer and LyTr in human CTR and NPC1 fibroblasts, treated with TPC2-A1-P (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's C, E, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( F - G ) Confocal images and statistical analysis showing LacCer / LyTr colocalization in MLIV patient fibroblasts which were mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 16h ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s G , multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Confocal images and statistical analysis showing LacCer/LyTr colocalization in MLIV patient fibroblasts which were mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector and treated with either DMSO or TPC2-A1-P (30 µM, 16h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's G, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( H - I ) Ca2 + chelation ( BAPTA - AM ) dose - dependently impairs LacCer trafficking in CTR fibroblasts . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s I ) multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-I) Ca2+ chelation (BAPTA-AM) dose-dependently impairs LacCer trafficking in CTR fibroblasts. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's I) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( J - K ) Confocal images ( J ) and statistical analysis ( K ) of NPC1 patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO or TPC2 - A1 - P ( 30 µM ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , Tukey ' s ( K ) multiple comparisons test . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-EXP_ASSAY O O O O O O O O O O O B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-K) Confocal images (J) and statistical analysis (K) of NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, Tukey's (K) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( A - B ) Confocal images ( A ) and statistical analysis ( B ) of cholesterol accumulation in human CTR , MLIV , JNCL , and NPC1 fibroblasts . Cholesterol accumulation was evident for NPC1 and MLIV fibroblasts , but not for JNCL fibroblasts . The images show filipin staining to visualize cholesterol accumulation and TO - PRO3 as nuclear staining . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test ( B , * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-DISEASE O B-DISEASE O O B-DISEASE B-CELL_TYPE O B-SMALL_MOLECULE O O O O B-DISEASE O B-DISEASE B-CELL_TYPE O O O O B-DISEASE B-CELL_TYPE O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Confocal images (A) and statistical analysis (B) of cholesterol accumulation in human CTR, MLIV, JNCL, and NPC1 fibroblasts. Cholesterol accumulation was evident for NPC1 and MLIV fibroblasts, but not for JNCL fibroblasts. The images show filipin staining to visualize cholesterol accumulation and TO-PRO3 as nuclear staining. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test (B, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


( C - D ) TPC2 - A1 - P ( 30 µM , 48h ) rescued NPC1 and MLIV cholesterol accumulation . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test D , * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-DISEASE O B-DISEASE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) TPC2-A1-P (30 µM, 48h) rescued NPC1 and MLIV cholesterol accumulation. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test D, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images ( E - F ) of MLIV patient fibroblasts mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector ( white arrow heads ) and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 48h ) .

B-EXP_ASSAY O O O O O O O B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

Confocal images (E-F) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or TPC2-A1-P (30 µM, 48h).


statistical analysis ( G ) of MLIV patient fibroblasts mock - electroporated and treated with DMSO or electroporated with a gain - of - function hTPC2 ( M484L / G734E ) : mCherry TOPO 3 . 1 vector ( white arrow heads ) and treated with either DMSO or TPC2 - A1 - P ( 30 µM , 48h ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

statistical analysis (G) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or TPC2-A1-P (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images and statistical analysis of human CTR patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) two - tailed Student ' s t - test * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

B-EXP_ASSAY O O O O O B-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images and statistical analysis of human CTR patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells) two-tailed Student's t-test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images and statistical analysis of human NPC1 patient fibroblasts treated with 50 nM mock siRNA ( siSCR ) or siRNA targeting TPCN2 ( siTPC2 ) for 72 hours . Cells were then treated with DMSO or TPC2 - A1 - P ( 30 µM ) . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; two - tailed Student ' s t - test K ) . * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

B-EXP_ASSAY O O O O O B-ORGANISM B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images and statistical analysis of human NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); two-tailed Student's t-test K). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Statistics ( L ) of human CTR , MLIV , JNCL , and NPC1 fibroblasts . Effect of the treatment with TPC2 agonist ( 30 µM , 48h ) was examined in NPC1 and MLIV cells . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ) ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test , L ) * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O B-ORGANISM O O B-DISEASE O B-DISEASE O O B-DISEASE B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Statistics (L) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test , L) *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


electron microscopy images ( M ) of human CTR , MLIV , JNCL , and NPC1 fibroblasts . Effect of the treatment with TPC2 agonist ( 30 µM , 48h ) was examined in NPC1 and MLIV cells .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O B-DISEASE O B-DISEASE O O B-DISEASE B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O B-DISEASE O B-DISEASE O O

electron microscopy images (M) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells.


( A ) Confocal images of CTR and JNCL fibroblasts . Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength , respectively , corresponding to the lipofuscin autofluorescence spectrum . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) following cell cycle arrest ( 2h mitomycin C treatment ) . ( B ) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength ( used for LAMP1 staining ) .

O O O B-EXP_ASSAY O O O O B-DISEASE B-CELL_TYPE O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O

(A) Confocal images of CTR and JNCL fibroblasts. Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength, respectively, corresponding to the lipofuscin autofluorescence spectrum. Cells were treated with DMSO or TPC2-A1-P (30 µM) following cell cycle arrest (2h mitomycin C treatment). (B) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength (used for LAMP1 staining).


( C ) Mean autofluorescence intensity in LAMP1 + area . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mean autofluorescence intensity in LAMP1+ area. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells; one-way ANOVA, post hoc Bonferroni's multiple comparisons test. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Mean intensity of shigatoxin ( STX ) in CTR and JNCL fibroblasts . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) after cell cycle arrest . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells ; one - way ANOVA , post hoc Bonferroni ' s multiple comparisons test . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-DISEASE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean intensity of shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells; one-way ANOVA, post hoc Bonferroni's multiple comparisons test. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Confocal images of Gb3 accumulation stained with shigatoxin ( STX ) in CTR and JNCL fibroblasts . Cells were treated with DMSO or TPC2 - A1 - P ( 30 µM ) after cell cycle arrest .

B-EXP_ASSAY O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-DISEASE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

Confocal images of Gb3 accumulation stained with shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( C - D ) Western blot analysis of cathepsin B ( CtsB ) in CTR and MCOLN1IVS3 - 2A > G neurons treated with TPC2 - A1 - P ( 30 µM ) or DMSO . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; two - tailed Student ' s t - test ( C ) . * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O B-DISEASE I-DISEASE I-DISEASE B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (C-D) Western blot analysis of cathepsin B (CtsB) in CTR and MCOLN1IVS3-2A>G neurons treated with TPC2-A1-P (30 µM) or DMSO. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); two-tailed Student's t-test (C). **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker ( LyTr ) . Endolysosomal expansion was observed in MCOLN1IVS3 - 2A > G , CLN3D416G and CLN3ΔEx4 - 7 neurons , which was ameliorated by TPC2 - A1 - P ( 30 µM ) treatment . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O B-DISEASE I-DISEASE I-DISEASE B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker (LyTr). Endolysosomal expansion was observed in MCOLN1IVS3-2A>G, CLN3D416G and CLN3ΔEx4-7 neurons, which was ameliorated by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


Cortical neurons were differentiated from iPSCs , generating lysosomal storage disease neurons and isogenic controls . ( H - I ) Electron microscopy analysis of neuronal rosettes ( neuronal progenitor cells , NPC ) treated with DMSO or TPC2 - A1 - P . TPC2 - A1 - P treatment significantly decreased the number of inclusion bodies ( black arrow heads ) in MCOLN1IVS3 - 2A > G . CLN3D416G and CLN3ΔEx4 - 7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies . However , CLN3ΔEx4 - 7 NPC showed significantly more mitochondria with aberrant cristae numbers ( white arrow heads ) , a phenotype which was rescued by TPC2 - A1 - P ( 30 µM ) treatment . Statistics : Shown are mean values ± SEM . n > 3 technical and biological replicates for each tested condition ( each dot represents an imaged frame containing several cells , obtained from at least three distinct neuronal differentiations ) ; one - way ANOVA , post hoc Tukey ' s multiple comparisons test * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE O O B-DISEASE I-DISEASE I-DISEASE B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O B-CELL_TYPE O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (H-I) Electron microscopy analysis of neuronal rosettes (neuronal progenitor cells, NPC) treated with DMSO or TPC2-A1-P. TPC2-A1-P treatment significantly decreased the number of inclusion bodies (black arrow heads) in MCOLN1IVS3-2A>G. CLN3D416G and CLN3ΔEx4-7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies. However, CLN3ΔEx4-7 NPC showed significantly more mitochondria with aberrant cristae numbers (white arrow heads), a phenotype which was rescued by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.


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

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


Immunoblot analysis of endogenous LC3 ( LC3I - II ) following TPC2 - A1 - P or ML - SA1 ( 30 µM , each ) treatment , alone or with BafA1 , under fed ( complete media ) or starvation ( HBSS ) conditions in CTR , MLIV and NPC1 patientfibroblasts .

B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-DISEASE O B-DISEASE B-ORGANISM O

Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts.


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

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-DISEASE O B-DISEASE B-ORGANISM O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O B-DISEASE B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


( A 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

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(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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-TISSUE O O B-ORGANISM B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O

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


( M 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 B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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


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

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

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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 ) .

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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 .

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(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 .

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(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

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(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 .

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(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 ) .

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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

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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 .

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(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 .

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(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 B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE 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 ) .

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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 B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE 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 B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O 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-GENEPROD O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE 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

B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY

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

B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD 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