B ) TRBP , PACT Ago2 partially co - localize with ER marker mCherry - CLIMP63 . Hippocampal neurons ( 10 DIV ) expressing mCherry - CLIMP63 were immunostained with TRBP , PACT or Ago2 antibodies . Boxed insets are magnifications of dendrites as depicted by adjacent letters ( scale bars ; 10μm ) .

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

B) TRBP, PACT and Ago2 partially co-localize with the ER marker mCherry-CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry-CLIMP63 were immunostained with TRBP, PACT or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10μm).


C ) GFP - Dicer partially co - localizes with mCherry - CLIMP63 at neuronal soma dendrites of hippocampal neurons . Neurons were imaged at 10 DIV using total internal reflection ( TIRF ) microscopy using a penetration depth of 150nm . Boxed insets are magnifications of dendrites as depicted by adjacent letters ( scale bars ; 2μm or 10μm ) .

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

C) GFP-Dicer partially co-localizes with mCherry-CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2μm or 10μm).


D ) majority of Dicer , TRBP PACT are associated with neuronal membranes . Sequential detergent extraction was used to separate from fractions in 7 DIV cortical neurons . relative quantity of depicted was analyzed in three independent experiments ( p < 0 . 005 , t - type 3 , n = 3 , error bars s . d . ) . Actin ribophorin I were used as loading controls for fractions , respectively .

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

D) The majority of Dicer, TRBP and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (p<0.005, t-test type 3, n=3, error bars s.d.). Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions, respectively.


E ) TRBP is in astrocytes . Sequential detergent extraction was performed in astrocyte cultures . Actin calnexin were used as loading controls for fractions , respectively .

O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD O O O O O O O O O O

E) TRBP is cytoplasmic in primary astrocytes. Sequential detergent extraction was performed in primary astrocyte cultures. Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions, respectively.


F ) majority of Dicer , TRBP PACT are present in fractions in HEK293T , isolated using sequential detergent extraction .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O

F) The majority of Dicer, TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells, isolated using sequential detergent extraction.


A ) BDNF stimulation leads to a decrease in TRBP from ER microsomes . Cortical neurons were stimulated BDNF with or vehicle control ER microsomes were isolated using differential centrifugation . values were normalized to calnexin in each of three independent experiments ( p = 0 . 008 , t - type 3 , n = 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O

A) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes. Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation. Protein intensity values were normalized to calnexin in each of three independent experiments (p=0.008, t-test type 3, n=3, error bars; s.d.).


B ) Endogenous TRBP partially co - localizes with calnexin at neuronal soma dendrites . Hippocampal neurons were transfected with GFP at 7DIV fixed at 10DIV following BDNF or control stimulation . Boxed insets are magnifications of dendrite ( 15μm in ) . Arrows point to overlapping ( scale bars ; 20μm ( GFP ) or 10μm ) .

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

B) Endogenous TRBP partially co-localizes with calnexin at the neuronal cell soma and primary dendrites. Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation. Boxed insets are magnifications of the primary dendrite (15μm in length). Arrows point to overlapping puncta (scale bars; 20μm (GFP) or 10μm).


C ) BDNF leads to a decrease in co - localization between TRBP calnexin at soma dendrites of hippocampal neurons . Manders co - localization co - efficients between TRBP Calnexin were measured in soma or dendrites ( 15μm ) using GFP as a morphological mask . Average co - localization values were normalized to controls in each experiment ( p < 0 . 008 , t - type 3 , n = 4 , error bars ; s . d . ) .

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

C) BDNF leads to a decrease in co-localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons. Manders co-localization co-efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites (15μm) using GFP as a morphological mask. Average co-localization values were normalized to controls in each experiment (p<0.008, t-test type 3, n=4, error bars; s.d.).


D ) BDNF decreases mCherry - CLIMP63 co - localization with TRBP , but not with PACT or Ago2 . Hippocampal neurons were transfected with GFP mCherry - CLIMP63 at 7DIV immunostained three days later , following BDNF or control stimulation . Manders co - localization was measured at soma using GFP as a mask as indicated ( p = 0 . 01 , t - type 3 , n = 4 , error bars ; s . d . ) .

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

D) BDNF decreases mCherry-CLIMP63 co-localization with TRBP, but not with PACT or Ago2. Hippocampal neurons were transfected with GFP and mCherry-CLIMP63 at 7DIV and immunostained three days later, following BDNF or control stimulation. Manders co-localization was measured at the cell soma using GFP as a mask as indicated (p=0.01, t-test type 3, n=4, error bars; s.d.).


E ) BDNF leads to an increase in of TRBP ( p = 0 . 03 ) PACT ( p = 0 . 004 ) in fraction . Cortical neurons ( 7 DIV ) were control - or BDNF - stimulated fractions were isolated using digitonin . values were normalized to actin in each experiment . position of star corresponds to TRBP band used for ( ~ 43kDa ) ( t - type 3 , n = 4 , error bars ; s . d . ) .

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

E) BDNF leads to an increase in the levels of TRBP (p=0.03) and PACT (p=0.004) in the cytoplasmic fraction. Cortical neurons (7 DIV) were control- or BDNF-stimulated and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each experiment. The position of the star corresponds to the TRBP band used for analysis (~43kDa) (t-test type 3, n=4, error bars; s.d.).


F ) BDNF - induced increase in TRBP is dependent on intracellular calcium . Cortical neurons ( 7 DIV ) were treated with vehicle control , BDNF BAPTA - AM as indicated , fractions were isolated using digitonin . values were normalized to actin in each independent experiment ( p = 0 . 03 , n = 3 , error bars ; s . d . ) .

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

F) The BDNF-induced increase in cytoplasmic TRBP is dependent on intracellular calcium. Cortical neurons (7 DIV) were treated with vehicle control, BDNF and BAPTA-AM as indicated, and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each independent experiment (p=0.03, n=3, error bars; s.d.).


BDNF stimulation causes a decrease in TRBP - Dicer interaction . Young cortical neurons were treated with BDNF or vehicle control co - immunoprecipitations ( co - IPs ) were performed using 2μg control IgG or polyclonal anti - TRBP antibodies . Dicer binding in co - IP was normalized to inputs in each experiment ( p = 0 . 001 , t - type 2 , n = 3 , error bars represent s . d . ) .

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

BDNF stimulation causes a decrease in the TRBP-Dicer interaction. Young cortical neurons were treated with BDNF or vehicle control and co-immunoprecipitations (co-IPs) were performed using 2μg control IgG or polyclonal anti-TRBP antibodies. Dicer binding in co-IP was normalized to inputs in each experiment (p=0.001, t-test type 2, n=3, error bars represent s.d.).


B ) Short BDNF stimulation changes of only a few microRNAs , such as miR - 16 - 5p miR - 551b - 5p . Volcano plot of small sequencing data from BDNF - or control - treated cortical neurons ( 6DIV ) was on differential expression using edgeR package . points represent average fold change obtained in three independent experiments for each miRNA , plotted against obtained p - values . MiRNAs above red dashed are significant ( p < 0 . 05 , t - type 2 , n = 3 ) .

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

B) Short BDNF stimulation changes the levels of only a few microRNAs, such as miR-16-5p and miR-551b-5p. Volcano plot of small RNA sequencing data from BDNF- or control-treated cortical neurons (6DIV) was based on differential expression analysis using the edgeR package. Individual points represent the average fold change obtained in three independent experiments for each miRNA, and plotted against obtained p-values. MiRNAs above the red dashed line are significant (p<0.05, t-test type 2, n=3).


C ) BDNF stimulation leads to a transient decrease in mature miR - 16 - 5p . 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes mature miRNA were measured in rtPCR . Ct values for each miRNA were normalized to U6 to 20 minutes of control stimulation ( n = 3 , p = 0 . 02 , t - type 3 , error bars ; s . d . ) .

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

C) BDNF stimulation leads to a transient decrease in mature miR-16-5p levels. 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR. Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation (n=3, p=0.02, t-test type 3, error bars; s.d.).


D ) BDNF stimulation leads to a transient increase in of pre - miR16 after 10 minutes of BDNF stimulation . Ct values obtained in rtPCR were normalized to U6 to 20 minutes control stimulation ( n = 4 , t - type 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY 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

D) BDNF stimulation leads to a transient increase in the levels of pre-miR16 after 10 minutes of BDNF stimulation. Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation (n=4, t-test type 3, error bars; s.d.).


E ) BDNF causes a decrease in pre - miR16 binding to TRBP . Cortical neurons were treated with BDNF or vehicle control IPs were performed using a mouse monoclonal anti - TRBP antibody . Ct values measured in rtPCR were normalized to respective inputs ( n = 3 , p = 0 . 002 , t - type 3 , error bars represent s . d . ) .

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

E) BDNF causes a decrease in pre-miR16 binding to TRBP. Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti-TRBP antibody. Ct values measured in rtPCR were normalized to respective inputs (n=3, p=0.002, t-test type 3, error bars represent s.d.).


A ) BDNF changes canonical isomiR proportion of a subset of miRNAs . IsomiR were compared to total amount of corresponding miRNA . Shown are miRNAs that had significant changes in canonical isomiR ( annotated as ' 0 ' , p < 0 . 05 , t - type 2 , n = 3 ) . Results are shown in order of decreasing significance . Shown in red are miRNAs of miR16 family , miR - 16 - 5p miR - 15b - 5p . Black squares correspond non - existing isomiRs . Numbers on top of table represent trimming ( negative ) or addition ( positive ) of nucleotides to 3 ' - or 5 ' - end of canonical 5p or 3p isomiRs , respectively . Only templated nucleotide additions trimmings , which might reflect Dicer - mediated pre - miRNA cleavage , were considered for .

O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD 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

A) BDNF changes the canonical isomiR proportion of a subset of miRNAs. IsomiR levels were compared to the total amount of the corresponding miRNA. Shown are the miRNAs that had significant changes in the canonical isomiR (annotated as '0', p<0.05, t-test type 2, n=3). Results are shown in order of decreasing significance. Shown in red are miRNAs of the miR16 family, miR-16-5p and miR-15b-5p. Black squares correspond non-existing isomiRs. Numbers on the top of the table represent the trimming (negative) or addition (positive) of nucleotides to the 3'- or 5'-end of the canonical 5p or 3p isomiRs, respectively. Only templated nucleotide additions and trimmings, which might reflect Dicer-mediated pre-miRNA cleavage, were considered for analysis.


B ) BDNF stimulation leads to a decrease in canonical ( 22 nucleotides ) isomiRs of miR16 family , miR - 16 - 5p ( p = 0 . 005 ) miR - 15b - 5p ( p = 0 . 02 ) . In both cases , this is accompanied by an increase in of shorter isomiRs ; 19 - nucleotide isoform of miR - 16 - 5p ( p = 0 . 04 ) 21 - nucleotide isoform of miR - 15b - 5p ( p = 0 . 007 ) . relative isomiRs proportion was calculated as described in A ( t - type 2 , n = 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-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-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 O O O O O O O O O O O O

B) BDNF stimulation leads to a decrease in the canonical (22 nucleotides) isomiRs of the miR16 family, miR-16-5p (p=0.005) and miR-15b-5p (p=0.02). In both cases, this is accompanied by an increase in the levels of shorter isomiRs; the 19-nucleotide isoform of miR-16-5p (p=0.04) and the 21-nucleotide isoform of miR-15b-5p (p=0.007). The relative isomiRs proportion was calculated as described in A (t-test type 2, n=3, error bars; s.d.).


B ) Endogenous miR - 16 - 5p activity in developing hippocampal neurons . Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV with or without LNA control ( ' LNA Ctrl ' ) or anti - sense miR - 16 - 5p inhibitor ( ' LNA - miR16 ' ) . were fixed at 7 DIV number of neurons expressing GFP not dsRed ( ' GFP - only ' ) was compared to total number of transfected neurons . LNA - miR16 reduces amount of repression of miR16 sensor compared to LNA - Ctrl ( n = 4 , p = 0 . 02 , t - type 2 , error bars represent s . d . ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE 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 B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD 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

B) Endogenous miR-16-5p activity in developing hippocampal neurons. Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ('LNA Ctrl') or the anti-sense miR-16-5p inhibitor ('LNA-miR16'). Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ('GFP-only') was compared to the total number of transfected neurons. LNA-miR16 reduces the amount of repression of the miR16 sensor compared to LNA-Ctrl (n=4, p=0.02, t-test type 2, error bars represent s.d.).


C ) Short - term BDNF stimulation inhibits endogenous miR - 16 - 5p activity . Hippocampal neurons were transfected at 4 DIV treated with BDNF either 24h or 20 minutes before fixation neurons were counted as in B . Repression index was obtained by normalizing miR16 sensor to control sensor condition in each experiment ( n = 4 , p = 0 . 02 , t - type 3 , error bars ; s . d . ) .

O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O

C) Short-term BDNF stimulation inhibits endogenous miR-16-5p activity. Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B. Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).


D & E ) Inhibition of miR - 16 - 5p increases dendritic complexity occludes BDNF - induced dendritogenesis . D ) Hippocampal neurons were transfected with control sensor ( A ) , stimulated with control or BDNF 24 hours before fixation . Pictures were obtained on a confocal microscope thresholds were set to dsRed signal in order to distinguish dendrites ( scale bars are 10μm ) . E ) To quantify dendritic complexity total number of dendritic intersections was calculated using Sholl . Values were normalized to LNA - Ctrl control treatment ( n = 3 , p < 0 . 04 , t - type 3 , error bars represent s . d . ) .

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

D&E) Inhibition of miR-16-5p increases dendritic complexity and occludes BDNF-induced dendritogenesis. D) Hippocampal neurons were transfected with the control sensor (A), and stimulated with control or BDNF 24 hours before fixation. Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites (scale bars are 10μm). E) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis. Values were normalized to LNA-Ctrl and control treatment (n=3, p<0.04, t-test type 3, error bars represent s.d.).


F & G ) Overexpression of miR - 16 - 5p blocks BDNF - induced dendritogenesis . F ) Hippocampal neurons were transfected with control sensor either control miRNA mimic ( miR - Ctrl ) or a miR - 16 - 5p mimic ( miR16 ) treated two days later with BDNF or control vehicle . DsRed signal was used to set a threshold on obtained on a confocal microscope ( scale bars ; 10μm ) . G ) Total number of intersections was quantified in Sholl values were normalized to miR - Ctrl , control treatment in each experiment ( n = 4 , p = 0 . 02 , t - type 3 , error bars ; s . d . ) .

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

F&G) Overexpression of miR-16-5p blocks BDNF-induced dendritogenesis. F) Hippocampal neurons were transfected with control sensor and either control miRNA mimic (miR-Ctrl) or a miR-16-5p mimic (miR16) and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). G) Total number of intersections was quantified in Sholl analysis and values were normalized to miR-Ctrl, control treatment in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).


H & I ) Overexpression of Dicer - binding deficient deletion mutant of TRBP ( GFP - TRBP Δ ) occludes BDNF - induced dendritogenesis . H ) Hippocampal neurons were transfected with 200ng dsRed either 50ng GFP or 200ng GFP - TRBP Δ , treated two days later with BDNF or control vehicle . DsRed signal was used to set a threshold on obtained on a confocal microscope ( scale bars ; 10μm ) . I ) Total number of intersections was quantified in Sholl values were normalized to control treatment in each experiment ( n = 5 , p < 0 . 02 , t - type 3 , error bars ; s . d . ) .

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

H&I) Overexpression of the Dicer-binding deficient deletion mutant of TRBP (GFP-TRBP Δ) occludes BDNF-induced dendritogenesis. H) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP-TRBP Δ, and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). I) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment (n=5, p<0.02, t-test type 3, error bars; s.d.).


B ) MiR - 16 - 5p targets 3 ' UTR of BDNFmRNA . Cortical neurons were transfected with 100ng pmiRGlo dual - luciferase reporter consisting of 3 ' UTR of BDNF ( 3 ' UTR WT ) or a miR - 16 - 5p - binding site mutant ( 3 ' UTR Mut ) either miR - Ctrl or miR - 16 . Firefly to Renilla luciferase was measured values were normalized to respective reporter expression without miRNA mimic co - transfection ( n = 3 , p = 0 . 02 , t - type 2 ) .

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B) MiR-16-5p targets the 3'UTR of the BDNFmRNA. Cortical neurons were transfected with 100ng pmiRGlo dual-luciferase reporter consisting of the 3'UTR of BDNF (3'UTR WT) or a miR-16-5p-binding site mutant (3'UTR Mut) and either miR-Ctrl or miR-16. The Firefly to Renilla luciferase ratio was measured and values were normalized to the respective reporter expression without miRNA mimic co-transfection (n=3, p=0.02, t-test type 2).


C ) BDNFstimulation leads to an increase in translation of 3 ' UTR WT ( p = 0 . 02 , t - type 3 ) but not 3 ' UTR Mut luciferase reporter , which is rescued by miR - 16 - 5p overexpression ( p = 0 . 002 ; t - type 2 ) . Cortical neurons were transfected as in ( B ) , stimulated with BDNF or vehicle control for 20 minutes , lysed two hours after stimulation ( n = 4 ) .

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C) BDNFstimulation leads to an increase in the translation of 3'UTR WT (p=0.02, t-test type 3) but not 3'UTR Mut luciferase reporter, which is rescued by miR-16-5p overexpression (p=0.002; t-test type 2). Cortical neurons were transfected as in (B), stimulated with BDNF or vehicle control for 20 minutes, and lysed two hours after stimulation (n=4).


D ) Pre - miR16 is present in neuronal soma processes of hippocampal neurons . Neurons were plated on compartmentalized chambers was isolated from neuronal processes separately . Pre - miR137 pre - miR341 were used to somatic neurite enrichment , respectively . Raw Ct values were normalized to GAPDH mRNA in each experiment ( n = 3 ) .

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D) Pre-miR16 is present in the neuronal cell soma and processes of hippocampal neurons. Neurons were plated on compartmentalized chambers and RNA was isolated from the neuronal cell body and processes separately. Pre-miR137 and pre-miR341 were used to assay somatic and neurite enrichment, respectively. Raw Ct values were normalized to GAPDH mRNA in each experiment (n=3).


More Dicer binds to - targeted GFP - TRBP ( mGFP - TRBP ) compared to GFP - TRBP . HEK293T were transfected with GFP , GFP - TRBP or mGFP - TRBP lysates were processed for co - IP using anti - GFP antibody . Dicer binding was normalized to respective input lanes in each condition ( n = 4 , p = 0 . 02 ; t - type 2 , error bars are s . d . ) .

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More Dicer binds to membrane-targeted GFP-TRBP (mGFP-TRBP) compared to GFP-TRBP. HEK293T cells were transfected with GFP, GFP-TRBP or mGFP-TRBP and cell lysates were processed for co-IP using anti-GFP antibody. Dicer binding was normalized to respective input lanes in each condition (n=4, p=0.02; t-test type 2, error bars are s.d.).


B ) mGFP - TRBP but not GFP - TRBP blocks BDNF - induced decrease in miR - 16 - 5p activity in dual - sensor . Hippocampal neurons were transfected with 200ng miR16 - or control - sensor ( see Fig 5A ) plus 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - TRBP . Neurons were treated with BDNF or vehicle control prior to fixation immunostained with anti - GFP antibody . BDNF led to a decrease in miR - 16 - 5p activity in GFP - transfected neurons ( p = 0 . 03 , n = 4 , t - type 3 ) , which was significantly different from mGFP - TRBP - expressing neurons ( p = 0 . 04 , n = 4 , t - type 2 , error bars are s . d . ) .

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B) mGFP-TRBP but not GFP-TRBP blocks the BDNF-induced decrease in miR-16-5p activity in dual-fluorescence sensor assay. Hippocampal neurons were transfected with 200ng miR16- or control-sensor (see Fig 5A) plus 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-TRBP. Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti-GFP antibody. BDNF led to a decrease in miR-16-5p activity in GFP-transfected neurons (p=0.03, n=4, t-test type 3), which was significantly different from mGFP-TRBP-expressing neurons (p=0.04, n=4, t-test type 2, error bars are s.d.).


C & D ) BDNF - induced dendritogenesis is blocked by mGFP - TRBP overexpression . C ) Hippocampal neurons ( 4DIV ) were transfected for three days with 200ng dsRed 50ng GFP , 400ng GFP - TRBP or 400ng mGFP - BDNF . Neurons were treated with BDNF or vehicle control 24 hours before fixation . Pictures were obtained on a confocal microscope threshold was set for dsRed to distinguish dendrites ( scale bars are 10μm ) . D ) total number of intersections was calculated in Sholl , values were normalized to control treatment in each of three independent experiments . BDNF led to a significant increase in dendritic complexity ( p = 0 . 04 ; t - type 3 ) , which was significantly different in transfected with mGFP - TRBP , but not GFP - TRBP ( n = 3 , p = 0 . 03 ; t - type 2 , error bars ; s . d . ) .

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C&D) BDNF-induced dendritogenesis is blocked by mGFP-TRBP overexpression. C) Hippocampal neurons (4DIV) were transfected for three days with 200ng dsRed and 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-BDNF. Neurons were treated with BDNF or vehicle control 24 hours before fixation. Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites (scale bars are 10μm). D) The total number of intersections was calculated in Sholl analysis, and values were normalized to control treatment in each of three independent experiments. BDNF led to a significant increase in dendritic complexity (p=0.04; t-test type 3), which was significantly different in cells transfected with mGFP-TRBP, but not GFP-TRBP (n=3, p=0.03; t-test type 2, error bars; s.d.).


B - D Left ventricular LXRα expression in Wt LXRα - Tg mice aged 12 . ( B ) Western blot of LXRα in liver LV tissue .

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B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue.


B - D Left ventricular LXRα expression in Wt LXRα - Tg mice aged 12 . ( B ) Western blot of LXRα in liver LV tissue . of ( C ) mRNA expression ( normalized to 36b4 ; n = 6 - 8 / group , * P < 0 . 00001 versus Wt ) ( D ) expression ( normalized to GAPDH ; n = 4 / group , * P = 0 . 0001 versus Wt ) .

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B-D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue. Quantification of (C) mRNA expression (normalized to 36b4; n = 6-8/group, *P < 0.00001 versus Wt) and (D) protein expression (normalized to GAPDH; n = 4/group, *P = 0.0001 versus Wt).


E LV to tibia ratios ( LV / tibia ) ; n = 15 / group . * P = 0 . 02 .

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E LV weight to tibia length ratios (LV/tibia); n = 15/group. *P = 0.02.


F Assessment of mean arterial pressure ( MAP ) with in situ catheterization shows no significant differences .

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F Assessment of mean arterial pressure (MAP) with in situ catheterization shows no significant differences.


G Representative Masson ' s trichrome wheat germ agglutinin ( WGA ) - FITC in cross - sectional LV , scale bars = 100 μm 10 μm , respectively .

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G Representative Masson's trichrome and wheat germ agglutinin (WGA)-FITC staining in cross-sectional LV, scale bars = 100 μm and 10 μm, respectively.


H Relative mRNA of known LXRα - associated target genes assessed with RT - PCR ; n = 8 / group . * P = 0 . 0003 , # P = 0 . 0004 , § P = 0 . 043 , † P = 0 . 0009 versus Wt .

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H Relative mRNA levels of known LXRα-associated and target genes assessed with RT-PCR; n = 8/group. *P = 0.0003, #P = 0.0004, §P = 0.043, †P = 0.0009 versus Wt.


LV / tibia ratios in sham - TAC - operated Wt LXRα - Tgmice ; n = 21 Wt sham , n = 24 LXRα - Tg sham , n = 24 Wt TAC , n = 26 LXRα - TgTAC . * P < 0 . 00001 versus respective sham , # P = 0 . 00004 .

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LV/tibia ratios in sham- and TAC-operated Wt and LXRα-Tgmice; n = 21 Wt sham, n = 24 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. *P < 0.00001 versus respective sham, #P = 0.00004.


of cardiomyocyte cross - sectional from WGA - FITC - stained histological sections ; n = 5 Wt sham , n = 4 LXRα - Tg sham , n = 5 Wt TAC , n = 3 LXRα - TgTAC . * * P = 0 . 00001 versus Wt sham , * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 01 .

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Quantification of cardiomyocyte cross-sectional area from WGA-FITC-stained histological sections; n = 5 Wt sham, n = 4 LXRα-Tg sham, n = 5 Wt TAC, n = 3 LXRα-TgTAC. **P = 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.01.


Representative LV sections stained with WGA - FITC Masson ' s trichrome to assess cardiomyocyte hypertrophy fibrosis , respectively ; scale bars = 1 mm , 100 μm , 10 μm , from top to bottom .

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Representative LV sections stained with WGA-FITC and Masson's trichrome to assess cardiomyocyte hypertrophy and fibrosis, respectively; scale bars = 1 mm, 100 μm, 10 μm, from top to bottom.


Whole of fibrosis in Masson ' s trichrome - stained sections ; n = 8 per group , except n = 7 in Wt TAC group . * P = 0 . 02 versus Wt sham , # P = 0 . 02 .

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Whole area percentage of fibrosis in Masson's trichrome-stained sections; n = 8 per group, except n = 7 in Wt TAC group. *P = 0.02 versus Wt sham, #P = 0.02.


Measurement of mRNA with RT - PCR to assess induction of gene program , as well as hypertrophy ( Rcan1 ) fibrosis ( Col1a1 , Ctgf ) ; n = 8 per group , except n = 7 in Wt TAC group . Myh6 / Myh7 : * P = 0 . 0001 versus Wt sham , # P = 0 . 00001 ; Anp : * P = 0 . 0005 versus LXRα - Tg sham , # P = 0 . 02 ; Bnp : * P = 0 . 0006 versus Wt sham , * * P = 0 . 009 versus LXRα - Tg sham , # P = 0 . 03 ; Acta1 : § P < 0 . 00001 versus Wt sham , * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 001 ; Rcan1 : * * P = 0 . 00004 versus Wt sham , * P = 0 . 05 versus LXRα - Tg sham , # P = 0 . 04 ; Col1a1 : * P = 0 . 00003 versus Wt sham ; Ctgf : § P < 0 . 00001 versus Wt sham , # P = 0 . 00001 .

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Measurement of mRNA levels with RT-PCR to assess induction of fetal gene program, as well as hypertrophy (Rcan1) and fibrosis (Col1a1, Ctgf); n = 8 per group, except n = 7 in Wt TAC group. Myh6/Myh7: *P = 0.0001 versus Wt sham, #P = 0.00001; Anp: *P = 0.0005 versus LXRα-Tg sham, #P = 0.02; Bnp: *P = 0.0006 versus Wt sham, **P = 0.009 versus LXRα-Tg sham, #P = 0.03; Acta1: §P < 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.001; Rcan1: **P = 0.00004 versus Wt sham, *P = 0.05 versus LXRα-Tg sham, #P = 0.04; Col1a1: *P = 0.00003 versus Wt sham; Ctgf: §P < 0.00001 versus Wt sham, #P = 0.00001.


A Echocardiographic assessment of fractional shortening in mice subjected to 5 of TAC ; n = 19 Wt sham , n = 22 LXRα - Tg sham , n = 24 Wt TAC , n = 26 LXRα - TgTAC . * * P < 0 . 00001 versus Wt sham , * P = 0 . 0005 versus LXRα - Tg sham , # P = 0 . 009 .

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A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC; n = 19 Wt sham, n = 22 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-TgTAC. **P < 0.00001 versus Wt sham, *P = 0.0005 versus LXRα-Tg sham, #P = 0.009.


B - D Hemodynamic measurements obtained in situ in sham - TAC - operated mice ; n = 6 - 8 / group . ( B ) Mean arterial pressure ( MAP ) showed no significant differences . ( C ) LV end - systolic pressure ( LVESP ) . * * P < 0 . 00001 versus Wt sham , * P = 0 . 00004 versus LXRα - Tg sham , * P = 0 . 003 . ( D ) LV end - diastolic pressure ( LVEDP ) . * P = 0 . 02 versus Wt sham .

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B-D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6-8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham.


Representative 18F - FDG with microPET in coronal axial planes in Wt LXRα - Tg mice 5 post - TAC .

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Representative 18F-FDG images with microPET in the coronal and axial planes in Wt and LXRα-Tg mice 5 weeks post-TAC.


Myocardial FDG uptake measured as standard uptake value ( SUV ) ; n = 4 - 6 / group . * P = 0 . 04 versus Wt sham , * * P = 0 . 02 versus LXRα - Tg sham , # P = 0 . 03 , # # P = 0 . 01 .

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Myocardial FDG uptake measured as standard uptake value (SUV); n = 4-6/group. *P = 0.04 versus Wt sham, **P = 0.02 versus LXRα-Tg sham, #P = 0.03, ##P = 0.01.


of myocardial glycogen content shows no significant differences ; n = 5 per sham group , n = 7 per TAC group .

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Quantification of myocardial glycogen content shows no significant differences; n = 5 per sham group, n = 7 per TAC group.


GLUT expression in LV tissue normalized to GAPDH ; n = 6 / group . Glut1 : * P = 0 . 01 versus LXRα - Tg sham , # P = 0 . 03 ; Glut4 : * P = 0 . 03 versus LXRα - Tg sham , # P = 0 . 01 .

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GLUT protein expression in LV tissue normalized to GAPDH; n = 6/group. Glut1: *P = 0.01 versus LXRα-Tg sham, #P = 0.03; Glut4: *P = 0.03 versus LXRα-Tg sham, #P = 0.01.


A LV / tibia ratios in sham - TAC - operated WT LXRα − / − mice ; n = 10 WT sham , n = 8 LXRα − / − sham , n = 12 WT TAC , n = 10 LXRα − / − TAC . * P = 0 . 006 versus WT sham , * * P = 0 . 00008 versus LXRα − / − sham .

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A LV/tibia ratios in sham- and TAC-operated WT and LXRα−/− mice; n = 10 WT sham, n = 8 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P = 0.006 versus WT sham, **P = 0.00008 versus LXRα−/−sham.


B , C Cardiac functional assessment at 5 post - TAC . ( B ) fractional shortening determined with echocardiography ; n = 10 WT sham , n = 7 LXRα − / − sham , n = 12 WT TAC , n = 10 LXRα − / − TAC . * P < 0 . 00001 versus respective sham , § P = 0 . 06 . ( C ) LV end - diastolic pressure ( LVEDP ) recorded in situ ; n = 10 WT sham , n = 7 LXRα − / − sham , n = 10 WT TAC , n = 9 LXRα − / − TAC . * P = 0 . 001 versus WT sham , * * P = 0 . 00005 versus LXRα − / − sham .

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B, C Cardiac functional assessment at 5 weeks post-TAC. (B) Percent fractional shortening determined with echocardiography; n = 10 WT sham, n = 7 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P < 0.00001 versus respective sham, §P = 0.06. (C) LV end-diastolic pressure (LVEDP) recorded in situ; n = 10 WT sham, n = 7 LXRα−/− sham, n = 10 WT TAC, n = 9 LXRα−/−TAC. *P = 0.001 versus WT sham, **P = 0.00005 versus LXRα−/− sham.


D - G Relative mRNA gene expression as determined by RT - PCR , normalized to 36b4 ; n = 8 / group . Anp : no significant differences ; Bnp : * P = 0 . 01 versus WT sham , * * P = 0 . 03 versus LXRα − / − sham ; Myh6 / Myh7 : * P = 0 . 0002 versus WT sham , * * P = 0 . 0003 versus LXRα − / − sham ; Acta1 : * P = 0 . 001 versus respective sham .

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D-G Relative mRNA gene expression as determined by RT-PCR, normalized to 36b4; n = 8/group. Anp: no significant differences; Bnp: *P = 0.01 versus WT sham, **P = 0.03 versus LXRα−/− sham; Myh6/Myh7: *P = 0.0002 versus WT sham, **P = 0.0003 versus LXRα−/− sham; Acta1: *P = 0.001 versus respective sham.


H Western blot detection of GLUT expression in LV tissue normalized to GAPDH , expressed as fold change , shows no significant differences among groups ; n = 6 - 8 / group .

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H Western blot detection of GLUT protein expression in LV tissue normalized to GAPDH, expressed as fold change, shows no significant differences among groups; n = 6-8/group.


I Representative 18F - FDG - microPET scans .

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I Representative 18F-FDG-microPET scans.


J Myocardial FDG uptake quantified as standard uptake value ( SUV ) ; n = 6 / group . * P = 0 . 0001 versus WT sham , # P = 0 . 047 .

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J Myocardial FDG uptake quantified as standard uptake value (SUV); n = 6/group. *P = 0.0001 versus WT sham, #P = 0.047.


A Assessment of 2 - deoxyglucose ( 2 - DG ) uptake from 4 independent experiments . * P = 0 . 03 versus Ad - cont .

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A Assessment of 2-deoxyglucose (2-DG) uptake from 4 independent experiments. *P = 0.03 versus Ad-cont.


B , C Glut mRNA expression determined by

B, C Glut mRNA expression determined by RT


D Western blot indicating Ad - LXRα - PE - induced increases in global O - GlcNAcylation , which was abrogated following inhibition of HBP with DON . LXRα expression is shown , GAPDH served as a loading control .

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D Western blot indicating Ad-LXRα- and PE-induced increases in global protein O-GlcNAcylation, which was abrogated following inhibition of HBP with DON. LXRα protein expression is shown, and GAPDH served as a loading control.


E , F Modulation of Anp Bnp mRNA by Ad - LXRα - induced O - GlcNAc signaling . Gene expression as determined by RT - PCR normalized to 36b4 , n = 5 per condition in absence of PE , n = 4 per condition in presence of PE . * P = 0 . 02 versus Ad - cont , * * P = 0 . 008 versus Ad - cont , # P = 0 . 03 , # # P = 0 . 02 .

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

E, F Modulation of Anp and Bnp mRNA levels by Ad-LXRα-induced O-GlcNAc signaling. Gene expression as determined by RT-PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.02.


G Measurement of , n = 5 per condition . * P = 0 . 008 versus Ad - cont , # P = 0 . 02 , # # P = 0 . 03 .

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G Measurement of cell size, n = 5 per condition. *P = 0.008 versus Ad-cont, #P = 0.02, ##P = 0.03.


H Representative for determination of . were stained with an antibody specific for LXRα ( green , indicated by arrow ) , DAPI for nuclei ( blue ) , rhodamine - phalloidin for F - actin ( red ) ; scale bar = 50 μm .

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H Representative images for the determination of cell size. Cells were stained with an antibody specific for LXRα (green, indicated by arrow), DAPI for nuclei (blue), and rhodamine-phalloidin for F-actin (red); scale bar = 50 μm.


A synthesis determined via leucine incorporation , n = 2 - 4 independent experiments . * P = 0 . 03 versus Ad - cont , § P = 0 . 06 versus Ad - cont .

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A Protein synthesis determined via leucine incorporation, n = 2-4 independent experiments. *P = 0.03 versus Ad-cont, §P = 0.06 versus Ad-cont.


B Global O - GlcNAc expression with Western blot , GAPDH served as a control .

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B Global O-GlcNAc protein expression with Western blot, GAPDH served as a control.


C - F mRNA expression , normalized to 36b4 , n = 6 per condition , except n = 5 for si - LXRα control . Anp : * P = 0 . 009 versus Ad - cont , # P = 0 . 004 , # # P = 0 . 009 ; Bnp : * P = 0 . 004 versus Ad - cont , * * P = 0 . 002 versus Ad - cont ; Acta1 : * P = 0 . 002 versus Ad - cont , § P = 0 . 06 versus Ad - cont , # P = 0 . 02 ; Rcan1 : * P = 0 . 03 versus Ad - cont , * * P = 0 . 004 versus Ad - cont , # P = 0 . 004 .

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C-F mRNA expression, normalized to 36b4, n = 6 per condition, except n = 5 for si-LXRα control. Anp: *P = 0.009 versus Ad-cont, #P = 0.004, ##P = 0.009; Bnp: *P = 0.004 versus Ad-cont, **P = 0.002 versus Ad-cont; Acta1: *P = 0.002 versus Ad-cont, §P = 0.06 versus Ad-cont, #P = 0.02; Rcan1: *P = 0.03 versus Ad-cont, **P = 0.004 versus Ad-cont, #P = 0.004.


A , B Western blot analyses of global O - GlcNAc in left ventricles of mice with either ( A ) cardiac - specific LXRα overexpression or ( B ) LXRα deficiency subjected to 5 TAC .

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

A, B Western blot analyses of global protein O-GlcNAc levels in left ventricles of mice with either (A) cardiac-specific LXRα overexpression or (B) LXRα deficiency and subjected to 5 weeks TAC.


C , D extracts were precipitated with agarose WGA in absence or presence of GlcNAc , a competitor , analyzed by Western blot with antibodies against Mef2c , GATA4 , or Nkx - 2 . 5 , known transcription factors of natriuretic peptides ; bands represent 3 pooled hearts per Wt LXRα - Tg lanes , ( D ) is for n = 2 , expressed as fold change .

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C, D Nuclear protein extracts were precipitated with agarose WGA in the absence or presence of GlcNAc, a competitor, and analyzed by Western blot with antibodies against Mef2c, GATA4, or Nkx-2.5, known transcription factors of natriuretic peptides; bands represent 3 pooled hearts per Wt and LXRα-Tg lanes, and (D) quantification is for n = 2, expressed as fold change.


C ) Results of a competitive uptake showing inhibition of 0 . 2 µM L - [ 3H ] Trp or L - [ 3H ] Leu uptake in MhsT - WT - expressing MQ614 , measured for 30 seconds in absence ( - ) or presence of 12 µM of natural ( one - letter code is used ) or 4 - fluoro - phenylalanine ( 4FP ) . Data are mean ± S . E . M . of three independent experiments each performed as technical triplicates .

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

C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.


A ) Uptake of 0 . 1 μM L - [ 3H ] Leu was measured for 10 - second periods in presence or absence of indicated natural 4 - fluorophenylalanine ( 4FP , compounds tested at 100 μM final ) in MQ614 expressing MhsT - WT or - M236F .

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A) Uptake of 0.1 μM L-[3H]Leu was measured for 10-second periods in the presence or absence of the indicated natural amino acids and 4-fluorophenylalanine (4FP, all compounds tested at 100 μM final concentration) in MQ614 expressing MhsT-WT or -M236F.


B , C ) Time course of 0 . 1 µM L - [ 3H ] Leu ( B ) or 0 . 1 µM L - [ 3H ] Trp ( C ) by MQ614 expressing MhsT - WT ( ) or - M236F ( ) , or MQ614 transformed with control plasmid , pQE60 ( ) .

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B, C) Time course of 0.1 µM L-[3H]Leu (B) or 0.1 µM L-[3H]Trp (C) by MQ614 expressing MhsT-WT () or -M236F (), or MQ614 transformed with the control plasmid, pQE60 ().


D , E ) Kinetics of L - [ 3H ] Leu ( D ) or L - [ 3H ] Trp ( E ) uptake .

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D, E) Kinetics of L-[3H]Leu (D) or L-[3H]Trp (E) uptake.


( A ) EGFR degradation was followed in MSD , MPS ‐ IIIA WT MEFs by treating with EGF for indicated time to stimulate EGFR internalization . were then immediately lysed subjected to anti ‐ EGFR blotting . amount of remaining EGFR was quantified by densitometry ( Image J ) of blot expressed in chart as % of EGFR amount present at time T0 ( 100 % ) . values in chart represent mean ± s . e . m . values of three independent experiments .

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(A) EGFR degradation was followed in MSD, MPS‐IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti‐EGFR blotting. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T0 (100%). The values in the chart represent the mean±s.e.m. values of three independent experiments.


( B ) MSD , MPS ‐ IIIA WT MEFs loaded with dextran ( alexafluor ‐ 594 conjugated ) were labelled with anti ‐ LAMP1 antibody of dextran co ‐ localizing with LAMP1 was evaluated . chart displays merge values ( means ± s . e . m . ) that represent of dextran co ‐ localizing with LAMP1 measured in 15 different of triplicate experiments .

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

(B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments.


( C ) rate of lysosome fusion with autophagosomes was monitored in MSD , MPS ‐ IIIA WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al , 2007 ) . rate of autophagosome maturation reflected of LC3 ' unfused ' ( green / red ) at each time ( 1 3 h ) after bafilomycin removal ( T0 ) . of LC3 ' unfused ' was displayed versus value at T0 ( assumed to be 100 % ) . Values are represented as means ± s . e . m . of triplicate experiments . * P0 . 05 , Student ' s t ‐ : ( A ) : WT versus MSD WT versus MPS ‐ IIIA ; ( B , C ) : WT versus MSD WT versus MPS ‐ IIIA at each time point . Scale bar : 10 μm ( B , C ) .

O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD 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 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 B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O

(C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 'unfused' (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 'unfused' was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).


( A ) Cholesterol were measured in indicated endolysosomal samples containing equal amount of expressed as ng of cholesterol per μg of ( see Materials methods section for details ) .

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(A) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein (see Materials and methods section for details).


( B ) Filipin showing cholesterol accumulation in endolysosomal compartment of MSD MPS ‐ IIIA MEFs ( arrowheads enlarged ) .

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(B) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS‐IIIA MEFs (arrowheads and enlarged images).


( C , D ) Total lipids extracted from indicated endolysosomal samples ( 30 μg of ) were either ( C ) subjected to a phosphate to quantify bulk of phospholipids or ( D ) separated by TLC . Phospholipids cholesterol on TLC plates were revealed by molybdenum blue . CHOL , cholesterol ; LBPA lysobisphosphatidic acid ; PC , phosphatidylcoline ; PE phosphatidylethanolamine ; PI phosphatidyinositol ; SM sphingomyelin . ( A , C ) Values represent mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ : ( A , C ) : WT versus MSD WT versus MPS ‐ IIIA . Scale bar , 10 μm ( B ) .

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(C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal cholesterol measurements Filipin were carried out in either ( A ) WT MEFs loaded with cholesterol or in

B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal cholesterol measurements Filipin were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads enlarged show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments rate of autophagosome maturation ( B , E ) transport of dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale

B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O B-SUBCELLULAR O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar


Cholesterol accumulation inhibits lysosomal fusion . Endolysosomal cholesterol measurements Filipin were carried out in either ( A ) WT MEFs loaded with cholesterol or in MSD ( D ) MPS ‐ IIIA MEFs treated with MβCD . Arrowheads enlarged show cholesterol accumulation in endolysosomes of cholesterol ‐ loaded WT MEFs . After treatments rate of autophagosome maturation ( B , E ) transport of dextran to lysosomes ( C , F ) were also analysed as in Figure 1 . WT controls for autophagosome maturation dextran transport experiments were performed as shown in Figure 1 . ( A - F ) Values represent mean ± s . e . m . values of three independent experiments . * P0 . 05 , Student ' s t ‐ : ( A , C ) : WT versus WT + cholesterol ; ( B ) : WT versus WT + cholesterol for each time point ; ( D , F ) : MSD versus MSD + MβCD MPS ‐ IIIA versus MPS ‐ IIIA + MβCD ; ( E ) : MSD versus MSD + MβCD MPS ‐ IIIA versus MPS ‐ IIIA + MβCD for each time point . Scale bar : 10 μm ( A , C , D , F ) .

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Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).


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( A ) Endolysosomal membranes from MSD , MPS ‐ IIIA WT MEFs were treated with 1 % Triton X ‐ 114 loaded on a sucrose gradient . Immunoblots with Flotillin ‐ 1 identified DRMs in fractions 2 , 3 4 ( arrows ) . fractions at bottom of gradient ( 12 13 ) correspond to high ‐ density detergent soluble fractions , whereas remaining ones were defined as intermediate fractions ( intermediate ‐ I : 5 , 6 , 7 8 ; intermediate ‐ II : 9 , 10 11 ) . of Flotillin ‐ 1 in DRMs was calculated from densitometric of immunoblots .

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(A) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots.


( B ) Immuno ‐ EM of GM1 lipid was carried out in WT , MSD MPS ‐ IIIA MEFs by with anti ‐ cholera toxin B antibodies ( see Materials methods section ) . number of GM1 ‐ positive dots was measured in 25 from three independent experiments displayed as fold to WT .

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(B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT.


( C ) Endolysosomal membranes from MSD , MPS ‐ IIIA WT MEFs were stained with C ‐ laurdan subsequently analysed by spectrophotometry to calculate GP value ( see Materials methods section for details ) . Distribution of cholesterol was also measured throughout gradient ad expressed as of total cholesterol in raft ( DRMs ) soluble fractions .

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

(C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions.


( D ) Equal aliquots from either DRMs or soluble fractions were pooled , content determined displayed as of total in DRM soluble gradient regions .

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(D) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions.


( E ) Immunoblotting profiles of transferrin receptor LAMP1 in sucrose gradient . Values represent mean ± s . e . m . values of three experiments ( A - D ) . * P0 . 05 , Student ' s t ‐ : ( A - C ) : WT versus MSD WT versus MPS ‐ IIIA ; ( D ) : WT versus MSD WT versus MPS ‐ IIIA for each fraction . Scale bar : 0 . 3 μm ( B ) .

O O O B-EXP_ASSAY O O O O B-GENEPROD 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 B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O

(E) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient. Values represent the mean±s.e.m. values of three experiments (A-D). *P0.05, Student's t‐test: (A-C): WT versus MSD and WT versus MPS‐IIIA; (D): WT versus MSD and WT versus MPS‐IIIA for each fraction. Scale bar: 0.3 μm (B).


( A ) VAMP7 , Vti1b syntaxin 7 distributions in endo ‐ lysosomal membranes from MSD , MPSIIIA WT MEFs was evaluated by immonoblotting of gradient fractions . To simplify DRM , intermediate ‐ I , intermediate ‐ II soluble fractions of gradient were pooled separately then subjected to immunoblotting . SNARE distribution was also analysed after loading WT with cholesterol after MβCD treatment . In MSD MPS ‐ IIIA MEFs , analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes . Cholesterol modulation results in a change of SNARE distribution .

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(A) VAMP7, Vti1b and syntaxin 7 distributions in endo‐lysosomal membranes from MSD, MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions. To simplify the analysis the DRM, the intermediate‐I, the intermediate‐II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting. SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment. In MSD and MPS‐IIIA MEFs, all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes. Cholesterol modulation results in a change of SNARE distribution.


( B ) of each analysed SNARE observed in DRM fractions was quantified from blots ( ImageJ densitometry ) displayed as relative amount versus WT .

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(B) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots (ImageJ densitometry analysis) and displayed as relative amount versus WT.


( C , E ) Immunoblots relative showing SNARE along with LAMP1 in ( C ) endolysosomal membranes ( E ) total lysates from WT ( untreated cholesterol treated ) MSD MEFs ( untreated MβCD treated ) . In graphs , were displayed as relative amount versus WT .

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(C, E) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in (C) endolysosomal membranes and (E) total cell lysates from WT (untreated and cholesterol treated) and MSD MEFs (untreated and MβCD treated). In the graphs, the protein levels were displayed as relative amount versus WT.


( D ) WT MSD MEFs transfected with GFP - VAMP7 were stained with anti ‐ GFP for immuno ‐ EM . enlarged image shows internalization of GFP - VAMP7 particles ( arrow ) .

O O O O B-ORGANISM B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O

(D) WT and MSD MEFs transfected with GFP-VAMP7 were stained with anti‐GFP for immuno‐EM. The enlarged image shows internalization of GFP-VAMP7 particles (arrow).


( F ) Syntaxin 5 , Sec22 SNAP23 distribution in total derived from control WT MSD MEFs . Syntaxin 5 immunoblot shows two bands ( * , 35 kDa * * , 42 kDa ) corresponding to two isoforms of .

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(F) Syntaxin 5, Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs. Syntaxin 5 immunoblot shows two bands (*, 35 kDa and **, 42 kDa) corresponding to the two isoforms of the protein.


( G ) Distribution profile of Rab7 in WT MSD lysosomal membranes . Values represent mean ± s . e . m . values of three independent experiments ( B , C , E ) . * P0 . 05 , Student ' s t ‐ : ( B ) : WT versus MSD , WT versus MPS ‐ IIIA , WT versus WT + cholesterol , MSD versus MSD + MβCD , MPS ‐ IIIA versus MβCD for each analysed SNARE ; ( C , E ) : WT versus MSD , WT versus WT + cholesterol MSD versus MSD + MβCD for each analysed . Scale bar : 0 . 3 μm ( D ) .

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(G) Distribution profile of Rab7 in WT and MSD lysosomal membranes. Values represent the mean±s.e.m. values of three independent experiments (B, C, E). *P0.05, Student's t‐test: (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD, and MPS‐IIIA versus MβCD for each analysed SNARE; (C, E): WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each analysed protein. Scale bar: 0.3 μm (D).


( A ) SDS ‐ resistant complexes containing Vti1b were detected by immunoblotting of nonboiled samples corresponding to total , detergent insoluble ( DRM ) detergent soluble ( Sol . ) endo ‐ lysosomal fractions derived from MSD WT MEFs . SDS ‐ resistant complexes were also visualized after loading WT MEFs with cholesterol after treating MSD MEFs with MβCD . Immunoblots revealed presence of low molecular complexes ( * , 50 - 60 kDa ) high molecular complexes ( * * , > 80 kDa ) . of Vti1b in SDS ‐ resistant complexes in total endolysosomal membranes ( bottom ‐ left chart ) amount of Vti1b ‐ containing SDS ‐ resistant complexes in DRM soluble fractions ( bottom ‐ right chart ) were calculated by densitometric of correspondent immunoblots ( ImageJ ) . Values represent mean ± s . e . m . values of three independent measurements . * P0 . 05 , Student ' s t ‐ : WT versus MSD , WT versus WT + cholesterol MSD versus MSD + MβCD ( bottom ‐ left chart ) ; WT versus MSD , WT versus WT + cholesterol MSD versus MSD + MβCD for each fraction ( bottom ‐ right chart ) .

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

(A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50-60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart).


( B ) Syntaxin 7 VAMP7 were co ‐ immunoprecipitated with Vti1b using anti ‐ Vti1b antibodies in WT ( untreated or cholesterol treated ) in MSD ( not treated or MβCD treated ) MEFs . amount of Vti1b precipitated in each is also shown .

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

(B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown.


( C ) SDS ‐ resistant complexes are decorated by anti ‐ syntaxin 7 antibodies in total endolysosomal fraction from WT MSD MEFs .

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(C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs.


( D ) ‐ associated α ‐ SNAP its release in cytosol were evaluated by western blot on total lysates ( total ) , intracellular membranes recovered after centrifugation from a post ‐ supernatant fraction ( associated ) lysates devoid of membranes ( released ) derived from MSD ( untreated or MβCD treated ) WT ( untreated or cholesterol treated ) MEFs .

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(D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.


( A ) MSD , MPS ‐ IIIA WT MEFs were subjected to a triple labelling with anti ‐ VAMP7 , anti ‐ Vti1b anti ‐ LAMP1 antibodies . merges between VAMP7 Vti1b ( double merges in yellow ) between VAMP7 , Vti1b LAMP1 ( triple merges in ) are shown ( see also enlarged showing extent of co ‐ localization in different regions of ) . VAMP7 - Vti1b co ‐ localization was quantified in 15 different displayed as % of Vti1b co ‐ localizing with VAMP7 ( means ± s . e . m . ) .

O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-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 B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O

(A) MSD, MPS‐IIIA and WT MEFs were subjected to a triple labelling with anti‐VAMP7, anti‐Vti1b and anti‐LAMP1 antibodies. The merges between VAMP7 and Vti1b (double merges in yellow) and between VAMP7, Vti1b and LAMP1 (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The VAMP7-Vti1b co‐localization was quantified in 15 different cells and displayed as % of Vti1b co‐localizing with VAMP7 (means±s.e.m.).


( B ) Co ‐ localization of Vti1b with epsinR was quantified by double ‐ labelling experiments in MSD MPS ‐ IIIA ( untreated MβCD treated ) in control WT ( untreated cholesterol treated ) MEFs . chart displays merge values ( means ± s . e . m . ) that represent of Vti1b co ‐ localizing with epsinR measured in 15 different .

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(B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells.


( C ) Vti1b trafficking was monitored by FRAP in WT ( untreated cholesterol treated ) MSD ( untreated MβCD treated ) MEFs transfected with GFP - Vti1b ( see Materials methods section for details ) . FRAP data are displayed as of recovery with respect to before bleach ( 100 % ) are representative of 10 recordings from different .

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(C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.


( A ) Blood ammonia in 8 - 9 - week - old C57BL / 6J wild - type ( WT ) mice ( n = 8 ) Becn1F121A mice with constitutive activation of autophagy ( n = 8 ) at baseline 30 min after i . p . injection of NH4Cl ( 10 mmol / kg ) . * * p < 0 . 01 ( Unpaired t - ) . ns : not statistically significant difference .

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD B-ORGANISM 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

(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.


( B ) Urinary orotic acid of 12 - week - old spf - ash mice treated with TB - 1 ( 15 mg / kg , i . p ) or vehicle at various times as indicated by arrows ( n = 5 mice / group ) . * p < 0 . 05 ( Two - way ANOVA ) .

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(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).


( C ; D ) Western blotting densitometric quantifications of autophagy markers ( LC3II : autophagosomes ; p62 NBR1 : cargo receptors ) in livers of spf - ash mice harvested after ten days of treatment with TB - 1 or vehicle . β - actin was used as loading control . n = 5 mice / group . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - ) .

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

(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).


( E ) Survival curves of spf - ash mice fed with a high diet ( HPD ) for ten days treated with TB - 1 alone or combined with scavenger drug L - Arginine , or treated with scavenger drug L - Arginine , or left untreated . WT control were included ( n = 5 / group ) . * * * p < 0 . 001 ( Log - rank Mantel - Cox ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were - , gender - ( C3H ) - matched . values are shown as averages ± S . E . M .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O 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

(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( F ) Blood ammonia determined after 4 days under HPD ( n = 5 mice / group ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were - , gender - ( C3H ) - matched . values are shown as averages ± S . E . M .

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O 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

(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( A ) Survival curves of AslNeo / Neo mice - matched wild - type ( WT ) controls treated with TB - 1 ( 15 mg / kg , i . p . , every 48 hours starting at day 10 of ) or vehicle . WT + Vehicle n = 8 ; WT + TB - 1 n = 8 ; AslNeo / Neo + Vehicle n = 20 ; AslNeo / Neo + TB - 1 n = 16 . * p < 0 . 05 ( Log - rank Mantel - Cox ) . Data information : values are shown as averages ± S . E . M . ns : not statistically significant difference .

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

(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( B ) Isotopic enrichment of 15N - labeled urea in blood , 20 min after i . p . injection of 15NH4Cl tracer ( 4 mmol / kg ) in AslNeo / Neo mice treated with TB - 1 ( n = 15 ) or vehicle ( n = 9 ) . * p < 0 . 05 ( Unpaired t - ) . Data information : values are shown as averages ± S . E . M . ns : not statistically significant difference .

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

(B) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( C ) Representative Western blotting bands of LC3 , p62 NBR1 in livers of AslNeo / Neo mice treated with TB - 1 or vehicle . GAPDH β - actin were used as loading controls . ( D ) Densitometric quantifications . AslNeo / Neo + Vehicle n = 5 ; AslNeo / Neo + TB - 1 n = 9 . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - ) . Data information : values are shown as averages ± S . E . M . ns : not statistically significant difference .

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

(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. (D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( E ) Argininosuccinate in dried blood spots ( DBS ) of WT AslNeo / Neo mice injected with TB - 1 or vehicle ( n = 4 - 14 mice / group ) . * * * p < 0 . 001 , * p < 0 . 05 ( One - way ANOVA ) . Data information : values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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 O O O

(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( F ) Isotopic enrichment of 15N - labeled argininosuccinate in livers of AslNeo / Neo mice treated with TB - 1 ( n = 11 ) or vehicle ( n = 5 ) . * p < 0 . 05 ( Unpaired t - ) . Data information : values are shown as averages ± S . E . M . ns : not statistically significant difference .

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

(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( A ) Hematoxylin eosin ( H & E , upper panels ) periodic acid Schiff ( PAS , lower panels ) of liver samples harvested from wild - type ( WT ) AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bars : 500 µm .

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

(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.


( B ) Computational of PAS ( n ≥ 4 mice / group ) . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : values are shown as averages ± S . E . M .

O O O B-EXP_ASSAY O B-EXP_ASSAY 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

(B) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.