Relative expression of leptin ( C ) , TNF - α ( D ) and IL - 6 ( E ) , respectively , in white adipose tissue of mice ( n = 7 Veh ; 9 AβOs ) 7 days after i . c . v . injection of vehicle or AβOs . In ( C ) , * P = 0 . 0394 ; in ( D ) , * * P = 0 . 0038 ; in ( E ) , * P = 0 . 0305 ; Student ' s t - test .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative expression of leptin (C), TNF-α (D) and IL-6 (E), respectively, in white adipose tissue of mice (n = 7 Veh; 9 AβOs) 7 days after i.c.v. injection of vehicle or AβOs. In (C), *P = 0.0394; in (D), **P = 0.0038; in (E), *P = 0.0305; Student's t-test.


p - JNK ( F ; n = 5 animals / group ) and IRS - 1pSer312 ( G ; n = 6 animals / group ) levels ( normalized by total JNK and total IRS - 1 , respectively ) in skeletal muscle of mice 7 days after i . c . v . injection of vehicle or AβOs . In ( F ) , * P = 0 . 0464 ; in ( G ) , * P = 0 . 0081 ; Student ' s t - test .

O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

p-JNK (F; n = 5 animals/group) and IRS-1pSer312 (G; n = 6 animals/group) levels (normalized by total JNK and total IRS-1, respectively) in skeletal muscle of mice 7 days after i.c.v. injection of vehicle or AβOs. In (F), *P = 0.0464; in (G), *P = 0.0081; Student's t-test.


Representative images of GLUT - 4 immunofluorescence in insulin - stimulated skeletal muscle from mice that were i . c . v . - injected with vehicle ( Veh ) or 10 pmol AβOs . Bar graphs show quantification of GLUT - 4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin ( 1 IU / kg body weight ) 7 days after i . c . v . injection of vehicle or AβOs , as indicated ( n = 5 animals / group ) . Scale bar = 25 μm . * P = 0 . 0144 , one - way ANOVA followed by Bonferroni post hoc test .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of GLUT-4 immunofluorescence in insulin-stimulated skeletal muscle from mice that were i.c.v.-injected with vehicle (Veh) or 10 pmol AβOs. Bar graphs show quantification of GLUT-4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin (1 IU/kg body weight) 7 days after i.c.v. injection of vehicle or AβOs, as indicated (n = 5 animals/group). Scale bar = 25 μm. *P = 0.0144, one-way ANOVA followed by Bonferroni post hoc test.


GLUT - 4 mRNA ( n = 4 animals / group ) and total protein levels ( normalized to actin levels ; n = 5 Veh ; 6 AβOs ) were unchanged in skeletal muscle of Swiss mice injected with vehicle ( Veh ) or 10 pmol AβOs .

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GLUT-4 mRNA (n = 4 animals/group) and total protein levels (normalized to actin levels; n = 5 Veh; 6 AβOs) were unchanged in skeletal muscle of Swiss mice injected with vehicle (Veh) or 10 pmol AβOs.


Plasma levels of insulin ( K ; n = 12 animals / group ) , leptin ( L ; n = 11 Veh ; 12 AβOs ) , cholesterol ( M ; n = 8 Veh ; 6 AβOs ) , triglycerides ( N ; n = 8 Veh ; 6 AβOs ) or noradrenaline ( O ; n = 7 Veh ; 8 AβOs ) measured 7 days after i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs . In ( O ) , * P = 0 . 0361 , Student ' s t - test .

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

Plasma levels of insulin (K; n = 12 animals/group), leptin (L; n = 11 Veh; 12 AβOs), cholesterol (M; n = 8 Veh; 6 AβOs), triglycerides (N; n = 8 Veh; 6 AβOs) or noradrenaline (O; n = 7 Veh; 8 AβOs) measured 7 days after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. In (O), *P = 0.0361, Student's t-test.


Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle ( Veh ) or AβOs ( 500 nM ) for 3 h . Binding of AβOs was detected using anti - oligomer monoclonal antibody NU4 ( red ) . Neurons were double - labeled using MAP - 2 antibody ( green ) . Images represent typical results from experiments with three independent hypothalamic cultures ( three coverslips / experimental condition per independent experiment ) . Scale bar = 30 and 10 μm for main panels and insets , respectively .

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Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h. Binding of AβOs was detected using anti-oligomer monoclonal antibody NU4 (red). Neurons were double-labeled using MAP-2 antibody (green). Images represent typical results from experiments with three independent hypothalamic cultures (three coverslips/experimental condition per independent experiment). Scale bar = 30 and 10 μm for main panels and insets, respectively.


Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs ( 500 nM ) and immunolabeled with anti - GFAP ( green ) and NU4 ( red ) antibodies . Insets show AβOs binding to neuronal dendrites , whereas no binding was detected to GFAP - positive cells .

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Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs (500 nM) and immunolabeled with anti-GFAP (green) and NU4 (red) antibodies. Insets show AβOs binding to neuronal dendrites, whereas no binding was detected to GFAP-positive cells.


Representative DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs ( 500 nM , 4 h ) . Insets show optical zoom images of the indicated areas . Scale bars = 100 and 50 μm for main panels and insets , respectively . Graph shows integrated DCFfluorescence intensities ( relative units ; see Materials and Methods ) ( n = 3 independent hypothalamic cultures ; three wells / experimental condition per experiment ; three images acquired per well ) . Bars represent means ± SEM . # P = 0 . 0604 , one - sample t - test compared with a fixed value of 100 RUs .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs (500 nM, 4 h). Insets show optical zoom images of the indicated areas. Scale bars = 100 and 50 μm for main panels and insets, respectively. Graph shows integrated DCFfluorescence intensities (relative units; see Materials and Methods) (n = 3 independent hypothalamic cultures; three wells/experimental condition per experiment; three images acquired per well). Bars represent means ± SEM. #P = 0.0604, one-sample t-test compared with a fixed value of 100 RUs.


LDH activity ( IU / l ) in culture media of hypothalamic cultures exposed to vehicle or AβOs ( 500 nM , 3 h ) .

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LDH activity (IU/l) in culture media of hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h).


Representative immunofluorescence images of eIF2α - P in hypothalamic cultures exposed to vehicle or AβOs ( 500 nM , 3 h ) in the absence or presence of infliximab ( 1 μg / ml ) . Scale bar = 30 μm . Graph represents integrated immunofluorescence intensities of eIF2α - P levels from three independent hypothalamic cultures ( three coverslips / experimental condition per experiment , 20 images per coverslip ) . Bars represent means ± SEM . * P = 0 . 0489 , one - way ANOVA followed by Bonferroni post hoc test comparing AβO - treated versus vehicle - treated cultures .

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

Representative immunofluorescence images of eIF2α-P in hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Scale bar = 30 μm. Graph represents integrated immunofluorescence intensities of eIF2α-P levels from three independent hypothalamic cultures (three coverslips/experimental condition per experiment, 20 images per coverslip). Bars represent means ± SEM. *P = 0.0489, one-way ANOVA followed by Bonferroni post hoc test comparing AβO-treated versus vehicle-treated cultures.


Representative images of hypothalamic cultures exposed to AβOs ( 500 nM , 3 h ) and double - labeled with NU4 ( oligomer - sensitive ) and eIF2α - P antibodies . Arrow points to a neuron presenting high levels of eIF2α - P in the absence of AβO binding . Nuclear staining ( DAPI ) is shown in blue . Scale bar = 30 μm .

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Representative images of hypothalamic cultures exposed to AβOs (500 nM, 3 h) and double-labeled with NU4 (oligomer-sensitive) and eIF2α-P antibodies. Arrow points to a neuron presenting high levels of eIF2α-P in the absence of AβO binding. Nuclear staining (DAPI) is shown in blue. Scale bar = 30 μm.


Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs ( 500 nM , 3 h ) in the absence or presence of infliximab ( 1 μg / ml ) . Similar patterns of AβO binding were observed in both conditions . Scale bar = 20 μm .

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Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Similar patterns of AβO binding were observed in both conditions. Scale bar = 20 μm.


Western blot analysis of eIF2α - P levels in the hypothalamus of mice 4 h ( A ; n = 4 animals / group ) or 7 days ( B ; n = 6 Veh ; 5 AβOs ) after a single i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs . Graphs show densitometric data normalized by total eIF2α levels . * P = 0 . 0213 .

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

Western blot analysis of eIF2α-P levels in the hypothalamus of mice 4 h (A; n = 4 animals/group) or 7 days (B; n = 6 Veh; 5 AβOs) after a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. Graphs show densitometric data normalized by total eIF2α levels. *P = 0.0213.


Western blot analysis of ATF4 levels in the hypothalamus of mice 4 h after i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs ; graph shows densitometric data normalized by β - actin ( n = 7 Veh ; 8 AβOs ) . # P = 0 . 0731 ; Student ' s t - test .

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Western blot analysis of ATF4 levels in the hypothalamus of mice 4 h after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs; graph shows densitometric data normalized by β-actin (n = 7 Veh; 8 AβOs). #P = 0.0731; Student's t-test.


Western blot analysis of hypothalamic phospho - IKKβ levels in the hypothalamus of mice 4 h ( C ; n = 6 animals / group ) or 7 days ( D ; n = 4 Veh ; 5 AβOs ) after i . c . v . injection of vehicle or 10 pmol AβOs . Graphs show densitometric data normalized by total IKKβ levels . In ( D ) , * P = 0 . 0437 ; in ( E ) , * P = 0 . 0444 ; Student ' s t - test .

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

Western blot analysis of hypothalamic phospho-IKKβ levels in the hypothalamus of mice 4 h (C; n = 6 animals/group) or 7 days (D; n = 4 Veh; 5 AβOs) after i.c.v. injection of vehicle or 10 pmol AβOs. Graphs show densitometric data normalized by total IKKβ levels. In (D), *P = 0.0437; in (E), *P = 0.0444; Student's t-test.


Western blot analysis of IκBα ( F ; n = 6 animals / group ) and cytoplasmic phospho - p65 - NF - κB ( G ; n = 4 Veh ; 5 AβOs ) in the hypothalamus of mice 4 h after i . c . v . injection of vehicle or 10 pmol AβOs . Graphs show densitometric data normalized by actin ( F ) or total NF - κB levels ( G ) . * P = 0 . 0207 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

Western blot analysis of IκBα (F; n = 6 animals/group) and cytoplasmic phospho-p65-NF-κB (G; n = 4 Veh; 5 AβOs) in the hypothalamus of mice 4 h after i.c.v. injection of vehicle or 10 pmol AβOs. Graphs show densitometric data normalized by actin (F) or total NF-κB levels (G). *P = 0.0207.


Nuclear NF - κB levels in the hypothalamus 6 h after i . c . v . injection of vehicle or 10 pmol AβOs in mice . Graphs show NF - κB levels normalized by nuclear marker , lamin B ( n = 6 animals / group ) . * * P = 0 . 0024 ; Student ' s t - test .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Nuclear NF-κB levels in the hypothalamus 6 h after i.c.v. injection of vehicle or 10 pmol AβOs in mice. Graphs show NF-κB levels normalized by nuclear marker, lamin B (n = 6 animals/group). **P = 0.0024; Student's t-test.


IRS - 1pSer636 ( I ; n = 4 animals / group ) and pTyr465 ( J ; n = 6 Veh ; 4 AβOs ) levels in the hypothalamus 7 days after i . c . v . injection of vehicle or AβOs in mice . Graphs show IRS - 1pSer or IRS - 1pTyr levels normalized by total IRS - 1 . In ( I ) , * P = 0 . 0043 ; in ( J ) , * P = 0 . 0275 ; Student ' s t - test .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRS-1pSer636 (I; n = 4 animals/group) and pTyr465 (J; n = 6 Veh; 4 AβOs) levels in the hypothalamus 7 days after i.c.v. injection of vehicle or AβOs in mice. Graphs show IRS-1pSer or IRS-1pTyr levels normalized by total IRS-1. In (I), *P = 0.0043; in (J), *P = 0.0275; Student's t-test.


Twelve - hour food intake after a single i . c . v . infusion of insulin ( 200 mU ) in mice . Experiment was performed 7 days after i . c . v . injection of vehicle or AβOs ( n = 5 PBS ; 5 Veh + Insulin ; 9 AβOs + Insulin ) , data are representative of two independent experiments with similar results . * * * P < 0 . 0001 , one - way ANOVA followed by Bonferroni post hoc test comparing Veh - Insulin versus PBS groups .

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Twelve-hour food intake after a single i.c.v. infusion of insulin (200 mU) in mice. Experiment was performed 7 days after i.c.v. injection of vehicle or AβOs (n = 5 PBS; 5 Veh + Insulin; 9 AβOs + Insulin), data are representative of two independent experiments with similar results. ***P < 0.0001, one-way ANOVA followed by Bonferroni post hoc test comparing Veh-Insulin versus PBS groups.


Representative images of AβO immunoreactivity ( detected using anti - oligomer monoclonal antibody NU4 ) in the hypothalamus of control , sham - operated adult cynomolgus macaques ( Sh ; n = 3 ) or macaques that received i . c . v . injections of AβOs ( n = 3 ; see Materials and Methods ) . Nuclear staining ( DAPI ) is shown in blue . Insets show optical zoom images of the areas indicated by white dashed rectangles in the main panels . Scale bars = 100 and 20 μm for main panels and insets , respectively .

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Representative images of AβO immunoreactivity (detected using anti-oligomer monoclonal antibody NU4) in the hypothalamus of control, sham-operated adult cynomolgus macaques (Sh; n = 3) or macaques that received i.c.v. injections of AβOs (n = 3; see Materials and Methods). Nuclear staining (DAPI) is shown in blue. Insets show optical zoom images of the areas indicated by white dashed rectangles in the main panels. Scale bars = 100 and 20 μm for main panels and insets, respectively.


Representative images showing eIF2α - P ( B ) , phospho - IKKβ ( C ) and IκBα ( D ) immunoreactivities in the hypothalamus of cynomolgus macaques that received i . c . v . injections of AβOs or control ( sham - operated ; Sh ) macaques ( n = 3 animals / group ) . Graphs show immunolabeling optical density analysis from three images acquired in the hypothalamus of each macaque ( three control versus three AβO - injected animals ) . In ( B ) , # P = 0 . 0523 ; in ( C ) # P = 0 . 1123 ; unpaired Student ' s t - test with Welch ' s correction for unequal variances ; AβO - injected monkeys compared to sham - operated monkeys . Scale bars = 50

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

Representative images showing eIF2α-P (B), phospho-IKKβ (C) and IκBα (D) immunoreactivities in the hypothalamus of cynomolgus macaques that received i.c.v. injections of AβOs or control (sham-operated; Sh) macaques (n = 3 animals/group). Graphs show immunolabeling optical density analysis from three images acquired in the hypothalamus of each macaque (three control versus three AβO-injected animals). In (B), #P = 0.0523; in (C) #P = 0.1123; unpaired Student's t-test with Welch's correction for unequal variances; AβO-injected monkeys compared to sham-operated monkeys. Scale bars = 50 μm


Accumulated chow intake ( normalized by body weight ) measured during 7 days following a single i . c . v . injection of vehicle or 10 pmol AβOs in mice ( n = 13 Veh ; 10 AβOs ; data are representative of two independent experiments with similar results ) . * * * P < 0 . 0001 ; Student ' s t - test .

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Accumulated chow intake (normalized by body weight) measured during 7 days following a single i.c.v. injection of vehicle or 10 pmol AβOs in mice (n = 13 Veh; 10 AβOs; data are representative of two independent experiments with similar results). ***P < 0.0001; Student's t-test.


Daily body weight measured during 7 days after i . c . v . injection of vehicle or AβOs ( n = 7 animals / group ; data are representative of two independent experiments with similar results ) .

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Daily body weight measured during 7 days after i.c.v. injection of vehicle or AβOs (n = 7 animals/group; data are representative of two independent experiments with similar results).


Adult Swiss mice received a single i . c . v . injection of vehicle or 10 pmol AβOs , and hypothalamic levels of mRNA for AgRP ( C ; n = 6 Veh ; 5 AβOs ) , NPY ( D ; n = 6 Veh ; 5 AβOs ) and POMC ( E ; n = 7 animals / group ) were analyzed 7 days after injection . In ( C ) , * P = 0 . 0191 ; in ( D ) , * P = 0 . 0115 ; Student ' s t - test .

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Adult Swiss mice received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (C; n = 6 Veh; 5 AβOs), NPY (D; n = 6 Veh; 5 AβOs) and POMC (E; n = 7 animals/group) were analyzed 7 days after injection. In (C), *P = 0.0191; in (D), *P = 0.0115; Student's t-test.


Swiss mice received a single i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs , and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection . Representative images of Fluorojade staining in the hypothalamus of vehicle - or AβO - injected mice ( n = 4 / group ) . Scale bar = 100 μm in left panels ( top and bottom ) and 20 μm in right panels ( top and bottom ) . Positive control ( bottom left panel ) was the hippocampus of a mouse that received one i . c . v . injection of quinolinic acid ( 36 . 8 nmol ) and was analyzed 24 h after .

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Swiss mice received a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs, and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection. Representative images of Fluorojade staining in the hypothalamus of vehicle- or AβO-injected mice (n = 4/group). Scale bar = 100 μm in left panels (top and bottom) and 20 μm in right panels (top and bottom). Positive control (bottom left panel) was the hippocampus of a mouse that received one i.c.v. injection of quinolinic acid (36.8 nmol) and was analyzed 24 h after.


Glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) in mice that received i . c . v . injections of vehicle , vehicle + TUDCA , AβOs or AβOs + TUDCA ( when used , TUDCA was administered in 5 i . c . v . injections of 5 μg TUDCA each , before and after oligomer injection ; see Materials and Methods . Control groups received injections of saline ) . Glucose tolerance test ( GTT ) was performed 7 days after i . c . v . injection of vehicle or AβOs . Bar graph represents areas under the curves ( AUC ) in the time course plots ( n = 15 Veh ; 15 AβOs ; 10 Veh + TUDCA ; 16 AβOs + TUDCA ) . Data are representative of two independent experiments with similar results . Left panel : * * P = 0 . 0048 , * * P = 0 . 003 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0384 , one - way ANOVA followed by Bonferroni post hoc test .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice that received i.c.v. injections of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA (when used, TUDCA was administered in 5 i.c.v. injections of 5 μg TUDCA each, before and after oligomer injection; see Materials and Methods. Control groups received injections of saline). Glucose tolerance test (GTT) was performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 15 Veh; 15 AβOs; 10 Veh + TUDCA; 16 AβOs + TUDCA). Data are representative of two independent experiments with similar results. Left panel: **P = 0.0048, **P = 0.003, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0384, one-way ANOVA followed by Bonferroni post hoc test.


Plasma noradrenaline ( NA ) levels measured 7 days after i . c . v . injection of vehicle , vehicle + TUDCA , AβOs or AβOs + TUDCA in mice ( n = 7 animals / group ) . Data are representative of two independent experiments with similar results . * P = 0 . 0071 , one - way ANOVA followed by Bonferroni post hoc test .

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Plasma noradrenaline (NA) levels measured 7 days after i.c.v. injection of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA in mice (n = 7 animals/group). Data are representative of two independent experiments with similar results. *P = 0.0071, one-way ANOVA followed by Bonferroni post hoc test.


Glucose tolerance test ( 2 g glucose / kg body weight , i . p . ) in TNFR1 − / − mice or wild - type littermates performed 7 days after i . c . v . injection of vehicle or AβOs . Bar graph represents areas under the curves ( AUC ) in the time course plots ( n = 8 WT + Veh ; 7 WT + AβOs ; 7 TNFR − / − + Veh ; 8 TNFR − / − + AβOs ) . Left panel : * * P = 0 . 0049 , * * * P < 0 . 0001 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0001 , one - way ANOVA followed by Bonferroni post hoc test .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in TNFR1−/− mice or wild-type littermates performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 8 WT + Veh; 7 WT + AβOs; 7 TNFR−/− + Veh; 8 TNFR−/− + AβOs). Left panel: **P = 0.0049, ***P < 0.0001, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0001, one-way ANOVA followed by Bonferroni post hoc test.


Western blot analysis of phospho - IKKβ ( D ; n = 6 WT + Veh ; 6 WT + AβOs ; 4 TNFR − / − + Veh ; 6 TNFR − / − + AβOs ) and IRS - 1pSer636 levels ( E ; n = 6 WT + Veh ; 5 WT + AβOs ; 6 TNFR − / − + Veh ; 5 TNFR − / − + AβOs ) in the hypothalamus of wild - type ( WT ) or TNFR1 − / − mice 10 days after i . c . v . injection of vehicle or AβOs . Representative images from Western blot experiments were always run on the same gels but represent noncontiguous lanes . In ( D ) , * P = 0 . 0088 , Student ' s t - test ; in ( E ) , * P = 0 . 0428 , one - way ANOVA followed by Bonferroni post hoc test .

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Western blot analysis of phospho-IKKβ (D; n = 6 WT + Veh; 6 WT + AβOs; 4 TNFR−/− + Veh; 6 TNFR−/− + AβOs) and IRS-1pSer636 levels (E; n = 6 WT + Veh; 5 WT + AβOs; 6 TNFR−/− + Veh; 5 TNFR−/− + AβOs) in the hypothalamus of wild-type (WT) or TNFR1−/− mice 10 days after i.c.v. injection of vehicle or AβOs. Representative images from Western blot experiments were always run on the same gels but represent noncontiguous lanes. In (D), *P = 0.0088, Student's t-test; in (E), *P = 0.0428, one-way ANOVA followed by Bonferroni post hoc test.


Adult Swiss mice pre - treated with minocycline or PBS received a single i . c . v . injection of vehicle or 10 pmol AβOs , and hypothalamic levels of mRNA for AgRP ( F ; n = 5 Veh ; 6 AβOs ; 5 Veh + Mino ; 4 AβOs + Mino ) and NPY ( G ; n = 14 Veh ; 13 AβOs ; 8 Veh + Mino ; 9 AβOs + Mino ) were analyzed 7 days after injection . In ( F ) , * P = 0 . 0097 , one - way ANOVA followed by Bonferroni post hoc test ; in ( G ) , * P = 0 . 0219 , one - way ANOVA followed by Bonferroni post hoc test .

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Adult Swiss mice pre-treated with minocycline or PBS received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (F; n = 5 Veh; 6 AβOs; 5 Veh + Mino; 4 AβOs + Mino) and NPY (G; n = 14 Veh; 13 AβOs; 8 Veh + Mino; 9 AβOs + Mino) were analyzed 7 days after injection. In (F), *P = 0.0097, one-way ANOVA followed by Bonferroni post hoc test; in (G), *P = 0.0219, one-way ANOVA followed by Bonferroni post hoc test.


Glucose tolerance test ( GTT ) in APP / PS1 mice before and after i . c . v . injection of infliximab ( 0 . 2 μg daily for 4 days ) . Bar graph represents areas under the curves ( AUC ) in the time course plots ( n = 9 animals / group ) . Left panel : * P = 0 . 0177 , two - way ANOVA followed by Bonferroni post hoc test ; right panel : * P = 0 . 0327 , paired t - test .

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Glucose tolerance test (GTT) in APP/PS1 mice before and after i.c.v. injection of infliximab (0.2 μg daily for 4 days). Bar graph represents areas under the curves (AUC) in the time course plots (n = 9 animals/group). Left panel: *P = 0.0177, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0327, paired t-test.


B : Scatter Plot of mass spectrometry assay . Dots depict protein intensity ( IBAQ ) and relative significance obtained from mass spectrometry assay . X - axis : Difference of Che - 1 intensity versus IP control ; on the y - axis are the corresponding − log10 P - values . Significant Che - 1 interacting proteins relevant in this study are represented in blue .

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B: Scatter Plot of mass spectrometry assay. Dots depict protein intensity (IBAQ) and relative significance obtained from mass spectrometry assay. X-axis: Difference of Che-1 intensity versus IP control; on the y-axis are the corresponding −log10 P-values. Significant Che-1 interacting proteins relevant in this study are represented in blue.


C : Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies ( abs ) and analyzed by western blot ( WB ) with the indicated abs . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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C: Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies (abs) and analyzed by western blot (WB) with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.


D : ( Top ) qRT - PCR approach described in the schematic representation . ( Bottom ) RIP - qPCR analysis with anti - Che - 1 specific antibody or control rabbit IgG of Kms27 MM cells . Data are expressed as percent of Input . Error bars represent the standard error of three different biological experiments performed in technical duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 , n . s . = not significant ) .

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D: (Top) qRT-PCR approach described in the schematic representation. (Bottom) RIP-qPCR analysis with anti-Che-1 specific antibody or control rabbit IgG of Kms27 MM cells. Data are expressed as percent of Input. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ***P<0.005, ****P<0.001, n.s. = not significant).


E : Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization ( FISH ) staining of NEAT1 ( Stellaris ) with immunofluorescence of Che - 1 . Scale bar 10 μm .

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E: Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization (FISH) staining of NEAT1 (Stellaris) with immunofluorescence of Che-1. Scale bar 10 μm.


F : ( Left ) RT - qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in technical duplicate ( Two Tailed T test * * * * P < 0 . 001 ) . ( Right ) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs .

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F: (Left) RT-qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ****P<0.001). (Right) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs.


A : Quantitative RT - PCR ( RT - qPCR ) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments performed in duplicate ( Two Tailed T test * * * * P < 0 . 001 ) .

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A: Quantitative RT-PCR (RT-qPCR) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments performed in duplicate (Two Tailed T test ****P<0.001).


B : WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A .

B: WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A.


C : Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A , GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti - Che - 1 antibody . Scale bar 10 μm .

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C: Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A, GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti-Che-1 antibody. Scale bar 10 μm.


D : Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides , were subjected to WB with the indicated abs .

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D: Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides, were subjected to WB with the indicated abs.


E : Che - 1 ChIP - seq signal intensity of LNA GapmeR NEAT1A ( 2 replicates on right ) and GapmeR Control ( 2 replicates on left ) and at co - localizing Che - 1 / NEAT1 sites ( N = 14654 sites ) . Signal intensity from 1 ( weak ) to 12 ( strongest ) .

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E: Che-1 ChIP-seq signal intensity of LNA GapmeR NEAT1A (2 replicates on right) and GapmeR Control (2 replicates on left) and at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 12 (strongest).


F : FISH images of Kms27 MM cells depleted or not for Che - 1 expression ( siChe - 1A ) labeled with Hoechst ( cell nuclei ) and Quasar 670 ( NEAT1 RNA ) . Scale bar 10 μm .

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F: FISH images of Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) labeled with Hoechst (cell nuclei) and Quasar 670 (NEAT1 RNA). Scale bar 10 μm.


G : NEAT1 ChIRP - seq signal intensity of Kms27 MM cells transiently transfected with siChe - 1A or siControl at co - localizing Che - 1 / NEAT1 sites ( N = 14654 sites ) . Signal intensity from 1 ( weak ) to 14 ( strongest ) .

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G: NEAT1 ChIRP-seq signal intensity of Kms27 MM cells transiently transfected with siChe-1A or siControl at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 14 (strongest).


B : Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H , were subjected to immunoprecipitation with S9 . 6 antibody and analyzed by WB with the indicated abs . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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B: Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H, were subjected to immunoprecipitation with S9.6 antibody and analyzed by WB with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.


C : Representative confocal images showing the interaction between Che - 1 and S9 . 6 revealed by PLA in Kms27 MM cells . Negative controls were performed by omitting Che - 1 or S9 . 6 primary antibody . Scale bar 10 μm .

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C: Representative confocal images showing the interaction between Che-1 and S9.6 revealed by PLA in Kms27 MM cells. Negative controls were performed by omitting Che-1 or S9.6 primary antibody. Scale bar 10 μm.


D : Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9 . 6 antibody and analyzed by WB with the indicated antibodies . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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D: Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9.6 antibody and analyzed by WB with the indicated antibodies. Input corresponds to 5% of the total extract used for immunoprecipitation.


E : ( Left ) Representative confocal images of Kms27 MM cells treated or not with RNase H , transiently transfected with siChe - 1A or siControl and immunostained with S9 . 6 antibody and . Scale bar 10 μm . ( Right ) Box plot showing S9 . 6 signal nuclear intensity per cell ( Arbitrary Unit - AU ) . siChe - 1A cells are described in green color , while siControl in grey color . 100 cells were counted in each replicate . Each dot represents a cell . Median scores per sample are written at the top of the median red line . P value : < 1 . 02 - 18 . ANOVA test was performed to evaluate statistical significance . The red center line denotes the median value ( 50th percentile ) while the black box contains the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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E: (Left) Representative confocal images of Kms27 MM cells treated or not with RNase H, transiently transfected with siChe-1A or siControl and immunostained with S9.6 antibody and. Scale bar 10 μm. (Right) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in green color, while siControl in grey color. 100 cells were counted in each replicate. Each dot represents a cell. Median scores per sample are written at the top of the median red line. P value: <1.02-18. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


F : ( Top ) S9 . 6 antibody was used for dot blot analysis to evaluate RNA : DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1 . 5 micrograms . ( Bottom ) Quantification of S9 . 6 levels by densitometric analysis . Values were normalized to a Methylene blue loading control . Error bars represent the standard error of three different biological experiments ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) .

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F: (Top) S9.6 antibody was used for dot blot analysis to evaluate RNA:DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1.5 micrograms. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ***P<0.005, ****P<0.001).


G : Enrichment density of DRIP - seq signals at NEAT1 / Che1 co - localizing sites in Kms27 MM cells identified in siRNA control ( siControl ) ( grey ) and siRNA Che - 1 ( siChe - 1A ) ( dark green ) samples . Enrichment density was calculated on the relative size of each peak ( start to end of each fragment ) extended 2000 bp upstream and downstream . Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments .

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G: Enrichment density of DRIP-seq signals at NEAT1/Che1 co-localizing sites in Kms27 MM cells identified in siRNA control (siControl) (grey) and siRNA Che-1 (siChe-1A) (dark green) samples. Enrichment density was calculated on the relative size of each peak (start to end of each fragment) extended 2000 bp upstream and downstream. Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments.


D : ( Top ) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9 . 6 antibody . Scale bar 10 μm . ( Bottom ) Box plot showing S9 . 6 signal nuclear intensity per cell ( Arbitrary Unit - AU ) . siChe - 1A cells are described in dark grey , siChe1A plus CX5461 in green color , while siControl in grey color . Median scores per sample are written at the top of the median red line . P value : < 1 . 30 - 54 . ANOVA test was performed to evaluate statistical significance . The red center line denotes the median value ( 50th percentile ) while the black box contains the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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D: (Top) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9.6 antibody. Scale bar 10 μm. (Bottom) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in dark grey, siChe1A plus CX5461 in green color, while siControl in grey color. Median scores per sample are written at the top of the median red line. P value: <1.30-54. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


E : ( Top ) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che - 1 expression ( siChe - 1A ) and treated with CX5461 , were probed using abs directed against RNA : DNA hybrids . ( Bottom ) Quantification of S9 . 6 levels by densitometric analysis . Values were normalized to a Methylene blue loading control . Error bars represent the standard error of three different biological experiments ( Two Tailed T test * * * * P < 0 . 001 ) .

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E: (Top) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) and treated with CX5461, were probed using abs directed against RNA:DNA hybrids. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ****P<0.001).


A : Differential analysis of siControl vs . siChe1A transcriptome in Kms27 MM cells . Volcano plot shows 135 significantly upregulated ( dark green ) and 392 downregulated genes ( dark grey ) . x - axis reports the base - 2 logarithm of fold change which is approximated to the B value coming from the statistical Wald test . y - axis reports the base - 10 logarithm of Q value of significant genes .

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A: Differential analysis of siControl vs. siChe1A transcriptome in Kms27 MM cells. Volcano plot shows 135 significantly upregulated (dark green) and 392 downregulated genes (dark grey). x-axis reports the base-2 logarithm of fold change which is approximated to the B value coming from the statistical Wald test. y-axis reports the base-10 logarithm of Q value of significant genes.


D : WB analysis of total extracts of Kms27 MM cells depleted or not for Che - 1 ( siChe - 1A ) and probed for the indicated abs .

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D: WB analysis of total extracts of Kms27 MM cells depleted or not for Che-1 (siChe-1A) and probed for the indicated abs.


E : ( Left ) Luciferase activity of human NF - kB promoter was measured in Kms27 MM cells transiently transfected with siChe - 1A or siControl oligonucleotides or with empty vector or p65 / RelA protein expressing vector . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) . ( Right ) WB analysis of total extracts of Kms27 MM cells transiently transfected as described in E and incubated with the indicated antibodies .

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E: (Left) Luciferase activity of human NF-kB promoter was measured in Kms27 MM cells transiently transfected with siChe-1A or siControl oligonucleotides or with empty vector or p65/RelA protein expressing vector. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001). (Right) WB analysis of total extracts of Kms27 MM cells transiently transfected as described in E and incubated with the indicated antibodies.


RT - qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe - 1A or siControl . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments in duplicate ( Two Tailed T test * * * P < 0 . 001 , * * * * P < 0 . 001 ) .

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RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).


G : RT - qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe - 1A or siControl . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments in duplicate ( Two Tailed T test * * * P < 0 . 001 , * * * * P < 0 . 001 ) .

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G: RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).


H : WB analysis for the indicated antibodies of total extracts from Kms27 MM cells with siChe - 1A or siControl and where indicated with GFP - tagged RNaseH expression vectors .

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H: WB analysis for the indicated antibodies of total extracts from Kms27 MM cells with siChe-1A or siControl and where indicated with GFP-tagged RNaseH expression vectors.


I : RT - qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * P < 0 . 01 , * * * P < 0 . 005 . )

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I: RT-qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test **P<0.01, ***P<0.005.)


A : Representative S9 . 6 immunofluorescence levels in healthy and tumor CD138 + plasma cells purified from patient bone marrow . Nuclei were visualized by staining with Hoechst dye . Scale bar 10 μm .

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A: Representative S9.6 immunofluorescence levels in healthy and tumor CD138+ plasma cells purified from patient bone marrow. Nuclei were visualized by staining with Hoechst dye. Scale bar 10 μm.


B : Dot blot performed to evaluate the enrichment of RNA : DNA hybrids formation using the S9 . 6 antibody by serial dilutions of genomic DNA starting at 1 . 0 micrograms . Methylene blue normalization of S9 . 6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd .

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B: Dot blot performed to evaluate the enrichment of RNA:DNA hybrids formation using the S9.6 antibody by serial dilutions of genomic DNA starting at 1.0 micrograms. Methylene blue normalization of S9.6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd.


C : Boxplot showing relative S9 . 6 nuclear mean intensity in CD138 + plasma cells from 15 MM patients ( red ) and 5 healthy ( light blue ) . Dot plot representing each cell considered overlay the boxplot . Y - axis : S9 . 6 Nuclear Mean Intensity ( AU ) . The center line denotes the median value ( 50th percentile ) while the boxes contain the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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C: Boxplot showing relative S9.6 nuclear mean intensity in CD138+ plasma cells from 15 MM patients (red) and 5 healthy (light blue). Dot plot representing each cell considered overlay the boxplot. Y-axis: S9.6 Nuclear Mean Intensity (AU). The center line denotes the median value (50th percentile) while the boxes contain the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


D : Representative WB analysis with the indicated antibodies of healthy and neoplastic CD138 + purified plasma cells from patients .

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D: Representative WB analysis with the indicated antibodies of healthy and neoplastic CD138+ purified plasma cells from patients.


F : Unsupervised clustering heatmap showing enrichment profiles of 9 IFN signature genes . From the top : ISS stages 1 , 2 , 3 of each patient ( N = 687 ) . IFN signature High ( red , N = 71 ) and Low ( white , N = 614 ) assigned to each patient . Color scheme representing up - ( red ) and down - ( blue ) regulation of the selected genes across the patient dataset . Colors are proportional to normalized ( cpm ) read enrichment - the relative read enrichment median of each gene within the full patient cohort .

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F: Unsupervised clustering heatmap showing enrichment profiles of 9 IFN signature genes. From the top: ISS stages 1,2,3 of each patient (N=687). IFN signature High (red, N=71) and Low (white, N=614) assigned to each patient. Color scheme representing up- (red) and down- (blue) regulation of the selected genes across the patient dataset. Colors are proportional to normalized (cpm) read enrichment - the relative read enrichment median of each gene within the full patient cohort.


A : WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs .

A: WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs.


B : ( Left ) Kms27 and U266 MM cells were subjected to immunostaining with S9 . 6 antibody Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 nuclear mean intensity cell in the nucleus . 100 cells were counted in each replicate . Median scores per sample are written at the top of the median red line . P value : 3 . 3 - 13 . T - test was performed to evaluate statistical significance .

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B: (Left) Kms27 and U266 MM cells were subjected to immunostaining with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity cell in the nucleus. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 3.3-13. T-test was performed to evaluate statistical significance.


C : Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT - qPCR . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * P < 0 . 005 ) .

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C: Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT-qPCR. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005).


D : WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin ( THAP ) 0 . 5 μg / ml for 16 hours . G : WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours .

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D: WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin (THAP) 0.5 μg/ml for 16 hours. G: WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours.


E : ( Left ) U266 MM cells treated or not with THAP as in D and immunostained with S9 . 6 antibody Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 nuclear mean intensity per cell . 100 cells were counted in each replicate . Median scores per sample are written at the top of the median red line . P value : 1 . 1 - 20 . T - test was performed to evaluate statistical significance . H : ( Left ) Immunostaining with S9 . 6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 nuclear mean intensity per cell . 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line . P value : 4 . 7 - 23 . T - test was performed to evaluate statistical significance .

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E: (Left) U266 MM cells treated or not with THAP as in D and immunostained with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 1.1-20. T-test was performed to evaluate statistical significance. H: (Left) Immunostaining with S9.6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line. P value: 4.7-23. T-test was performed to evaluate statistical significance.


F : RT - qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) I : RT - qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * * P < 0 . 001 ) .

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F: RT-qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001) I: RT-qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ****P<0.001).


( A , B ) Immunoblot analysis of the phosphorylation level of CLIP - 170 , AMPK and ACC in heart ( A ) and liver ( B ) along with the developmental stages . α - tubulin was used as a loading control . ( C - E ) Quantitative analysis of western blotting shown in Fig 1A . ( F - H ) Quantitative analysis of western blotting shown in Fig 1B .

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(A, B) Immunoblot analysis of the phosphorylation level of CLIP-170, AMPK and ACC in heart (A) and liver (B) along with the developmental stages. α-tubulin was used as a loading control. (C-E) Quantitative analysis of western blotting shown in Fig 1A. (F-H) Quantitative analysis of western blotting shown in Fig 1B.


( I ) Immunostained images of adult mouse heart tissue . These were stained with a phosphorylated AMPK antibody ( I , upper ) , a phosphorylated CLIP - 170 antibody ( I , lower ) and a plakoglobin antibody ( middle ) . The representative images from more than 5 analyzed tissues were shown .

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(I) Immunostained images of adult mouse heart tissue. These were stained with a phosphorylated AMPK antibody (I, upper), a phosphorylated CLIP-170 antibody (I, lower) and a plakoglobin antibody (middle). The representative images from more than 5 analyzed tissues were shown.


( A , B ) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , middle row ) and 4 hours after washing out of MYK - 461 ( Wash out , bottom row ) . These cells were stained with a phosphorylated AMPK antibody , an AMPKβ2 antibody and a N - cadherin antibody .

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(A, B) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, middle row) and 4 hours after washing out of MYK-461 (Wash out, bottom row). These cells were stained with a phosphorylated AMPK antibody, an AMPKβ2 antibody and a N-cadherin antibody.


( C , D ) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) and stained with a connexin43 antibody , a plakoglobin antibody , an LKB - 1 antibody and an N - cadherin antibody . The representative images from 4 independent experiments were shown .

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(C, D) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row) and stained with a connexin43 antibody, a plakoglobin antibody, an LKB-1 antibody and an N-cadherin antibody. The representative images from 4 independent experiments were shown.


( E ) Quantification of pAMPK , AMPKβ2 , Connexin43 or LKB1 that was localized with a cell - cell junction marker ( N - cadherin or Plakoglobin ) . Box and whisker plots showed the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Numbers in the brackets indicate the cells that were analyzed for quantification . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . Two - tailed Student ' s t - test was used to analyze differences between two groups . * , P < 0 . 05 * * , P < 0 . 01 . n . s . , not significant .

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(E) Quantification of pAMPK, AMPKβ2, Connexin43 or LKB1 that was localized with a cell-cell junction marker (N-cadherin or Plakoglobin). Box and whisker plots showed the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Numbers in the brackets indicate the cells that were analyzed for quantification. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. Two-tailed Student's t-test was used to analyze differences between two groups. *, P<0.05 **, P<0.01. n.s., not significant.


( A ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 min and 15 min after treatment with 20 μM Compound C ( left side panel ) and expressing EGFP - CLIP - 170 S311A ( right side panel ) . Higher magniﬁcation of white square ( upper row ) showing CLIP - 170 migrated longitudinally toward the cell - cell junctions .

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(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0 min and 15 min after treatment with 20 μM Compound C (left side panel) and expressing EGFP-CLIP-170 S311A (right side panel). Higher magniﬁcation of white square (upper row) showing CLIP-170 migrated longitudinally toward the cell-cell junctions.


( B ) Beeswarm plots of comet length of EGFP - CLIP - 170 . The comet length from multiple cells in different fields was analyzed . Number of comets analyzed , Pre : n = 32 , Cpd . C : n = 36 , CLIP S311A : n = 68 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Pre .

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(B) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n=32, Cpd. C: n=36, CLIP S311A: n=68. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Pre.


( C ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT treated with control siRNA ( siCL , left ) or siRNA targeting both AMPKα1 and α2 ( siAMPKα1α2 , right ) . White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP - CLIP - 170 WT .

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(C) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT treated with control siRNA (siCL, left) or siRNA targeting both AMPKα1 and α2 (siAMPKα1α2, right). White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP-CLIP-170 WT.


( D ) Beeswarm plots of comet length of EGFP - CLIP - 170 . The comet length from multiple cells from different fields was analyzed . Number of comets analyzed , siCL : n = 178 , siAMPKα1α2 : n = 184 . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus siCL .

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(D) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells from different fields was analyzed. Number of comets analyzed, siCL: n=178, siAMPKα1α2: n=184. Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus siCL.


( A ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 , 2 and 3 hours after treatment with 2 μM MYK - 461 .

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(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0, 2 and 3 hours after treatment with 2 μM MYK-461.


( B ) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) . These cells were stained with an α - tubulin ( green ) and a plakoglobin ( red ) antibody .

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(B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody.


( C ) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK - 461 , showing the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Adenovirus expressing EGFP - CLIP - 170 ( adCLIP ) mutant was used ( WT , S311A , S311D ) . Numbers in the graph indicate n number of the cells from 2 independent experiments . Control ( - / - ) : n = 784 , MYK - 461 : n = 939 , CLIP WT : n = 656 , CLIP WT + MYK - 461 : n = 619 , CLIP S311A : n = 405 , CLIP S311A + MYK - 461 : n = 635 , CLIP S311D : n = 613 , CLIP S311D + MYK - 461 : n = 735 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Control , † † , P < 0 . 01 versus CLIP WT , n . s . , not significant .

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(C) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK-461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Adenovirus expressing EGFP-CLIP-170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (-/-): n=784, MYK-461: n=939, CLIP WT: n=656, CLIP WT + MYK-461: n=619, CLIP S311A: n=405, CLIP S311A + MYK-461: n=635, CLIP S311D: n=613, CLIP S311D + MYK-461: n=735. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Control, ††, P<0.01 versus CLIP WT, n.s., not significant.


( A ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) of individual Cre control ( CLIP - 170 S311A + / + ; α - MHC - MerCreMer + / − ) , Control mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer − / − ) , and CLIP - 170 S311A overexpressing mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer + / − ) , before ( Pre ) , 2 weeks ( Tx2w ) , 8 weeks ( Tx8w ) , 26 weeks ( Tx26w ) and over 1 year ( Tx1y ) after tamoxifen induction . Number of mice , ( Cre control , Control , S311A ) , Pre ( 6 , 2 , 3 ) , Tx 2w ( 9 , 5 , 5 ) , Tx 8w ( 9 , 10 , 14 ) , Tx1 2w ( 7 , 10 , 14 ) , Tx 26w ( 3 , 7 , 8 ) , Tx 52w ( 3 , 7 , 9 ) .

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(A) Beeswarm plots of the echocardiographic parameter (ejection fraction) of individual Cre control (CLIP-170 S311A +/+; α-MHC-MerCreMer+/−), Control mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer−/−), and CLIP-170 S311A overexpressing mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer+/−), before (Pre), 2 weeks (Tx2w), 8 weeks (Tx8w), 26 weeks (Tx26w) and over 1 year (Tx1y) after tamoxifen induction. Number of mice, (Cre control, Control, S311A), Pre (6, 2, 3), Tx 2w (9, 5, 5), Tx 8w (9, 10, 14), Tx1 2w (7, 10, 14), Tx 26w (3, 7, 8), Tx 52w (3, 7, 9).


( B ) Beeswarm plots of another line of CLIP - 170 S311A overexpressing mice ( line 9 ) . Number of mice , ( Control , S311A ) , Pre ( 7 , 3 ) , Tx 2w ( 4 , 7 ) , Tx 8w ( 6 , 4 ) , Tx 12w ( 5 , 4 ) , Tx 52w ( 3 , 3 ) . Data means ± S . D . Statistical significance was determined by two - way ANOVA followed by post hoc Tukey ' s multiple comparison test . # # : p < 0 . 01 vs Duration - matched Cre Control , * : p < 0 . 05 , * * : p < 0 . 01 vs Duration - matched Control ; † : p < 0 . 05 , † † : p < 0 . 01 , S311A TG mice 12 weeks vs 52 weeks .

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(B) Beeswarm plots of another line of CLIP-170 S311A overexpressing mice (line 9). Number of mice, (Control, S311A), Pre (7, 3), Tx 2w (4, 7), Tx 8w (6, 4), Tx 12w (5, 4), Tx 52w (3, 3). Data means ±S.D. Statistical significance was determined by two-way ANOVA followed by post hoc Tukey's multiple comparison test. ##: p < 0.01 vs Duration-matched Cre Control, *: p < 0.05, **: p < 0.01 vs Duration-matched Control; †: p < 0.05, ††: p < 0.01, S311A TG mice 12 weeks vs 52 weeks.


( C ) Representative long axis four - chamber cardiac magnetic resonance images of CLIP - 170 S311A TG mice and control mice over 1 year after tamoxifen induction . Left column showed systole images and right column represented diastole images . Three mice from each group were analyzed for cardiac MRI . The representative images were shown .

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(C) Representative long axis four-chamber cardiac magnetic resonance images of CLIP-170 S311A TG mice and control mice over 1 year after tamoxifen induction. Left column showed systole images and right column represented diastole images. Three mice from each group were analyzed for cardiac MRI. The representative images were shown.


( D ) Beeswarm plots showed the ejection fraction from cardiac MRI . LV , left ventricular , RV , right ventricular . Number of mice , ( Control , S311A ) , 1y ( 3 , 3 ) . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus Control .

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(D) Beeswarm plots showed the ejection fraction from cardiac MRI. LV, left ventricular, RV, right ventricular. Number of mice, (Control, S311A), 1y (3, 3). Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus Control.


( E ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) from CLIP - 170 S311D overexpressing mice and the control . Number of mice , ( Control , S311D ) , Tx 2w ( 3 , 4 ) , Tx 8w ( 5 , 11 ) , Tx 26w ( 4 , 11 ) . Data means ± S . D . There was no significant difference .

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(E) Beeswarm plots of the echocardiographic parameter (ejection fraction) from CLIP-170 S311D overexpressing mice and the control. Number of mice, (Control, S311D), Tx 2w (3, 4), Tx 8w (5, 11), Tx 26w (4, 11). Data means ±S.D. There was no significant difference.


( A ) Masson ' s trichrome staining of the heart of CLIP - 170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment .

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(A) Masson's trichrome staining of the heart of CLIP-170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment.


( B ) Quantitative analysis of cardiac fibrosis . Quantification was made as percentage of fibrosis area ( in blue ) in the left ventricle . Number of mice , ( Control , S311A ) , 3m ( 7 , 6 ) , 1y ( 5 , 6 ) . * , P < 0 . 05 versus Control .

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(B) Quantitative analysis of cardiac fibrosis. Quantification was made as percentage of fibrosis area (in blue) in the left ventricle. Number of mice, (Control, S311A), 3m (7, 6), 1y (5, 6). *, P<0.05 versus Control.


( C ) Representative immunostained images with an α - tubulin antibody of CLIP - 170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment . ( D ) Quantitative analysis of intensity of α - tubulin in ( C ) . Number of mice , ( Control : n = 7 , S311A : n = 8 ) . Average of 5 sections per animal were analyzed . * * , P < 0 . 01 versus Control .

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(C) Representative immunostained images with an α-tubulin antibody of CLIP-170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment. (D) Quantitative analysis of intensity of α-tubulin in (C). Number of mice, (Control: n=7, S311A: n=8). Average of 5 sections per animal were analyzed. **, P<0.01 versus Control.


( E ) Representative images of western blot for an α - tubulin antibody . GAPDH was used as a loading control . ( F ) Quantitative analysis of intensity of α - tubulin in ( E ) . Number of mice , ( Control : n = 4 , S311A : n = 5 ) . * , P < 0 . 05 versus Control .

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(E) Representative images of western blot for an α-tubulin antibody. GAPDH was used as a loading control. (F) Quantitative analysis of intensity of α-tubulin in (E). Number of mice, (Control: n=4, S311A: n=5). *, P<0.05 versus Control.


( G ) Representative immunostained images of CLIP - 170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment . These were stained with WGA ( red ) and a plakoglobin antibody ( green ) .

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(G) Representative immunostained images of CLIP-170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment. These were stained with WGA (red) and a plakoglobin antibody (green).


( H ) Quantitative analysis of ( G ) Cell width , length and aspect ratio ( length / width ) were measured . Number of cells analyzed , Control , n = 251 , S311A , n = 310 , * * , P < 0 . 01 versus Control .

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(H) Quantitative analysis of (G) Cell width, length and aspect ratio (length/width) were measured. Number of cells analyzed, Control, n=251, S311A, n=310, **, P<0.01 versus Control.


( A ) Swelling of neurons in the vestibular nucleus due to fine vesiculation ( arrows ) and clear vacuolization ( arrowhead ) of the cytoplasm . HE , scale bar 100 μm .

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(A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm.


( B ) Clear cytoplasmic vacuolization ( arrows ) in cerebellar corticalPurkinje cells . HE , scale bar 100 μm .

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(B) Clear cytoplasmic vacuolization (arrows) in cerebellar corticalPurkinje cells. HE, scale bar 100 μm.


( C ) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells ( arrows ) and a dense granular cell layer . HE , scale bar 100 μm . ( D ) Marked neuronal loss is present the cerebellar cortex of an affected dog . The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain ( arrow ) . HE , scale bar 100 μm .

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(C) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm.


( E ) Axonal spheroids of varying quality were seen in the white matter ( arrows ) of cerebellum and brainstem . HE , scale bar 100 μm .

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(E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm.


( F ) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells . HE , scale bar 100 μm .

(F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm.


( G ) Purkinje cell with numerous single - membrane bound , cytoplasmic vacuoles tethering to each other ( arrows , inset ) . Electronmicrograph , scale bar 2 μm . Inset : scale bar 1 μm .

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(G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm.


( H ) Axonal spheroid containing aggregated degenerated mitochondria , occasional double - membrane - bound autophagosomes ( arrows ) and free electron dense material , compressed by a peripheral clear vacuolar space . Electronmicrograph , scale bar 0 . 5 μm . Abbreviations : n , nucleus ; ml , molecular layer ; pl , Purkinje cell layer ; gl , granular cell layer .

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(H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer.


( A ) Axonal spheroids stain diffusely positive for LC3B . IHCLC3B , scale bar 100 μm . ( B , C ) The granular cores of the spheroids are positive for ( B ) ubiquitin ( arrow ) and ( C ) p62 . IHCubiquitin and p62 , scale bars 20 and 100 μm , respectively . ( D ) Smooth axonal swellings in the cerebellar cortex contain ATG4D . Inset : control . IHCATG4D , scale bar 100 μm . ( E ) Affected neurons show increased perinuclear granular LC3B positivity . Inset : control . IHCLC3B , scale bar 100 μm . ( F ) Neuronalvacuoles are partially LAMP2 positive ( arrow ) and partially negative ( arrow head ) . Inset : control . IHCLAMP2 , scale bar 20 μm .

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(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.


( A - F ) Lateral views of control and morphant zebrafishembryos . ( A , B ) Control embryos appear normal at 1 dpf . ( C , D ) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf . Black arrowheads denote the developing cerebellum . Purple arrows indicate the hindbrain irregularities . ( E ) Control embryo at 2 dpf . ( F ) A morphant embryo at 2 dpf displaying hydrocephalus ( yellow arrow ) and small head and eye .

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(A-F) Lateral views of control and morphant zebrafishembryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye.


( G ) RT - PCR assay showing the efficiency of the atg4daSMO .

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(G) RT-PCR assay showing the efficiency of the atg4daSMO.


Immunostaining with ( A - C ) anti - Pvalb7 and ( D - F ) anti - zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants . ( A , D ) Control embryos at 4 . 5 dpf . ( B , E ) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells . ( C , F ) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons , which are laterally located in the cerebellum . ( G - I ) Labeling of cerebellar granule cells with anti - Vglut1 antibody in control and morphant embryos . ( H ) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum . ( I ) Vglut1 expression is strongly reduced in embryos showing severe phenotype . White arrowheads indicate the region of cerebellum . Abbreviations : hb , hindbrain .

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Immunostaining with (A-C) anti-Pvalb7 and (D-F) anti-zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants. (A,D) Control embryos at 4.5 dpf. (B,E) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells. (C,F) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons, which are laterally located in the cerebellum. (G-I) Labeling of cerebellar granule cells with anti-Vglut1 antibody in control and morphant embryos. (H) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum. (I) Vglut1 expression is strongly reduced in embryos showing severe phenotype. White arrowheads indicate the region of cerebellum. Abbreviations: hb, hindbrain.


C : FACS plots from a representative experiment showing gates for purification of the cell populations . Upper left : GFP + cells in the population of ESCs infected with pGIPZ - shRNA library lentiviruses . Upper right : Isolation of mesodermal ( SSEA1 - Flk1 + Cxcr4 - ) and endodermal ( SSEA1 - Flk1 - Cxcr4 + ) cells . Lower panels : Isolation of D20LS ( Lin - Sca1 + c - Kit - ) , D20LK ( Lin - Sca1 - c - Kit + ) , and D20LSK ( Lin - Sca1 + c - Kit + ) cells .

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C: FACS plots from a representative experiment showing gates for purification of the cell populations. Upper left: GFP+ cells in the population of ESCs infected with pGIPZ-shRNA library lentiviruses. Upper right: Isolation of mesodermal (SSEA1-Flk1+Cxcr4-) and endodermal (SSEA1-Flk1-Cxcr4+) cells. Lower panels: Isolation of D20LS (Lin-Sca1+c-Kit-), D20LK (Lin-Sca1-c-Kit+), and D20LSK (Lin-Sca1+c-Kit+) cells.


A : mRNA analysis of expression of endothelial ( Tie2 , CD31 , Cdh5 ) , epithelial ( Cdh1 ) , and early hematopoietic ( Scl , CD41 ) marker genes in the indicated sorted cell populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

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A: mRNA analysis of expression of endothelial (Tie2, CD31, Cdh5), epithelial (Cdh1), and early hematopoietic (Scl, CD41) marker genes in the indicated sorted cell populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


B : Confirmation of the purity of sorted populations by qRT - PCR : c - Kit is highly expressed only in the c - Kit + ( D20LSK and D20LK ) populations , and Sca1 is highly expressed only in the in Sca1 + ( D20LSK and D20LS ) populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

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B: Confirmation of the purity of sorted populations by qRT-PCR: c-Kit is highly expressed only in the c-Kit+ (D20LSK and D20LK) populations, and Sca1 is highly expressed only in the in Sca1+ (D20LSK and D20LS) populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


C : qRT - PCR analysis of Gata1 , Gata2 , Lyz2 , and Scl expression in the sorted cell populations on day 20 . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

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C: qRT-PCR analysis of Gata1, Gata2, Lyz2, and Scl expression in the sorted cell populations on day 20. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


D : FACS analysis of ESC - derived cells confirming that D20LS , D20LK , and D20LSK cell populations express the early hematopoietic marker CD41 and the pan - hematopoietic marker CD45 .

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D: FACS analysis of ESC-derived cells confirming that D20LS, D20LK, and D20LSK cell populations express the early hematopoietic marker CD41 and the pan-hematopoietic marker CD45.


A : Clustering and enrichment analyses of shRNA reads in D6C ( endoderm ) , D6F ( HM / E ) , D20LS , D20LK , and D20LSK ( HSPC ) populations compared with ESCs . Specifically enriched / depleted shRNAs are grouped according to their target gene function ( I - X ) . Color coding bar is shown below .

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A: Clustering and enrichment analyses of shRNA reads in D6C (endoderm), D6F (HM/E), D20LS, D20LK, and D20LSK (HSPC) populations compared with ESCs. Specifically enriched/depleted shRNAs are grouped according to their target gene function (I-X). Color coding bar is shown below.


B : Heat map of shRNA reads in D6C and D6F populations compared with ESCs . shRNAs specifically depleted in D6C and D6F are labeled as Groups XI and XII , respectively .

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B: Heat map of shRNA reads in D6C and D6F populations compared with ESCs. shRNAs specifically depleted in D6C and D6F are labeled as Groups XI and XII, respectively.


E : Heat map showing depletion of shRNAs targeting Cdc and Cdk genes in all day 20 samples .

E: Heat map showing depletion of shRNAs targeting Cdc and Cdk genes in all day 20 samples.