( A - K ) C2C12 were transfected for 48 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) , YFP ‐ Jumpy C330S ( Jumpy CS ) , YFP ‐ Jumpy Y462C ( Jumpy YC ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , fixed immunostained with anti ‐ p62 antibody ( red ) . p62 were quantitated by confocal microscopy . Representative confocal of YFP ( A ) , YFP ‐ Jumpy WT ( B ) , YFP ‐ Jumpy C330S ( C ) , YFP ‐ Jumpy Y462C ( D ) YFP ‐ Jumpy R336Q ( E ) transfected , immunostained for p62 ( F ) , ( G ) , ( H ) , ( I ) ( J ) , respectively . Scale bars , 5 μm . Quantitation of p62 per ( K ) . Bars , s . e . m . ( n = 3 independent experiments , 30 per experiments ) . * P0 . 05 ( t ‐ ) , ns : nonsignificant .

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

(A-K) C2C12 cells were transfected for 48 h with YFP, YFP‐Jumpy WT (Jumpy WT), YFP‐Jumpy C330S (Jumpy CS), YFP‐Jumpy Y462C (Jumpy YC) or YFP‐Jumpy R336Q (Jumpy RQ), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of YFP (A), YFP‐Jumpy WT (B), YFP‐Jumpy C330S (C), YFP‐Jumpy Y462C (D) and YFP‐Jumpy R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P0.05 (t‐test), ns: nonsignificant.


( L ) HeLa were transfected for 24 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , incubated with or without 50 ng / μl rapamycin ( BafA1 ( 100 nM ) was present in both control rapamycin treated ) for 2 h , lysed analysed for LC3 , YFP actin by immunoblotting . Densitometric LC3 ‐ II / actin ratios are shown underneath blot .

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(L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.


( C ) Example of yeast for each of 21 subcellular endocytic identified in this study . relevant markers are listed to left of . Scale bar : 5 µm . ( D ) Pie charts showing proportion of specific phenotype mutants ( SPMs ) that have one or more distinct aberrant , non - phenotype - specific mutants for each of compartments screened .

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(C) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study. The relevant markers are listed to the left of the micrographs. Scale bar: 5 µm. (D) Pie charts showing the proportion of specific phenotype mutants (SPMs) that have one or more distinct aberrant phenotypes, and non-phenotype-specific mutants for each of the compartments screened.


Representative of wild - type mutant organized by marker phenotype ( labels on left of each panel ) . For each phenotype , three labelled for training set ( labelled single ) identified by 2NN classifier ( identified single ) are shown . table to right of shows ( from left to right ) : [ 1 ] occurrence of each phenotype in a wild - type population ( % in WT ) ; [ 2 ] number of specific phenotype mutants ( ) stringent specific phenotype mutants ( str ) for each of 17 mutant morphologies ; [ 3 ] most significantly enriched GO Slim biological process ; [ 4 ] most significantly enriched . # denotes term below statistical significance .

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 O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O 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 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

Representative images of wild-type and mutant cells organized by marker and phenotype (labels on the left of each panel). For each phenotype, three cells labelled for the training set (labelled single cells) and cells identified by the 2NN classifier (identified single cells) are shown. The table to the right of the images shows (from left to right): [1] the occurrence of each phenotype in a wild-type population (% in WT); [2] the number of specific phenotype mutants (all) and stringent specific phenotype mutants (str) for each of the 17 mutant morphologies; [3] the most significantly enriched GO Slim biological process and; [4] the most significantly enriched protein complex. # denotes term below statistical significance.


Endocytic patch formation dynamics in ydl176wΔ ( ipf1Δ ) . Patch dynamics were examined using time - lapse microscopy of wild - type ( WT ) ipf1Δ deletion strains carrying reporters for coat ( Sla1 - GFP ; green ) , actin ( Sac6 - tdTomato ; red ) modules . Left : Representative kymographs for WT ipf1Δ strains . Right : Box plot illustrating distribution of lifetimes of Sla1 - GFP Sac6 - tdTomato patches . Significance was determined using unpaired t - . Scale bar : 10 seconds .

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Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds.


Penetrance as a function of replicative . Top : Bar graph showing fraction of outliers in populations of increasing replicative ( # of divisions ) for wild - type ( WT ) , 5 mutant strains ( rrd2Δ , rpl20bΔ , cka2Δ , vac8Δ vac17Δ ) . Data are presented as mean of 3 biological replicates + / - SD . Bottom : Box plot with distribution of sizes for same populations of . Whiskers extend to 5th 95th percentile .

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Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile.


of young ( top row of ) older ( bottom row of ) wild - type ( WT ) expressing Vph1 - EGFP ( green vacuole ) stained with CF640R WGA ( magenta bud scars ) . Scale bar : 5 µm .

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Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.


Combined effect of replicative a vacuole inheritance defect on penetrance . of wild - type vac17Δ expressing Vph1 - EGFP ( green vacuole ) Hta2 - mCherry ( red nucleus ) , stained with CF640R WGA ( magenta bud scars ) . with increasing bud - scar ( replicative ) are shown from left to right . Scale bar : 5 µm .

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

Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.


Relationship between stress response penetrance . Single - UPRE - RFP were measured in ~ 60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole . were binned into equal - sized bins , from low to high stress response , assessed as outlier or inlier , clustered on their penetrance profile ( composed from fraction of outliers in each stress - response bin ) . Each plot represents a penetrance profile . * denotes cluster with a profile most similar to wild - type . Insert pie charts show proportion of mutant strains in each cluster .

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Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.


( C ) Blood brain tissue of CNP520 in rat after a 30 µmol / kg ( 15 . 4 mg / kg ) oral dose . Shown are unbound fraction ( main graph ) total ( insert ) of CNP520 in blood brain , using values for rat plasma binding of 97 . 8 % unspecific binding to brain homogenate of 99 % . Values are mean ± SD , with n = 5 per time point .

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(C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point.


( A ) Subjective assessment of hair depigmentation ( score 0 - 5 , with 0 = completely black 5 = completely ) at 2 doses of either CNP520 or BACE - 1 / - 2 inhibitor NB - 360 ( mean ± , n = 8 / group ) , statistical comparisons were made for every scoring day , versus vehicle using Kruskal - Wallis with Dunn ' s post - hoc .

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(A) Subjective assessment of hair depigmentation (score 0-5, with 0 = completely black and 5 = completely white) at 2 doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (mean ± SEM, n=8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal-Wallis with Dunn's post-hoc test.


( B ) Summary of doses , steady - state effects on Aβ40 in brain ( % compared to vehicle - treated mice ) ratios between total skin concentrations of CNP520 NB - 360 their respective IC50 values for inhibition of BACE - 2 in vitro .

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

(B) Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro.


( A ) Doses from 1 . 5 mg / kg ( 3 µmol / kg ) to 51 . 3 mg / kg ( 100 µmol / kg ) were given by oral gavage to rats ( n = 5 per dose ) ; were killed 4 hours later for determination of Aβ40 in brain tissue . Values are mean ± as determined by ANOVA ( GraphPad Prism ) .

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(A) Doses from 1.5 mg/kg (3 µmol/kg) to 51.3 mg/kg (100 µmol/kg) were given by oral gavage to rats (n = 5 per dose); animals were killed 4 hours later for determination of Aβ40 in brain tissue. Values are mean ± SEM as determined by ANOVA (GraphPad Prism).


( B ) Time - dependent reduction of Aβ40 in rat brain CSF after a single 15 . 4 mg / kg ( 30 µmol / kg ) oral dose of CNP520 .

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(B) Time-dependent reduction of Aβ40 in rat brain and CSF after a single 15.4 mg/kg (30 µmol/kg) oral dose of CNP520.


( C ) CNP520 in dog blood CSF , following a 3 . 1 mg / kg ( 6 µmol / kg ) oral dose ( mean ± SD = 4 ) .

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(C) CNP520 levels in dog blood and CSF, following a 3.1 mg/kg (6 µmol/kg) oral dose (mean ± SD = 4).


( D ) Time - dependent reduction of Aβ40 42 in dog CSF , after a 3 . 1 mg / kg oral CNP520 dose ( mean ± s . d . ) .

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(D) Time-dependent reduction of Aβ40 and 42 in dog CSF, after a 3.1 mg/kg oral CNP520 dose (mean ± s.d.).


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( A ) CNP520 in blood cerebellum .

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

Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (A) CNP520 levels in blood and cerebellum.


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( B ) Total cortical plaque ( normalized to total sample ( * 10 − 2 ) , stained with antibody NT - 12 .

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

Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (B) Total cortical plaque area (normalized to total sample area (*10−2), stained with antibody NT-12.


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( C ) Formic acid - soluble Aβ40 in forebrain . ( D ) Formic acid - soluble Aβ42 in forebrain .

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

Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (C) Formic acid-soluble Aβ40 in forebrain. (D) Formic acid-soluble Aβ42 in forebrain.


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( E ) Double - immunofluorescence stained brain sections with NT12 ( against amyloid - β , green ) Iba1 ( against microglia , red ) or GFAP ( astrocytes , red ) for different treatment groups as indicated . Scale bar : 500 µm .

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

Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (E) Double-immunofluorescence stained brain sections with NT12 (against amyloid-β, green) and Iba1 (against microglia, red) or GFAP (astrocytes, red) for the different treatment groups as indicated. Scale bar: 500 µm.


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( F ) of total Iba1 positive microglia ( normalized to total ( * 10 − 2 ) , only a subset of samples analyzed .

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Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (F) Quantification of total Iba1 positive microglia (normalized to total area (*10−2), only a subset of samples analyzed.


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( G ) of total GFAP positive astrocyte ( normalized to total ( * 10 − 2 ) . Data were analyzed with Dunnett ' s multiple comparison .

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

Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (G) Quantification of total GFAP positive astrocyte area (normalized to total area (*10−2). Data were analyzed with Dunnett's multiple comparison test.


( A ) Mean plasma vs time profiles after single dose .

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(A) Mean plasma concentration vs time profiles after single dose.


( B ) Mean plasma profiles for 91 days of dosing post - dose concentrations up to day 121 .

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(B) Mean plasma concentration profiles for 91 days of dosing and post-dose concentrations up to day 121.


( C ) Time vs CSF profiles after single dose of CNP520 ( Mean ± , n = 6 - 8 ) .

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(C) Time vs CSF concentration profiles after single dose of CNP520 (Mean ± SEM, n = 6-8).


( D ) Dose - CSF profiles after 3 months of dosing ( Mean ± , n = 22 - 24 ) .

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(D) Dose-CSF concentration profiles after 3 months of dosing (Mean ± SEM, n = 22-24).


( A ) Changes in Aβ40 relative to baseline values . CSF was sampled via a lumbar catheter at time points from 2 hours pre - to 34 hours post - administration of a single dose of CNP520 ( mean ± , n = 6 - 8 ) .

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(A) Changes in Aβ40 relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).


( B ) Changes in sAPPβ relative to baseline values . CSF was sampled via a lumbar catheter at time points from 2 hours pre - to 34 hours post - administration of a single dose of CNP520 ( mean ± , n = 6 - 8 ) .

(B) Changes in sAPPβ relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).


( C ) Changes in sAPPα relative to baseline values . CSF was sampled via a lumbar catheter at time points from 2 hours pre - to 34 hours post - administration of a single dose of CNP520 ( mean ± , n = 6 - 8 ) .

(C) Changes in sAPPα relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).


( D ) Dose response of Aβ40 ( mean ± SD ) to CNP520 relative to baseline Aβ40 after 2 of dosing . ( D ) CNP520 was administered daily for 14 days ; CSF was sampled via a lumbar catheter 2 hours before dosing , 24 hours after last dose ( mean ± SD , n = 8 in treatment groups , n = 16 in placebo group ) .

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

(D) Dose response of Aβ40 (mean ± SD) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing. (D) CNP520 was administered daily for 14 days; CSF was sampled via a lumbar catheter 2 hours before dosing, and 24 hours after the last dose (mean ± SD, n = 8 in treatment groups, n = 16 in placebo group).


( A ) Changes of Aβ40 relative from baseline values up to 3 months of daily dosing . Shown are means ± SD , n = 21 - 25 for pre - dose day 91 , n = 2 - 7 for intermediate time points .

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(A) Changes of Aβ40 relative from baseline values up to 3 months of daily dosing. Shown are means ± SD, n = 21-25 for pre-dose and day 91, and n = 2 - 7 for the intermediate time points.


( B ) Dose - response for CSF Aβ40 at day 91 ( 24 hours after last dose ) change from baseline , shown as mean 95 % confidence interval , n = 21 - 25 per treatment group .

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(B) Dose-response for CSF Aβ40 at day 91 (24 hours after last dose) and individual percent change from baseline, shown as mean and 95% confidence interval, n = 21-25 per treatment group.


( A ) Mouse forebrain Aβ40 in homozygous APOE4 - TR mice with without CNP520 treatment , 4 hours after dosing , shown are mean ± , n = 6 / group .

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(A) Mouse forebrain Aβ40 levels in homozygous male and female APOE4-TR mice with and without CNP520 treatment, 4 hours after dosing, shown are mean ± SEM, n = 6/group.


( B ) Distribution of APOE4 genotypes in Phase IIa study , vs their baseline Aβ42 / 40 ratios . dashed at Aβ42 / 40 of 0 . 09 represents a cutoff , which separates a normal from an abnormally low .

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(B) Distribution of APOE4 genotypes in the Phase IIa study, vs their baseline Aβ42/40 ratios. The dashed line at Aβ42/40 ratio of 0.09 represents a cutoff, which separates a normal ratio from an abnormally low ratio.


( C ) Comparison of CSF Aβ40 reduction in APOE4 non - carriers APOE4 carriers .

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(C) Comparison of CSF Aβ40 reduction in APOE4 non-carriers and APOE4 carriers.


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(D) Comparison of CSFAβ40 reduction in participants with Aβ42/40 ≥ 0.09 and Aβ42/40 < 0.09.


( E ) Comparison of Aβ42 / 40 at baseline at day 91 for participants in placebo , 35 mg , 85 mg dose group .

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(E) Comparison of Aβ42/40 ratio at baseline and at day 91 for all participants in the placebo, 35 mg, and 85 mg dose group.


( F ) Change of Aβ42 / 40 for participants with Aβ42 / 40 ≤ 0 . 09 at baseline ; bars represent mean ± SD . Full circles : APOE4 carrier , open circles : APOE4 non - carrier .

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(F) Change of Aβ42/40 ratio for all participants with Aβ42/40 ≤ 0.09 at baseline; bars represent mean ± SD. Full circles: APOE4 carrier, open circles: APOE4 non-carrier.


293T were transfected with a plasmid encoding FLAG - Stx17 wild type ( WT ) or indicated constructs . At 24 h after transfection , lysates were immunoprecipitated ( IP ) with anti - FLAG M2 beads , analyzed by IB using antibodies against PGAM5 FLAG . Five of lysates was analyzed as input .

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293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.


HeLa stably expressing FLAG - Stx17 WT or K254C mutant were fixed subjected to PLA using antibodies against FLAG PGAM5 . Scale bar , 5 μm . Values are means ± ( n = 3 ) . * * * P < 0 . 001 as compared with WT ( paired Student ' s t - ) .

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HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).


MBP or MBP - Stx17 constructs attached to amylose resin were mixed with GST - PGAM5 , bound to resin were separated by SDS - PAGE blotted onto PVDF membranes . blots were detected by an anti - GST antibody ( upper panels ) or stained with Coomassie Brilliant Blue R - 250 ( lower panels ) . Ten of used for each experiment was analyzed as input . Asterisks double asterisk may represent MBP dimers degradation products .

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MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.


293T were cotransfected with plasmids encoding FLAG - Stx17 WT indicated PGAM5 - GFP constructs ,

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293T cells were cotransfected with plasmids encoding FLAG-Stx17 WT and the indicated PGAM5-GFP constructs, and analyzed


293T were cotransfected with plasmids encoding FLAG - Stx17 or Stx18 C - terminally GFP - tagged transmembrane domain of PGAM5 （ 1 - 35 ） constructs ,

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293T cells were cotransfected with plasmids encoding FLAG-Stx17 or Stx18 and the C-terminally GFP-tagged transmembrane domain of PGAM5（amino acids 1-35）constructs, and analyzed


HeLa were treated with DMSO ( Vehicle ) or 0 . 03 mg / ml digitonin ( + Digitonin ) , fixed then double immunostained for PGAM5 Tom20 . Scale bar , 5 μm . bar graph on right shows Manders ' coefficients for colocalization of PGAM5 Tom20 . Values are means ± ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student '

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HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's


HeLa stably expressing FLAG - Stx17 wild type ( WT ) were transfected with a plasmid encoding Su9 - GFP ( mitochondria ) or Sec61β - GFP ( ER ) . At 24 h after transfection , were subjected to PLA using antibodies against FLAG PGAM5 . Scale bar , 5 μm . bar graph on right shows Manders ' coefficients for colocalization of PLA dots Su9 - GFP or Sec61β - GFP . Values are means ± ( n = 3 ) . * * * P < 0 . 001 ( paired Student ' s t - ) .

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

HeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test).


HeLa were treated with DMSO ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed subjected to Percoll - fractionation . Equal amounts of were analyzed by IB using indicated antibodies . PNS , postnuclear supernatant ; MS , microsomes ; Mt , mitochondria . amounts of recovered on fractionation were as follows for vehicle CCCP treatment , respectively : PNS ( 6 . 6 mg 5 . 5 mg ) , cytosol ( 4 . 8 mg 4 . 9 mg ) , MS ( 2 . 0 mg 2 . 2 mg ) , MAM ( 0 . 55 mg 0 . 46 mg ) Mt ( 0 . 30 mg 0 . 28 mg ) .

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HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).


Electron microscopic of HeLa expressing PGAM5 - GFP APEX2 - GFP - binding peptide . Arrows indicate position of 3 , 3 ' - diaminobenzidine reaction at ER - mitochondria interface . Scale bar , 500 nm .

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Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.


HeLa stably expressing FLAG - Stx17 WT were mock - transfected or transfected with siRNA for Mfn1 , Mfn2 or PACS - 2 . At 72 h after transfection , were subjected to PLA using antibodies against FLAG PGAM5 . Scale bar , 5 μm . Values are means ± ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - ) .

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HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


HeLa were mock - transfected or transfected with siRNA for Mfn1 , Stx17 , Mfn2 or PACS - 2 . At 72 h after transfection , were subjected to PLA using antibodies against Drp1 PGAM5 . Scale bar , 5 μm . Values are means ± ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - ) .

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

HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


HeLa with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were fixed double immunostained for PGAM5 Tom20 . Scale bar , 5 μm .

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HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.


293T with mock treatment or depleted of Stx17 were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed then analyzed by IB using indicated antibodies .

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

293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.


293T transiently expressing FLAG - Stx17 wild type ( WT ) or K254C mutant were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed , immunoprecipitated with anti - FLAG M2 beads then analyzed by IB using indicated antibodies .

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

293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.


HeLa stably expressing FLAG - Stx17 WT were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , subjected to PLA using antibodies against FLAG PGAM5 . Scale bar , 5 μm . Values are means ± ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student ' s t - ) .

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

HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).


HeLa stably expressing GFP - Parkin with mock treatment ( Control ) or depleted ( KD ) of Stx17 , PGAM5 or Drp1 were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , analyzed by IB using indicated antibodies .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.


HeLa stably expressing GFP - Parkin with mock treatment ( Mock ) or depleted of Stx17 , PGAM5 or Drp1 were incubated in absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h analyzed by immunofluorescence microscopy . Scale bar , 5 μm . bar graph on right shows Manders ' coefficients for colocalization of GFP - Parkin Tom20 . Values are means ± ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - ) .

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 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 O O O O O O O O O

HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


PINK1 - FLAG GFP - Parkin stably expressing HeLa with mock treatment or depleted of Stx17 or PGAM5 were incubated in absence ( ethanol ) or presence of 20 μM CCCP for 2 h analyzed by IB using indicated antibodies . F , full - PINK1 ; C , PINK1 .

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PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.


GFP - Parkin stably expressing HeLa were mock - transfected ( Mock ) or transfected with siRNA ( KD ) for Stx17 or PGAM5 . At 48 h after siRNA transfection , were transfected with a plasmid encoding FLAG - DFCP1 , incubated for 24 h , treated with 20 μM CCCP for 2 h then immunostained for FLAG . Scale bars , 5 μm . bar graph below shows Manders ' coefficients for colocalization of GFP - Parkin FLAG - DFCP1 . Values are means ± ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - ) .

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GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).


GFP - Parkin stably expressing HeLa with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h immunostained for LC3 . Scale bar , 5 μm . bar graph below shows Manders ' coefficients for colocalization of GFP - Parkin LC3 . Values are means ± ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - ) .

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

GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).


GFP - Parkin stably expressing HeLa with mock treatment or depleted of Stx17 or PGAM5 were incubated in absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h analyzed by IB using indicated antibodies .

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GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.


HeLa stably expressing GFP - Parkin with mock treatment ( Control ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , analyzed by IB using indicated antibodies .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.


GFP - Parkin stably expressing HeLa with mock treatment ( Mock ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated with 20 μM CCCP for 2 h immunostained for Tom20 . Scale bar , 5 μm .

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GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.


293T with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were incubated in absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h , immunoprecipitated with anti - FLAG M2 beads then analyzed by IB using indicated antibodies . Three of lysates was analyzed as input . During Stx17 knockdown , were transfected with a plasmid encoding FLAG - FUNDC1 .

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

293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.


GFP - Parkin stably expressing HeLa were treated then subjected to PLA using antibodies against FLAG PGAM5 . During Stx17 knockdown , were transfected with a plasmid encoding FLAG - FUNDC1 . Scale bar , 5 μm . Values are means ± ( n = 3 ) . * P < 0 . 05 * * P < 0 . 01 as compared with Mock ( + CCCP ) by paired Student ' s t - .

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

GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.


A PGAM5 overexpression partially rescues mitochondrial defects by Stx17 loss . Transmission electron microscope of indirect flight muscle in indicated genotypes ( a , Control ; b , Stx17 - / - ; c , Stx17 - / - Stx17 overexpression ; d , Stx17 - / - PGAM5 overexpression ) of 7 - day - old adult flies are shown . bar graph on right shows frequency of healthy abnormal mitochondria presented as percentages ( mean ± ) using scoring system : Class 0 , normal ; Class 1 , fuzzy or dilated cristae ; Class 2 , fragmented cristae loss of electron density . * p < 0 . 05 , # p < 0 . 001 vs . same class of control ( Dunnett ' s ) . n = 50 - 119 mitochondria from three or four independent samples . Genotypes used were : + / y ; Act5c - GAL4 / + ( a ) , + / y ; Act5c - GAL4 / + ; Stx17LL06330 / Stx17LL06330 ( b ) , + / y ; Act5c - GAL4 / UAS - FLAG - Stx17 ; Stx17LL06330 / Stx17LL06330 ( c ) , + / y ; Act5c - GAL4 / UAS - PGAM5 ; Stx17LL06330 / Stx17LL06330 ( d ) . Scale bars , 200 nm .

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

A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17-/-; c, Stx17-/- and Stx17 overexpression; d, Stx17-/- and PGAM5 overexpression) of 7-day-old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system : Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *p < 0.05, #p < 0.001 vs. the same class of control (Dunnett's test). n = 50-119 mitochondria from three or four independent samples. Genotypes used were: +/y; Act5c-GAL4/+ (a), +/y; Act5c-GAL4/+; Stx17LL06330/Stx17LL06330 (b), +/y; Act5c-GAL4/UAS-FLAG-Stx17; Stx17LL06330/Stx17LL06330 (c), +/y; Act5c-GAL4/UAS-PGAM5; Stx17LL06330/Stx17LL06330 (d). Scale bars, 200 nm.


B Simultaneous reduction of Stx17 PGAM5 results in mitochondrial degeneration . a , Stx17 + / - ; b , PGAM5 - / - ; c , Stx17 + / - , PGAM5 - / - . bar graph on right represents mitochondrial classified p < 0 . 05 , * * p < 0 . 01 vs . same class of Stx17 + / - ( Dunnett ' s ) . n = 62 - 195 mitochondria from three independent samples . Scale bars = 1 µm . Genotypes used were : + / y ; Act5c - GAL4 / + ; Stx17LL06330 / + ( a ) , PGAM51 / y ; Act5c - GAL4 / + ( b ) , PGAM51 / y ; Act5c - GAL4 / UAS - Stx17 - FLAG ; Stx17LL06330 / + ( c ) .

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

B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).


A ) Single dose AR - 42 plasma tissue pharmacokinetic study . - free CD2F1 mice were administered a single dose of 10 , 20 or 50 mg / kg AR - 42 ( n = 3 ) plasma ( dashed ) gastrocnemius ( solid ) tissue were analyzed for AR - 42 content at different times using LC - MS / MS analyses according to Materials Methods ( Mean ± SD ) .

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

A) Single dose AR-42 plasma and tissue pharmacokinetic study. Tumor-free CD2F1 mice were administered a single dose of 10, 20 or 50 mg/kg AR-42 (n=3) and plasma (dashed) and gastrocnemius (solid) tissue were analyzed for AR-42 content at different times using LC-MS/MS analyses according to the Materials and Methods (Mean±SD).


B ) AR - 42 dose - response . Starting six days after C - 26 injection , received vehicle or AR - 42 orally at 1 , 3 , 10 or 20 mg / kg daily or 50 mg / kg every other day for 13 days . terminal ( Day 18 post - injection ) weights corrected for mass according to Materials Methods are compared to - free controls . Groups : 1mg / kg ( n = 8 ) , 3mg / kg ( n = 8 ) , 10mg / kg ( n = 13 ) , 20mg / kg ( n = 6 ) , 50mg / kg ( n = 5 ) . Dashed reference ( 100 % ) represents mean - free control value , solid represents non - linear fit of dose - response data .

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

B) AR-42 dose-response. Starting six days after C-26 cell injection, animals received vehicle or AR-42 orally at 1, 3, 10 or 20 mg/kg daily or 50 mg/kg every other day for 13 days. Individual animal terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor-free controls. Groups: 1mg/kg (n=8), 3mg/kg (n=8), 10mg/kg (n=13), 20mg/kg (n=6), 50mg/kg (n=5). Dashed reference line (100%) represents the mean tumor-free control value, solid line represents non-linear fit of dose-response data.


Study 1 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 . C ) : Terminal ( Day 18 post - injection ) weights corrected for mass according to Materials Methods compared to - free controls ( mean ± SD ) .

O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-ORGANISM 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-CELL_LINE I-CELL_LINE I-CELL_LINE 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

Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. C): Terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).


Study 1 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 . D ) Terminal volumes ( mean ± SD ) .

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

Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) Terminal tumor volumes (mean±SD).


Study 1 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 . E ) Terminal gastrocnemius ( mean ± SD ) F ) quadriceps muscle mass ( mean ± SD ) compared to - free controls .

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

Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. E) Terminal gastrocnemius (mean±SD) and F) quadriceps muscle mass (mean±SD) compared to tumor-free controls.


Study 2 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 . A ) Terminal weights corrected for mass according to Materials Methods compared to - free controls ( mean ± SD ) .

O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-ORGANISM 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-CELL_LINE I-CELL_LINE I-CELL_LINE 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

Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).


Study 2 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 . B ) Terminal gastrocnemius ( mean ± SD ) C ) quadriceps muscle mass ( mean ± SD ) compared to - free controls .

Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.


Study 2 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 . D ) grip strength ( mean ± SD ) compared to - free controls . Statistics : V , G , A indicate significant differences versus - bearing vehicle - treated , - bearing GTx - 024 - treated - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S2A - D , one - way ANOVA followed by Tukey ' s multiple comparison .

O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-ORGANISM 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-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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

Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S2A-D, one-way ANOVA followed by Tukey's multiple comparison test.


E ) Survival analyses of Study 2 performed as outlined in Materials Methods . Statistics : V , G indicate significant differences versus - bearing vehicle - treated - bearing GTx - 024 - treated groups , respectively . p = 0 . 0005 for Combo vs . V , p = 0 . 0064 for Combo vs . G , Logrank ( Mantel - Cox ) using Bonferroni corrected threshold for multiple comparisons .

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 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

E) Survival analyses of Study 2 animals performed as outlined in the Materials and Methods. Statistics: V, G indicate significant differences versus tumor-bearing vehicle-treated and tumor-bearing GTx-024-treated groups, respectively. p=0.0005 for Combo vs. V, p=0.0064 for Combo vs. G, Logrank (Mantel-Cox) test using Bonferroni corrected threshold for multiple comparisons.


Study 3 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 . A ) Terminal weights corrected for mass according to Materials Methods compared to - free controls ( mean ± SD ) .

O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE 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

Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD) .


Study 3 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 . B ) Terminal gastrocnemius ( mean ± SD ) C ) quadriceps muscle mass ( mean ± SD ) compared to - free controls .

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

Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.


Study 3 , - bearing mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 . D ) grip strength ( mean ± SD ) compared to - free controls . Statistics : V , G , A indicate significant differences versus - bearing vehicle - treated , - bearing GTx - 024 - treated - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S3A - D , one - way ANOVA followed by Tukey ' s multiple comparison .

O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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

Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S3A-D, one-way ANOVA followed by Tukey's multiple comparison test.


Study 4 , - bearing mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 DHT ( 10 mg / kg oral gavage 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after injection . A ) Terminal weights corrected for mass according to Materials Methods compared to - free controls ( mean ± SD ) .

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

Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).


Study 4 , - bearing mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 DHT ( 10 mg / kg oral gavage 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after injection . B ) Terminal gastrocnemius ( mean ± SD ) C ) quadriceps muscle mass ( mean ± SD ) compared to - free controls .

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

Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.


Study 4 , - bearing mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 DHT ( 10 mg / kg oral gavage 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after injection . D ) grip strength ( mean ± SD ) compared to - free controls . Statistics : V , T , A indicate significant differences versus - bearing vehicle - treated , - bearing TFM - 4AS - 1 - treated - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S4A - D , Tukey ' s multiple comparison .

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

Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, T, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing TFM-4AS-1-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S4A-D, Tukey's multiple comparison test.


A ) Gene expression of multiple cachexia - associated markers in gastrocnemius of from Study 1 ( n = 5 for - bearing groups ; n = 6 for - free groups ) . Expression was determined by qRT - PCR presented as described in Materials Methods ( Geometric Mean ± Geometric STD ) . B ) Androgen receptor ( AR ) mRNA expression in gastrocnemius from Study 1 . Statistics : p values provided in Appendix Table S5A - B . Dunnett ' s multiple comparison . CEBPδ , n = 4 , insufficient sample to analyze - bearing GTx - 024 - treated .

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

A) Gene expression of multiple cachexia-associated markers in gastrocnemius muscles of individual animals from Study 1 (n=5 for tumor-bearing groups; n=6 for tumor-free groups). Expression was determined by qRT-PCR and presented as described in the Materials and Methods (Geometric Mean ± Geometric STD). B) Androgen receptor (AR) mRNA expression in gastrocnemius muscles from Study 1. Statistics: p values provided in Appendix Table S5A-B. Dunnett's multiple comparison test. CEBPδ, n=4, insufficient sample to analyze all tumor-bearing GTx-024-treated animals.


A ) ELISA of serum IL - 6 in terminal samples from Study 2 . - free groups ( n = 6 ) , - bearing receiving Vehicle ( n = 7 ) , GTx - 024 ( n = 10 ) , AR - 42 ( n = 8 ) or Combination ( n = 9 ) . ND , not detected .

O O B-EXP_ASSAY O B-TISSUE 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 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

A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected.


B C ) Phospho ( p ) STAT3 ( Y705 ) / total ( t ) STAT3 western blot of gastrocnemius tissues from treated in Study 1 ( B ) Study 3 ( C ) . Black circle - - free , blue square - - free / GTx - 024 , black triangle - - bearing , blue diamond - / GTx - 024 , red triangle - / AR - 42 , green circle - / combo .

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

B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo.


D ) Spleen weights normalized to - corrected terminal weights of mice from Study 2 . - free groups ( n = 6 ) , - bearing Vehicle ( n = 7 ) , - bearing receiving GTx - 024 ( n = 10 ) , AR - 42 ( n = 9 ) or Combination ( n = 9 ) .

O O B-TISSUE B-EXP_ASSAY O O O O O B-EXP_ASSAY 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 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

D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9).


E ) ELISA of pSTAT3 ( Y705 ) / tSTAT3 within C - 26 tumors from Study 2 . - bearing Vehicle - treated ( n = 7 ) , - bearing receiving GTx - 024 , AR - 42 or Combination ( n = 8 ) .

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

E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8).


A ) Effect of GTx - 024 AR - 42 monotherapies on cachexia - related differentially regulated genes ( DEGs ) from - seq analyses of Study 1 gastrocnemius . three panels consist of genes plotted with respect to their log2 fold change - log10 Benjamini - Hochburg adjusted p - values from comparison of cachexia vs - free controls . Colors of points reflect DEG status of each gene for given comparison .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O 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-DISEASE O O O O O O O O O O O O O O O O O O

A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison.


C ) Significance values from GSEA using combined STAT3 gene sets identified in B . Each treatment group is compared to - bearing control ( cachexia ) transcriptomes .

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 B-DISEASE O B-EXP_ASSAY O

C) Significance values from GSEA using combined STAT3 gene sets identified in B. Each treatment group is compared to tumor-bearing control (cachexia) transcriptomes.


D ) Heat map of DEGs within combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq data . - free control ( black checkered ) , GTx - 024 - treated - free ( blue checkered ) , - bearing control ( black ) , GTx - 024 - treated - bearing ( blue ) , AR - 42 - treated - bearing ( red ) Combination - treated - bearing ( green ) .

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

D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).


E ) Results of GSEA using combined ATF - 1 gene sets identified in B across treatment groups versus - bearing control ( cachexia ) transcriptomes .

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 B-DISEASE O B-EXP_ASSAY O

E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes.


- free control ( black checkered ) , GTx - 024 - treated - free ( blue checkered ) , - bearing control ( black ) , GTx - 024 - treated - bearing ( blue ) , AR - 42 - treated - bearing ( red ) Combination - treated - bearing ( green ) . F ) Heat map of DEGs within combined ATF - 1 gene sets ( mean z score ) . Treatment groups are as in D .

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

Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D.


G ) mRNA expression of mediators of IL - 6 signaling upstream of STAT3 . Data presented as mean ± SD of per log - transformed fold change ( log2FC ) values versus - free controls . Groups : - free control ( n = 6 ) , - free GTx - 024 treated ( n = 5 ) , - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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

G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


A ) Volcano plot from - seq analyses of Study 1 gastrocnemius for - bearing combination - treated mice versus - bearing controls . Genes not differentially expressed in this comparison are indicated with blue . remaining genes ( red green ) are DEGs in combination - treated versus - bearing control comparison . green coloring indicates subset of these DEGs that are not also differentially expressed in comparison of AR - 42 - treated - bearing mice versus - bearing controls , suggesting these genes are responsive to only combination therapy . Log2 - transformed fold change ( FC ) in expression is plotted on x - axis - log10 transformed Benjamini - Hochburg adjusted p - values are plotted on y - axis .

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

A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis.


C ) Significance values from GSEA using combined CTNNB1 gene sets . Each treatment group was compared to - free control transcriptomes . * FDR < 1e - 5 determined , set to 1e - 5 for plot .

O O O O O O O O B-GENEPROD 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

C) Significance values from GSEA using combined CTNNB1 gene sets. Each treatment group was compared to tumor-free control transcriptomes. *FDR < 1e-5 determined, set to 1e-5 for plot.


D ) Enrichment plots from GSEA of CTNNB1 gene set for each treatment group versus - free control comparisons . GTx - 024 - treated - free ( blue checkered ) , - bearing control ( black ) , GTx - 024 - treated - bearing ( blue ) , AR - 42 - treated - bearing ( red ) Combination - treated - bearing ( green ) .

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

D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).


E ) mRNA expression of WNT effectors upstream of β - catenin . Data are presented as mean ± SD of log - transformed fold change ( log2FC ) values versus - free controls . Groups : - free control ( n = 6 ) , - free GTx - 024 treated ( n = 5 ) , - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

O O B-EXP_ASSAY I-EXP_ASSAY 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 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 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

E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


A) Neutrophils and lymphocytes count.


B ) of creatine kinase ( CK ) creatinine .

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

B) Levels of creatine kinase (CK) and creatinine.


C ) of procalcitonin ( PCT ) C - reactive ( CRP ) .

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

C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).


D ) of erythrocyte sedimentation rate ( ESR ) ferritin .

D) Levels of erythrocyte sedimentation rate (ESR) and ferritin.


E ) of D - dimer lactate dehydrogenase ( LDH ) .

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

E) Levels of D-dimer and lactate dehydrogenase (LDH).


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

F) Proportion of total CD3+ and CD4+ T cells.


G ) Proportion of CD8 + T CD4 + / CD8 + T .

G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio.


H ) Proportion of B natural killer ( NK ) .

H) Proportion of B and natural killer (NK) cells.


cytokine profile of COVID - 19 for IL - 2 , IL - 4 ( I )

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

cytokine profile of COVID-19 patients for IL-2, IL-4 (I)


cytokine profile of COVID - 19 for , IL - 6 , IL - 10 ( J ) TNF - α IFN - γ ( K ) .

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cytokine profile of COVID-19 patients for , IL-6, IL-10 (J) and TNF-α and IFN-γ (K).


Time from symptom onset to initial diagnosis ( TFSD ) .

Time from symptom onset to initial diagnosis (TFSD).


Time from symptom onset to development of pneumonia ( TFSP ) .

Time from symptom onset to development of pneumonia (TFSP).


Time from symptom onset to treatment ( TFST ) .

Time from symptom onset to treatment (TFST).


Time from development of pneumonia to recovery ( TFPR ) .

Time from development of pneumonia to recovery (TFPR).


Correlation between IL - 6 maximal during hospitalization .

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Correlation between IL-6 and maximal body temperature during hospitalization.


Correlation between IL - 6 C - reactive ( CRP ) .

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Correlation between IL-6 and C-reactive protein (CRP).


Correlation between IL - 6 lactate dehydrogenase ( LDH ) .

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Correlation between IL-6 and lactate dehydrogenase (LDH).


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Correlation between IL-6 and D-dimer.


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Correlation between IL-6 and ferritin.


of baseline IL - 6 in who received glucocorticoids , human immunoglobulin , high flow oxygen inhalation or mechanical ventilation during hospitalization versus who did not . Error bars , .

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Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.


IL - 6 before after treatment in three groups classified according to baseline IL - 6 of ≥ 40 pg / mL ( n = 11 ) , ≥ 20 pg / mL ( n = 6 ) or < 20 pg / mL ( n = 9 ) ( left ) . Two representative chest computed tomography ( CT ) scans before after treatment from each group ( right ) .

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IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right).


IL - 6 at diagnosis after disease progression in three exacerbated ( left ) . Progressed radiological findings were recorded in two ( right ) , while radiological assessment after treatment was not performed in third due to poor general condition .

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IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition.


baseline IL - 6 level was 197 . 39 pg / mL in a 69 - year - old who showed high fever dyspnea . IL - 6 decreased to 9 . 47 pg / mL after treatment ( day 8 ) , while symptoms were not relieved . C - reactive ( CRP ) rebounded procalcitonin ( PCT ) increased together with disease exacerbation . Follow - up chest computed tomography ( CT ) assessment was not performed due to poor general condition , whereas chest X - ray showed aggravated pneumonia . Follow - up sputum culture confirmed exacerbation was caused by infection .

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The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.


A ) chest computed tomography ( CT ) was normal five days after symptom onset . Disease aggravation was evidenced by progressed chest CT ten days after symptom onset displaying bilateral multiple patchy ground glass opacities . Disease alleviation was evidenced by improved chest CT 25 days after symptom onset .

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A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset.


B ) Procalcitonin ( PCT ) stayed within normal range throughout course of disease .

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B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.


C ) Lymphocyte was normal five days after symptom onset , while firstly decreasing then returning to normal range during course of disease .

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C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease.


Both abnormal C - reactive ( CRP ) ( D ) were detected five days after symptom onset when chest CT lymphocyte were still normal . In association with changes in chest CT scans , CRP further increased ten days after symptom onset returned to normal range 25 days after symptom onset .

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Both abnormal C-reactive protein (CRP) (D) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.


IL - 6 ( E ) were detected five days after symptom onset when chest CT lymphocyte were still normal . In association with changes in chest CT scans , IL - 6 further increased ten days after symptom onset returned to normal range 25 days after symptom onset .

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IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.


F ) was elevated when CRP IL - 6 were rising , but returned to normal when CRP IL - 6 . were declining .

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F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.