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 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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 .

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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

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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 .

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 .

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

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 .

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

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.


The location of causal candidate LN1 is indicated as is the position of the linked TAS rs2303565 associated with ALS . The structures of transcripts indicate the exons ( blue ) and introns ( gray with > ) . The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium . Chromatin states are shown for brain tissues , neural progenitor cells ( NPCs ) , and a variety of other tissues . Next to the color key , NCBI dbVar ID for TE , SNP ID for TAS , genic location of TAS , TE name , and r2 value are shown . * r2 value verified by genotyping PCR . ChIP - seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states .

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

The location of causal candidate LN1 is indicated as is the position of the linked TAS rs2303565 associated with ALS. The structures of transcripts indicate the exons (blue) and introns (gray with >). The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, neural progenitor cells (NPCs), and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states.


The location of causal candidate LN2 is indicated as is the position of the linked TAS rs11759769 associated with migraine disorder . The structures of transcripts indicate the exons ( blue ) and introns ( gray with > ) . L1MA5 indicates the position of an L1 element fixed in the genome . The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium . Chromatin states are shown for brain tissues , NPCs , and a variety of other tissues . Next to the color key , NCBI dbVar ID for TE , SNP ID for TAS , genic location of TAS , TE name , and r2 value are shown . * r2 value verified by genotyping PCR . ChIP - seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states and peaks for two transcription factors known for heterochromatin formation located around the site of LN2 insertion are labeled .

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

The location of causal candidate LN2 is indicated as is the position of the linked TAS rs11759769 associated with migraine disorder. The structures of transcripts indicate the exons (blue) and introns (gray with >). L1MA5 indicates the position of an L1 element fixed in the genome. The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, NPCs, and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states and peaks for two transcription factors known for heterochromatin formation located around the site of LN2 insertion are labeled.


The location of causal candidate LN16 is indicated as is the position of the linked TAS rs12185268 associated with PD . The structures of transcripts indicate the exons ( blue ) and introns ( gray with > ) . The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium . Chromatin states are shown for brain tissues , NPCs , and a variety of other tissues . Next to the color key , NCBI dbVar ID for TE , SNP ID for TAS , genic location of TAS , TE name , and r2 value are shown . * r2 value verified by genotyping PCR . ChIP - seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states . The position of the LN16 TE is enlarged in an inset .

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

The location of causal candidate LN16 is indicated as is the position of the linked TAS rs12185268 associated with PD. The structures of transcripts indicate the exons (blue) and introns (gray with >). The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, NPCs, and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states. The position of the LN16 TE is enlarged in an inset.


Approximately 500 bp of genomic sequence , with and without Alu inserts was assayed for transcription activity when inserted upstream of a minimal promoter in a luciferase vector transfected into NCRM1 cells . The orientation ( white triangle ) of the Alu and its associated genomic sequence ( yellow box ) is diagramed at top . Forward orientation ( Fwd ) indicates plus strand sequence of the reference genome . The minimal promoter is symbolized by a black rectangle with an arrow indicating the direction of transcription . The causal TE candidates tested , their linked TASs , and their ChromHMM status are indicated at right . Luciferase values are normalized to transfection efficiency ( renilla ) for each of four independent transfections and subsequently to luciferase expression from the minimal promoter alone . Mean values ( ± SD ) were determined , and statistical significance was analyzed by Student ' s two - sample unequal variance t - Test with a one - tailed distribution . Each locus was examined in four independent experiments and a representative dataset is shown here . ☨ : p < 0 . 05 in two experiments and p > 0 . 05 in two experiments . ns : P > 0 . 05 .

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

Approximately 500 bp of genomic sequence, with and without Alu inserts was assayed for transcription activity when inserted upstream of a minimal promoter in a luciferase vector transfected into NCRM1 cells. The orientation (white triangle) of the Alu and its associated genomic sequence (yellow box) is diagramed at top. Forward orientation (Fwd) indicates plus strand sequence of the reference genome. The minimal promoter is symbolized by a black rectangle with an arrow indicating the direction of transcription. The causal TE candidates tested, their linked TASs, and their ChromHMM status are indicated at right. Luciferase values are normalized to transfection efficiency (renilla) for each of four independent transfections and subsequently to luciferase expression from the minimal promoter alone. Mean values (± SD) were determined, and statistical significance was analyzed by Student's two-sample unequal variance t-Test with a one-tailed distribution. Each locus was examined in four independent experiments and a representative dataset is shown here. ☨: p < 0.05 in two experiments and p > 0.05 in two experiments. ns: P > 0.05.


B . Detection of ALDHbr cells by high content screening . The algorithm first detect nuclear region of interest ( ROI ) in the Hoechst channel ( rainbow areas ) . Then , it computes the averaged ALDH signal in the ROI to identify ALDHbr cells ( red nuclear ROIs ) and calculate their proportion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Detection of ALDHbr cells by high content screening. The algorithm first detect nuclear region of interest (ROI) in the Hoechst channel (rainbow areas). Then, it computes the averaged ALDH signal in the ROI to identify ALDHbr cells (red nuclear ROIs) and calculate their proportion.


C - D . Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death ( cell viability > 20 % , B - score > │ 2 . 58 │ ) . Data represent mean ± SD ( n = 3 ) .

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

C-D. Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death (cell viability>20%, B-score>│2.58│). Data represent mean ±SD (n=3).


E . Representative images of the high - content screening captures . ALDEFLUOR cellular staining is represented in green . ALDHbr cells are labelled in red .

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E. Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. ALDHbr cells are labelled in red.


F . Volcano plot of the hits . Scatter points represent genes , the x - axis is the log2 fold change of the proportion of ALDHbr cells compared to the control , and the y - axis is the B - score . Genes targeted by a siRNA inducing a significant reduction ( green dots ; B - score > 2 . 58 , log2 FC < 1 ) or increase ( red dots ; B - score > 2 . 58 , log2 FC < 1 ) of the ALDHbr cell proportion are highlighted .

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

F. Volcano plot of the hits. Scatter points represent genes, the x-axis is the log2 fold change of the proportion of ALDHbr cells compared to the control, and the y-axis is the B-score. Genes targeted by a siRNA inducing a significant reduction (green dots; B-score>2.58, log2 FC<1) or increase (red dots; B-score>2.58, log2 FC<1) of the ALDHbr cell proportion are highlighted.


B . Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping ( right ) and tumor SFE ( left ) using the 11 siRNAs pool ( 3 siRNAs / pool ) against the selected target genes and one scrambled siRNA pool used as control . Results for each siRNA pool ( from top to bottom ) are represented as opposite bars ( n = 3 ) . Correlations are measured using Spearman rank correlation ( R ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping (right) and tumor SFE (left) using the 11 siRNAs pool (3 siRNAs/pool) against the selected target genes and one scrambled siRNA pool used as control. Results for each siRNA pool (from top to bottom) are represented as opposite bars (n=3). Correlations are measured using Spearman rank correlation (R).


C - D . Examples of dose - dependent ALDHbr cells variation in response to drug treatments . ALDHbr cell proportion in response to Tazemetostat in SUM159 ( n = 10 ) ( C ) and JQ1 in SUM149 ( n = 10 ) ( D ) . The horizontal red dashed - line corresponds to a reduction of 50 % of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed - line corresponds to the target dose predicted by the K - score . On the bottom panel , corresponding flow charts for the ALDEFLUOR staining . DEAB is an ALDH inhibitor used as negative control .

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 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

C-D. Examples of dose-dependent ALDHbr cells variation in response to drug treatments. ALDHbr cell proportion in response to Tazemetostat in SUM159 (n=10) (C) and JQ1 in SUM149 (n=10) (D). The horizontal red dashed-line corresponds to a reduction of 50% of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed-line corresponds to the target dose predicted by the K-score. On the bottom panel, corresponding flow charts for the ALDEFLUOR staining. DEAB is an ALDH inhibitor used as negative control.


F . Heat map for the variation of the ALDHbr cells proportion under drug treatment , in five different cell lines . Red and blue indicate the K - score in treated conditions , respectively , above and below the K - score in the untreated condition .

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

F. Heat map for the variation of the ALDHbr cells proportion under drug treatment, in five different cell lines. Red and blue indicate the K-score in treated conditions, respectively, above and below the K-score in the untreated condition.


G - H . Primary ( n = 4 ) ( G ) and secondary ( n = 4 ) ( H ) tumorsphere - forming efficiency ( SFE ) of SUM159 cells under treated and untreated conditions . Grey dashed - line corresponds to the level of SFE in the untreated conditions . Statistical test used is Pairwise chi - square test . Data represent mean ± SD .

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

G-H. Primary (n=4) (G) and secondary (n=4) (H) tumorsphere-forming efficiency (SFE) of SUM159 cells under treated and untreated conditions. Grey dashed-line corresponds to the level of SFE in the untreated conditions. Statistical test used is Pairwise chi-square test. Data represent mean ±SD.


A . Interaction surface built using the estimated combination index ( CI ) for the Salinomycin and JQ1 drug combination , in four different breast cancer cell lines ( top panels ) . The CI is represented in 2D color code interaction surface . The color code spans from strong synergism ( dark blue , CI < 1 ) to strong antagonism ( red , CI > 1 ) . The concentrations of each particular drug in the combinations are denoted on each axis . On the bottom panels , neighborhood Z - core matrix estimating the significant synergistic interaction . The color code spans from a statistically significant synergism ( dark blue , CI < 1 , p < 0 . 01 ) to a statistically significant antagonism ( dark red , CI > 1 , p < 0 . 01 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Interaction surface built using the estimated combination index (CI) for the Salinomycin and JQ1 drug combination, in four different breast cancer cell lines (top panels). The CI is represented in 2D color code interaction surface. The color code spans from strong synergism (dark blue, CI<1) to strong antagonism (red, CI>1). The concentrations of each particular drug in the combinations are denoted on each axis. On the bottom panels, neighborhood Z-core matrix estimating the significant synergistic interaction. The color code spans from a statistically significant synergism (dark blue, CI<1, p<0.01) to a statistically significant antagonism (dark red, CI>1, p<0.01).


B . Representative examples of flow chart for the ALDEFLUOR staining following different JQ1 / Salinomycin drug combinations . DEAB is an ALDH inhibitor used as negative control . Data represent mean ± SD .

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

B. Representative examples of flow chart for the ALDEFLUOR staining following different JQ1/Salinomycin drug combinations. DEAB is an ALDH inhibitor used as negative control. Data represent mean ±SD.


C - D . Primary ( n = 4 ) ( C ) and secondary ( n = 4 ) ( D ) tumorsphere - forming efficiency ( SFE ) of SUM159 cells under mono - and combined - therapy compared to untreated conditions . Statistical test used is Pairwise chi - square test . .

C-D. Primary (n=4) (C) and secondary (n=4) (D) tumorsphere-forming efficiency (SFE) of SUM159 cells under mono- and combined-therapy compared to untreated conditions. Statistical test used is Pairwise chi-square test..


E . Bar plots representing the dose - dependent changes in the proportion of apoptotic cells ( YO - PRO - 3 + ) in each cell subpopulations , in response to drug treatments ( right panel ) . Cisplatin ( CisPt ) is used as a positive control . On the right panel , representative fluorescence images obtained in different treatment conditions ( n = 6 ) . Nucleus are in blue ( Hoechst ) , bCSCs are in green ( ALDH ) , and apoptotic cells in red ( YO - PRO - 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bar plots representing the dose-dependent changes in the proportion of apoptotic cells (YO-PRO-3+) in each cell subpopulations, in response to drug treatments (right panel). Cisplatin (CisPt) is used as a positive control. On the right panel, representative fluorescence images obtained in different treatment conditions (n=6). Nucleus are in blue (Hoechst), bCSCs are in green (ALDH), and apoptotic cells in red (YO-PRO-3).


G . Representation of the BFP + / RFP + cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone , or in combination . Blue bars : BFP + cells proportion , Red bars : RFP + cells proportion .

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G. Representation of the BFP+/RFP+ cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone, or in combination. Blue bars: BFP+ cells proportion, Red bars: RFP+ cells proportion.


H - I . Representation of the bCSC proportion in the BFP + ( H ) and RFP + ( I ) progenies in the control cells compared to the JQ1 - and Salinomycin - treated cells alone or in combination ( n = 4 ) . Statistical test used is a student ' s t - test . JQ1 Vs CTRL and Sal Vs JQ1 p = 0 . 03 ; JQ1 / Sal Vs CTRL p = 0 . 01 . Error bars represent mean ± SD .

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

H-I. Representation of the bCSC proportion in the BFP+ (H) and RFP+ (I) progenies in the control cells compared to the JQ1- and Salinomycin-treated cells alone or in combination (n=4). Statistical test used is a student's t-test. JQ1 Vs CTRL and Sal Vs JQ1 p=0.03; JQ1/Sal Vs CTRL p=0.01. Error bars represent mean ± SD.


B . Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 ( n = 10 ) The grey area corresponds to the period of drug treatments .

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

B. Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 (n=10) The grey area corresponds to the period of drug treatments.


C - E . Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle - treated tumors , measured by flow cytometry . Statistical test used is a student t - test . Data represent mean ± SD ( n = 10 ) .

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

C-E. Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10).


Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle - treated tumors , measured by flow cytometry . Statistical test used is a student t - test . Data represent mean ± SD ( n = 10 ) .

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

Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10).


Reimplantation assay ( CRCM434 ) . Two - week treated PDXs were reimplanted , in serial dilutions , into new recipient mice and tumor growth was monitored Each curve represents the growth kinetic from one individual injection .

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

Reimplantation assay (CRCM434). Two-week treated PDXs were reimplanted, in serial dilutions, into new recipient mice and tumor growth was monitored Each curve represents the growth kinetic from one individual injection.


G - I . Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis ( ELDA ) . Results are expressed as the estimated number of bCSCs for 10 , 000 tumor cells . Statistical test used is Pairwise chi - square test .

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

G-I. Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis (ELDA). Results are expressed as the estimated number of bCSCs for 10,000 tumor cells. Statistical test used is Pairwise chi-square test.


J - L . Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434 . Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs ( J ) . Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal / JQ1 treated mice compared to the control mice ( K , CRCM404 , p = 0 . 04 ; L , CRCM434 , p = 0 . 02 ) . Statistical test used is a Wilcoxon test . Error bars represent mean ± SD ( n = 10 ) .

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

J-L. Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434. Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs (J). Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal/JQ1 treated mice compared to the control mice (K, CRCM404, p=0.04; L, CRCM434, p=0.02). Statistical test used is a Wilcoxon test. Error bars represent mean ± SD (n=10).


C . Kaplan - Meier relapse - free survival curve according to subnetwork metagenes expression levels in breast tumors . Curves were compared with a log - rank test ( p - value ) .

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C. Kaplan-Meier relapse-free survival curve according to subnetwork metagenes expression levels in breast tumors. Curves were compared with a log-rank test (p-value).


A CD4 + CD127 - CD25hiFoxP3 + CTLA4 + Treg induction from PBMCs incubated with CVF , CLys and CSN ( 10µg / ml ) after 4h , 24h , 48h , 72h ( plots and graphs shown ) , 120h and 168h .

O O O O O O O O O O O O O O O O O O O O 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 CD4+CD127-CD25hiFoxP3+CTLA4+ Treg induction from PBMCs incubated with CVF, CLys and CSN (10µg/ml) after 4h, 24h, 48h, 72h (plots and graphs shown), 120h and 168h.


B Trafficking receptors ( CXCR3 , CCR4 , CCR6 and CCR7 ) evaluation on induced Treg ( CD25hiCD127 - ) and non - Treg cells ( CD127 + CD25 - ) .

O O O O O O O O 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 Trafficking receptors (CXCR3, CCR4, CCR6 and CCR7) evaluation on induced Treg (CD25hiCD127-) and non-Treg cells (CD127+CD25-).


C Induced Treg proliferation with Ki67 staining and Treg expression of IL - 10 and IFNγ .

C Induced Treg proliferation with Ki67 staining and Treg expression of IL-10 and IFNγ.


A Levels of pro - and anti - inflammatory cytokines in PBMC culture supernatants with CVF , CLys and CSN ( 10µg / ml ) after 72h determined using a LUMINEX - based technology .

A Levels of pro- and anti-inflammatory cytokines in PBMC culture supernatants with CVF, CLys and CSN (10µg/ml) after 72h determined using a LUMINEX-based technology.


B Composition and levels of bioactive lipid mediators ( LMs ) , including prostaglandins , leukotrienes , lipoxins , thromboxanes , and their precursors in culture with CVF , CLys and CSN quantified by LC - MS / MS - based profiling . Heat maps show relative amounts of LMs detected by LC - MS / MS in supernatants . Asterisks show statistical differences analyzed using two - way ANOVA followed by Turkey ' s multiple comparison test .

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

B Composition and levels of bioactive lipid mediators (LMs), including prostaglandins, leukotrienes, lipoxins, thromboxanes, and their precursors in culture with CVF, CLys and CSN quantified by LC-MS/MS-based profiling. Heat maps show relative amounts of LMs detected by LC-MS/MS in supernatants. Asterisks show statistical differences analyzed using two-way ANOVA followed by Turkey's multiple comparison test.


C Dynamic of PGE2 release correlated with Treg induction by CLys ( upper panel ) and CSN ( lower panel ) using a Pearson correlation .

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

C Dynamic of PGE2 release correlated with Treg induction by CLys (upper panel) and CSN (lower panel) using a Pearson correlation.


D Treg induction by CLys or CSN following PBMC pre - incubation with 10µM mPGES1 inhibitor 934 ( or DMSO as a control ) .

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D Treg induction by CLys or CSN following PBMC pre-incubation with 10µM mPGES1 inhibitor 934 (or DMSO as a control).


E Induction of Tregs with PGE2 analog ( DINP ) ( 5µM ) and / or recombinant human IL - 10 ( rhIL - 10 ) ( 10ng / ml ) as compared to CLys .

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E Induction of Tregs with PGE2 analog (DINP) (5µM) and/or recombinant human IL-10 (rhIL-10) (10ng/ml) as compared to CLys.


A Levels of bioactive LMs and IL - 10 released from monocyte and non - monocyte fractions .

A Levels of bioactive LMs and IL-10 released from monocyte and non-monocyte fractions.


B Non - monocyte fractions were pre - incubated with IL - 10R inhibitor and PGE2 receptor ( EP2 and EP4 ) antagonists ( or DMSO as a control ) and incubated with CLys ( left upper panel ) or CSN ( 10µg / ml ) ( left lower panel ) in presence or absence of monocytes and Treg induction was analysed after 72h . On the right panel , PGE2 receptors were first blocked on monocytes ( 2 ) or non - monocytes ( 3 ) or both ( 4 ) before addition of non - monocytes ( 2 ) or monocytes ( 3 ) and incubation with CSN for 72h and analysis of Treg induction compared to control condition ( 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Non-monocyte fractions were pre-incubated with IL-10R inhibitor and PGE2 receptor (EP2 and EP4) antagonists (or DMSO as a control) and incubated with CLys (left upper panel) or CSN (10µg/ml) (left lower panel) in presence or absence of monocytes and Treg induction was analysed after 72h. On the right panel, PGE2 receptors were first blocked on monocytes (2) or non-monocytes (3) or both (4) before addition of non-monocytes (2) or monocytes (3) and incubation with CSN for 72h and analysis of Treg induction compared to control condition (1).


C Treg induction by CLys and CSN in presence of monocyte / non - monocyte fractions and naïve CD4 + CD45RA + CD45RO - T cells .

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

C Treg induction by CLys and CSN in presence of monocyte/non-monocyte fractions and naïve CD4+CD45RA+CD45RO- T cells.


D Monocyte phenotyping in presence of CVF , CLys and CSN by FACS using CD68 , CD80 , CD163 and CD206 surface expression .

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

D Monocyte phenotyping in presence of CVF, CLys and CSN by FACS using CD68, CD80, CD163 and CD206 surface expression.


E Levels of LMs and PGE2 released by macrophages in presence of CVF , CLys and CSN Additionally , macrophages were lysed and gene expression of PTGS2 , PTGES , ALOX5 , ALOX15 and MaR1 quantified by RT - PCR .

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

E Levels of LMs and PGE2 released by macrophages in presence of CVF, CLys and CSN Additionally, macrophages were lysed and gene expression of PTGS2, PTGES, ALOX5, ALOX15 and MaR1 quantified by RT-PCR.


F Treg induction following naïve CD4 + CD45RA + CD45RO - T cells incubation with CLys - , CSN - monocyte conditioned media .

F Treg induction following naïve CD4+CD45RA+CD45RO- T cells incubation with CLys-, CSN-monocyte conditioned media.


A Induction of Treg following PBMC treatment with either native or proteinase K - treated CLys or CSN ( 10µg / ml ) for 72h .

O O 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 Induction of Treg following PBMC treatment with either native or proteinase K-treated CLys or CSN (10µg/ml) for 72h.


C PBMCs were treated with GDH inhibitor bithionol ( BT ) ( 20µM ) or IDH inhibitor IDH - 305 ( 50nM ) alone or in combination before incubation with CLys or CSN . Percentage of Treg was determined and PGE2 and IL - 10 levels were evaluated .

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

C PBMCs were treated with GDH inhibitor bithionol (BT) (20µM) or IDH inhibitor IDH-305 (50nM) alone or in combination before incubation with CLys or CSN. Percentage of Treg was determined and PGE2 and IL-10 levels were evaluated.


D Treg induction from PBMC by CLys , CSN and TsGDH ( 5µg / ml ) and levels of PGE2 and IL - 10 in culture supernatants .

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

D Treg induction from PBMC by CLys, CSN and TsGDH (5µg/ml) and levels of PGE2 and IL-10 in culture supernatants.


E Treg induction from monocyte - naïve CD4 + - TsGDH culture and levels of PGE2 and IL - 10 in monocyte - CD4 + - TsGDH and monocyte - TsGDH culture supernatants .

O O O O O O O O O O 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 Treg induction from monocyte-naïve CD4+-TsGDH culture and levels of PGE2 and IL-10 in monocyte-CD4+-TsGDH and monocyte-TsGDH culture supernatants.


F Treg induction from PBMC by CLys , CSN and rhGDH ( 10µg / ml ) .

F Treg induction from PBMC by CLys, CSN and rhGDH (10µg/ml).


Immunoprecipitation of SATB2 from mouse neonatal cortical lysates . LEMD2 was detected by Western blotting in the SATB2 - immunoprecipitate from control but not SATB2 - deficient cortical lysate . The equal input of total protein from control and SATB2 - deficient cortical lysates was controlled by ERK2 detection . Representative images of the immunoblots are shown ; I ( Input ) , F ( Flow - through ) , B ( Beads ) .

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

Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2-immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads).


Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody , followed by WB detection of LEMD2 and SATB2 . Lysates from primary cortical culture lysates were used .

Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used.


GST pull - down assays . Representative images of n = 3 independent experiments are shown . GST pull - down assay of transiently overexpressed V5 - tagged LEMD2 ( i ) , SATB2 ( ii ) , and LEMD2ΔLEM ( iii ) from HeLa cell lysates using GST , GST - SATB2 , and GST - LEMD2 ( 413 - 503 ) hybrid proteins .

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

GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins.


Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5 - tagged full - length SATB2 ( Satb21 - 733 ) and EGFP as control . The equal input of total protein was controlled by GAPDH detection . Representative images of the immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular weight marker ) .

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

Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21-733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


The CUT - like domain of SATB2 is required for the interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa cells transfected with V5 - tagged Satb21 - 247 and Satb21 - 157 deletion mutants . LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT - like domain ( Satb21 - 247 ) . The equal input of total protein was controlled by GAPDH detection . Representative images of the immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular weight marker ) .

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

The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21-247 and Satb21-157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21-247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


CUT1 and CUT2 domains are required for the interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from HeLa cells transfected with V5 - tagged Satb2346 - 733 and Satb2616 - 733 deletion mutants . LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant ( Satb2346 - 733 ) .

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

CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).


Representative images of z - axis projected stacks of confocal images of hippocampal neuronal nuclei immunostained for Lamin B2 . Examples of spherical and infolded nuclei are shown . Scale bars : 3 μm .

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

Representative images of z-axis projected stacks of confocal images of hippocampal neuronal nuclei immunostained for Lamin B2. Examples of spherical and infolded nuclei are shown. Scale bars: 3 μm.


Activity - induced formation of nuclear infoldings is abolished in SATB2 - defficient hippocampal neurons . Bic - induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx / flx mice but not from Satb2flx / flx : : Nes - Cre mice ( Satb2NesCre ) ( n = 6 independent primary cultures , two - way ANOVA , F1 , 20 = 9 . 51 , significant interaction p = 0 . 0058 , simple main effects analysis , Satb2flx / flx cultures , Bic - treated vs untreated p = 0 . 0000043 , Satb2NesCre cultures , Bic - treated vs untreated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , untreated - 680 , Satb2flx / flx cultures , Bic - treated - 711 , Satb2NesCre , untreated - 755 , Satb2NesCre , Bic - treated - 720 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

Activity-induced formation of nuclear infoldings is abolished in SATB2-defficient hippocampal neurons. Bic-induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx/flx mice but not from Satb2flx/flx::Nes-Cre mice (Satb2NesCre) (n = 6 independent primary cultures, two-way ANOVA, F1,20 = 9.51, significant interaction p = 0.0058, simple main effects analysis, Satb2flx/flx cultures, Bic-treated vs untreated p = 0.0000043, Satb2NesCre cultures, Bic-treated vs untreated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, untreated - 680, Satb2flx/flx cultures, Bic-treated - 711, Satb2NesCre, untreated - 755, Satb2NesCre, Bic-treated - 720). Data are presented as mean ± SEM, ***p < 0.001.


Activity - induced formation of nuclear infoldings is impaired in SATB2 - deficient cortical neurons . DIV14 cortical cultures form Satb2flx / flx and Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h . The percentage of infolded nuclei following AP bursting was increased in control Satb2flx / flx cultures but not in SATB2 - deficient cultures ( n = 3 - 4 independent primary cultures , two - way ANOVA , F1 , 10 = 39 . 124 , significant interaction p = 0 . 000094 , simple main effects analysis , Satb2flx / flx cultures : Bic - treated vs NBQX - treated p = 7 . 66E - 07 , Satb2NesCre cultures : Bic - treated vs NBQX - treated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , NBQX - 606 , Satb2flx / flx cultures , Bic - 910 ; Satb2NesCre cultures , NBQX - 604 , Satb2NesCre cultures , Bic - 835 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 .

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

Activity-induced formation of nuclear infoldings is impaired in SATB2-deficient cortical neurons. DIV14 cortical cultures form Satb2flx/flx and Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h. The percentage of infolded nuclei following AP bursting was increased in control Satb2flx/flx cultures but not in SATB2-deficient cultures (n = 3-4 independent primary cultures, two-way ANOVA, F1,10 = 39.124, significant interaction p = 0.000094, simple main effects analysis, Satb2flx/flx cultures: Bic-treated vs NBQX-treated p = 7.66E-07, Satb2NesCre cultures: Bic-treated vs NBQX-treated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, NBQX - 606, Satb2flx/flx cultures, Bic - 910; Satb2NesCre cultures, NBQX -604, Satb2NesCre cultures, Bic - 835). Data are presented as mean ± SEM, ***p < 0.001.


Confocal images ( z - axis - projected stacks ) of Lamin B2 stained neuronal nuclei from the CA1 pyramidal cell layer of adult Satb2flx / flx and Satb2flx / flx : : CamK2a - Cre ( Satb2CamkCre ) mice . Scale bars : 10 μm .

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

Confocal images (z-axis-projected stacks) of Lamin B2 stained neuronal nuclei from the CA1 pyramidal cell layer of adult Satb2flx/flx and Satb2flx/flx::CamK2a-Cre (Satb2CamkCre) mice. Scale bars: 10 μm.


Analysis of the percentage of infolded nuclei in the hippocampus of Satb2flx / flx mice vs Satb2CamkCre mice . In Satb2flx / flx mice , the number of infolded nuclei was significantly higher in the CA1 area compared to the CA3 area ( p = 0 . 000001 ) and DG ( p = 1 . 8 E - 12 ) . In Satb2CamkCre mice , the number of infolded nuclei in the CA1 area was significantly reduced compared to the corresponding number in littermate controls ( p = 1 . 9 E - 09 ) . No significant differences were observed in this number in the CA3 and DG between Satb2CamkCre mice and floxed controls ( n = 4 mice , two - way ANOVA , significant interaction , F2 , 18 = 47 . 81 , p = 0 . 00001 , simple main effects analysis , CA1 area , Satb2flx / flx vs CKO mice p = 1 . 9 E - 09 , CA3 area , Satb2flx / flx vs CKO mice p = 0 . 909 , DG , Satb2flx / flx vs CKO mice p = 0 . 262 , adjustment for multiple comparisons : Bonferroni ) . Number of analyzed nuclei : Satb2flx / flx mice : 469 ( CA1 ) , 225 ( CA3 ) , 665 ( DG ) ; Satb2CamkCre : 508 ( CA1 ) , 224 ( CA3 ) , 575 ( DG ) . Data are presented as mean ± SEM , * * * p < 0 . 001 .

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

Analysis of the percentage of infolded nuclei in the hippocampus of Satb2flx/flx mice vs Satb2CamkCre mice. In Satb2flx/flx mice, the number of infolded nuclei was significantly higher in the CA1 area compared to the CA3 area (p = 0.000001) and DG (p = 1.8 E-12). In Satb2CamkCre mice, the number of infolded nuclei in the CA1 area was significantly reduced compared to the corresponding number in littermate controls (p = 1.9 E-09). No significant differences were observed in this number in the CA3 and DG between Satb2CamkCre mice and floxed controls (n = 4 mice, two-way ANOVA, significant interaction, F2,18 = 47.81, p = 0.00001, simple main effects analysis, CA1 area, Satb2flx/flx vs CKO mice p = 1.9 E-09, CA3 area, Satb2flx/flx vs CKO mice p = 0.909, DG, Satb2flx/flx vs CKO mice p = 0.262, adjustment for multiple comparisons: Bonferroni). Number of analyzed nuclei: Satb2flx/flx mice: 469 (CA1), 225 (CA3), 665 (DG); Satb2CamkCre: 508 (CA1), 224 (CA3), 575 (DG). Data are presented as mean ± SEM, ***p < 0.001.


Analysis of the percentage of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice vs . littermate controls after stereotaxic injections of AAV - SATB2 - V5 or AAV8 - EGFP . Viral delivery of SATB2 resulted in restoration of the number of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice ( n = 3 - 5 mice , one - way ANOVA followed by Hochberg post hoc test , F2 , 9 = 33 . 1 , Satb2CamkCre : : AAV - SATB2 vs . Satb2flx / flx : : AAV - EGFP , p = 0 . 0012 ; Satb2flx / flx : : AAV - EGFP vs . Satb2CamkCre : : AAV - EGFP , p = 0 . 00008 , Satb2flx / flx : : AAV - EGFP vs . Satb2CamkCre : : AAV - SATB2 , p = 0 . 072 ) . Number of analyzed nuclei : 388 ( Satb2flx / flx : : AAV - EGFP ) , 560 ( Satb2CamkCre : : AAV - EGFP ) , 546 ( Satb2CamkCre : : AAV - SATB2 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 compared to Satb2flx / flx : : AAV - EGFP , # # p < 0 . 01 , compared to Satb2CamkCre : : AAV - EGFP .

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

Analysis of the percentage of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice vs. littermate controls after stereotaxic injections of AAV-SATB2-V5 or AAV8-EGFP. Viral delivery of SATB2 resulted in restoration of the number of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice (n = 3-5 mice, one-way ANOVA followed by Hochberg post hoc test, F2,9 = 33.1, Satb2CamkCre::AAV-SATB2 vs. Satb2flx/flx::AAV-EGFP, p = 0.0012; Satb2flx/flx::AAV-EGFP vs. Satb2CamkCre::AAV-EGFP, p = 0.00008, Satb2flx/flx::AAV-EGFP vs. Satb2CamkCre::AAV-SATB2, p = 0.072). Number of analyzed nuclei: 388 (Satb2flx/flx::AAV-EGFP), 560 (Satb2CamkCre::AAV-EGFP), 546 (Satb2CamkCre::AAV-SATB2). Data are presented as mean ± SEM, ***p < 0.001 compared to Satb2flx/flx::AAV-EGFP, ##p < 0.01, compared to Satb2CamkCre::AAV-EGFP.


Nuclear infolding triggered by SATB2 overexpression requires LEMD2 . Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression and Lemd2 knockdown . AAV - SATB2 - transduced neurons ( both non - transfected and scrambled siRNA - transfected , Scr ) exhibited higher percentage of infolded nuclei compared to AAV - EGFP - transduced neurons . In contrast , LEMD2 - depleted AAV - SATB2 - transduced cultures showed similar percentage of infolded nuclei to AAV - EGFP - transduced neurons ( n = 3 - 4 independent primary cultures ; one - way ANOVA F3 , 11 = 30 . 982 , p = 0 . 000011 ; Hochberg post hoc test , AAV - EGFP vs . AAV - SATB2 p = 0 . 0001 , AAV - EGFP vs . AAV - SATB2 + Scr p = 0 . 0001 , AAV - EGFP vs . AAV - SATB2 + siLemd2 p = 0 . 9999 , AAV - SATB2 + Scr vs . AAV - SATB2 + siLemd2 p = 0 . 0001 ) . Number of nuclei analyzed : 460 ( AAV - EGFP ) , 466 ( AAV - SATB2 ) , 537 ( AAV - SATB2 + Scr ) , 597 ( AAV - SATB2 + siLem2 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 compared to AAV - EGFP ; # # # p < 0 . 001 compared to AAV - SATB2 + Scr .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 infolding triggered by SATB2 overexpression requires LEMD2. Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression and Lemd2 knockdown. AAV-SATB2-transduced neurons (both non-transfected and scrambled siRNA-transfected, Scr) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, LEMD2-depleted AAV-SATB2-transduced cultures showed similar percentage of infolded nuclei to AAV-EGFP-transduced neurons (n = 3-4 independent primary cultures; one-way ANOVA F3,11 = 30.982, p = 0.000011; Hochberg post hoc test, AAV-EGFP vs. AAV-SATB2 p = 0.0001, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0001, AAV-EGFP vs. AAV-SATB2 + siLemd2 p= 0.9999, AAV-SATB2 + Scr vs. AAV-SATB2 + siLemd2 p = 0.0001). Number of nuclei analyzed: 460 (AAV-EGFP), 466 (AAV-SATB2), 537 (AAV-SATB2 + Scr), 597 (AAV-SATB2+siLem2). Data are presented as mean ± SEM, ***p < 0.001 compared to AAV-EGFP; ###p < 0.001 compared to AAV-SATB2 + Scr.


Activity - induced formation of nuclear infoldings is impaired in LEMD2 - depleted cortical neurons . Bic - triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in cortical neurons transduced with AAV - scrambled but not with AAV - shLemd2 virus ( n = 5 independent primary cultures , two - way ANOVA , F1 , 16 = 8 . 35 , significant interaction p = 0 . 0107 , simple main effects analysis : AAV - scrambled - transduced cultures , Bic - treated vs untreated p = 0 . 0049 , AAV - shLemd2 - transduced cultures , Bic - treated vs untreated p > 0 . 99 , adjustment for multiple comparisons : Bonferroni ) . Data are presented as mean ± SEM , * * p < 0 . 01 compared to NBQX - treated cultures .

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

Activity-induced formation of nuclear infoldings is impaired in LEMD2-depleted cortical neurons. Bic-triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in cortical neurons transduced with AAV-scrambled but not with AAV-shLemd2 virus (n = 5 independent primary cultures, two-way ANOVA, F1,16 = 8.35, significant interaction p = 0.0107, simple main effects analysis: AAV-scrambled-transduced cultures, Bic-treated vs untreated p = 0.0049, AAV-shLemd2-transduced cultures, Bic-treated vs untreated p > 0.99, adjustment for multiple comparisons: Bonferroni). Data are presented as mean ± SEM, **p < 0.01 compared to NBQX-treated cultures.


LEMD2 is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo . Top panel : representative confocal images ( z - axis - projected stack ) of Lamin B2 / DAPI - stained coronal brain sections from wild - type mice after stereotaxic injection of AAV - shRNA viruses ( AAV - shLemd2 - mCherry or AAV - scrambled - mCherry ) into the dorsal hippocampus . Merged , colocalization of the three signals . Scale bars : 50 μm . Middle panel : higher magnification images of AAV - shLemd2 - transduced CA1 pyramidal neurons . Arrows show examples of mCherry - positive nuclei that are devoid of nuclear infoldings , whereas stars denote non - transduced infolded nuclei . Lower panel : higher magnification images of AAV - scrambled siRNA - transduced CA1 pyramidal neurons . Arrows show examples of infolded , mCherry - positive nuclei ; stars denote infolded , non - transduced neurons . Scale bars ( middle and lower panels ) : 10 μm .

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

LEMD2 is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo. Top panel: representative confocal images (z-axis-projected stack) of Lamin B2/DAPI-stained coronal brain sections from wild-type mice after stereotaxic injection of AAV-shRNA viruses (AAV-shLemd2-mCherry or AAV-scrambled-mCherry) into the dorsal hippocampus. Merged, colocalization of the three signals. Scale bars: 50 μm. Middle panel: higher magnification images of AAV-shLemd2-transduced CA1 pyramidal neurons. Arrows show examples of mCherry-positive nuclei that are devoid of nuclear infoldings, whereas stars denote non-transduced infolded nuclei. Lower panel: higher magnification images of AAV-scrambled siRNA-transduced CA1 pyramidal neurons. Arrows show examples of infolded, mCherry-positive nuclei; stars denote infolded, non-transduced neurons. Scale bars (middle and lower panels): 10 μm.


Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression and Vps4a / Vps4b knockdown . AAV - SATB2 - transduced neurons ( non - transfected or scrambled siRNA ( Scr ) - transfected ) exhibited higher percentage of infolded nuclei compared to AAV - EGFP - transduced neurons . In contrast , VPS4 - depleted AAV - SATB2 - transduced cultures showed similar number of infolded nuclei to AAV - EGFP - transduced neurons ( n = 4 independent primary cultures , one - way ANOVA followed by Tuckey post hoc test , F3 , 11 = 15 . 866 , p = 0 . 00026 , AAV - EGFP vs . AAV - SATB2 p = 0 . 0104 , AAV - EGFP vs . AAV - SATB2 + Scr p = 0 . 0028 , AAV - EGFP vs . AAV - SATB2 + siVps4 p = 0 . 605 , AAV - SATB2 + Scr vs . AAV - SATB2 + siVps4 p = 0 . 0007 ) . Number of nuclei analyzed : 594 ( AAV - EGFP ) , 657 ( AAV - SATB2 ) , 612 ( AAV - SATB2 + Scr ) , 428 ( AAV - SATB2 + siVps4 ) . Data are presented as mean ± SEM , * * p < 0 . 01 compared with AAV - EGFP ; # # # p < 0 . 001 compared with AAV - SATB2 + Scr .

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

Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression and Vps4a/ Vps4b knockdown. AAV-SATB2-transduced neurons (non-transfected or scrambled siRNA (Scr)-transfected) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, VPS4-depleted AAV-SATB2-transduced cultures showed similar number of infolded nuclei to AAV-EGFP-transduced neurons (n = 4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,11 =15.866, p = 0.00026, AAV-EGFP vs. AAV-SATB2 p = 0.0104, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0028, AAV-EGFP vs. AAV-SATB2 + siVps4 p = 0.605, AAV-SATB2 + Scr vs. AAV-SATB2 + siVps4 p = 0.0007). Number of nuclei analyzed: 594 (AAV-EGFP), 657 (AAV-SATB2), 612 (AAV-SATB2 + Scr), 428 (AAV-SATB2 + siVps4). Data are presented as mean ± SEM, **p < 0.01 compared with AAV-EGFP; ###p < 0.001 compared with AAV-SATB2 + Scr.


Activity - induced formation of nuclear infoldings is impaired in VPS4 - depleted cortical neurons . Upon AP bursting , the percentage of infolded nuclei was increased in non - transfected cultures and in cultures transfected with scramble siRNA ( Scr ) but not with siVps4 ( n = 3 - 4 independent primary cultures , one - way ANOVA followed by Tuckey post hoc test , F3 , 8 = 90 . 525 , p = 1 . 63E - 06 , Bic - treated vs NBQX - treated p = 0 . 000012 , Scr Bic - treated vs NBQX - treated p = 0 . 000052 , siVps4 Bic - treated vs NBQX - treated p = 0 . 396 , siVps4 Bic - treated vs Scr Bic - treated p = 0 . 000015 ) . Number of analyzed nuclei : 474 ( NBQX ) , 456 ( Bic ) , 409 ( Bic + Scr ) , 383 ( Bic + siVps4 ) . Data are presented as mean ± SEM , * * * p < 0 . 001 compared to NBQX - treated , # # # p < 0 . 001 compared to Scr Bic - treated .

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

Activity-induced formation of nuclear infoldings is impaired in VPS4-depleted cortical neurons. Upon AP bursting, the percentage of infolded nuclei was increased in non-transfected cultures and in cultures transfected with scramble siRNA (Scr) but not with siVps4 (n = 3-4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,8 = 90.525, p = 1.63E-06, Bic-treated vs NBQX-treated p = 0.000012, Scr Bic-treated vs NBQX-treated p = 0.000052, siVps4 Bic-treated vs NBQX-treated p = 0.396, siVps4 Bic-treated vs Scr Bic-treated p = 0.000015). Number of analyzed nuclei: 474 (NBQX), 456 (Bic), 409 (Bic + Scr), 383 (Bic + siVps4). Data are presented as mean ± SEM, ***p < 0.001 compared to NBQX-treated, ###p < 0.001 compared to Scr Bic-treated.


Representative confocal images ( Z - axis projection ) of CA1 pyramidal neurons of : ( top panel ) Satb2 floxed mice ( n = 3 ) maintained in their home cages ( HC ) , ( middle panel ) Satb2 floxed mice ( n = 4 ) subjected to NEE for 1 . 5 h , ( bottom panel ) Satb2CamkCre mice ( n = 5 ) exposed to NE for 1 . 5 h . Brain sections were stained for cFos and Lamin B2 . Scale bars : 10 μm . Higher magnification views of boxed areas in A ( middle panel ) . Arrows show examples of infolded , cFos - positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE . Scale bars : 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal images (Z-axis projection) of CA1 pyramidal neurons of: (top panel) Satb2 floxed mice (n = 3) maintained in their home cages (HC), (middle panel) Satb2 floxed mice (n = 4) subjected to NEE for 1.5 h, (bottom panel) Satb2CamkCre mice (n = 5) exposed to NE for 1.5 h. Brain sections were stained for cFos and Lamin B2. Scale bars: 10 μm. Higher magnification views of boxed areas in A (middle panel). Arrows show examples of infolded, cFos-positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE. Scale bars: 5 μm.


( a ) Flow cytometry of LPS - stimulated B cells stained with LysoTracker Red .

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(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.


( b ) Autophagic flux of GFP - LC3 splenic B cells stimulated with LPS and then left untreated ( UT ) or treated for 1 h with bafimolycin A1 ( BafA1 ) , assessed by live - cell fluorescence microscopy ( left ) and automated quantification of spots ( right ) . Scale bar ( left ) , 5 μm .

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(b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm.


( c ) Immunoblot analysis of the processing of endogenous unconjugated LC3 - I to lipid - conjugated LC3 - II in LPS - stimulated B cells with ( + NH4Cl ) or without treatment with NH4Cl ( left ) . Actin serves as a loading control throughout . Right , quantification of LC3 - II band intensity in the absence of NH4Cl , presented relative to that of actin .

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

(c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin.


( d ) Quantitative RT - PCR analysis of transcripts encoding autophagy - related molecules in ASCs 3 d after stimulation with LPS , presented relative to that in unstimulated B cells .

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

(d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells.


( e ) Immunoblot analysis of p62 in LPS - stimulated B cells ( bottom ) . Above , quantification of band intensity ( as in c ) .

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

(e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c).


( f ) Live - cell fluorescence microscopy of CD138 + PCs purified from the spleen of GFP - LC3 mice 5 d after the injection of NP - Ficoll ( PCs ) and of splenic B cells ( B cells ) and bone marrow CD138 + PCs ( BM PCs ) purified from unstimulated GFP - LC3 mice ( right ) and automated quantification of spots ( left ) . Scale bar ( right ) , 5 μm . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) . Data are representative of three or more independent experiments ( a , c - e ) or one experiment or more with > 40 cells ( b , f ; average and s . e . m . ) .

(f) Live-cell fluorescence microscopy of CD138+ PCs purified from the spleen of GFP-LC3 mice 5 d after the injection of NP-Ficoll (PCs) and of splenic B cells (B cells) and bone marrow CD138+ PCs (BM PCs) purified from unstimulated GFP-LC3 mice (right) and automated quantification of spots (left). Scale bar (right), 5 μm. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test). Data are representative of three or more independent experiments (a,c-e) or one experiment or more with >40 cells (b,f; average and s.e.m.).


( a ) Proliferation of B cells purified from Atg5f / fCD19 - Cre and Atg5f / f control mice and activated for 3 d with LPS , assessed by flow cytometry analysis of dilution of the membrane dye CFSE . ( b ) Size of B cells purified as in a and activated for various times ( horizontal axis ) with LPS , assessed by the intensity of forward scatter ( FSC ) by flow cytometry . ( c ) CD138 expression of B cells purified and activated as in a . APC , allophycocyanin ; PE , phycoerythrin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin.


( d ) Immunoblot analysis of ER proteins in lysates of Atg5f / f and Atg5f / fCD19 - Cre B cells 3 d after stimulation with LPS ( left ) , and quantification of band intensity ( right ; as in Fig . 1c ) . IgM - H , IgM heavy chain . NS , not significant ; * P 0 . 05 and * * P 0 . 01 ( Student ' s t - test ) .

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

(d) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test).


( a ) Electron microscopy of Atg5f / f and Atg5f / fCD19 - Cre B cells 3 d after stimulation with LPS ( left and middle ) , and quantification of the ER by stereology ( right ) . Scale bars ( left and middle ) , 1 μm .

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

(a) Electron microscopy of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left and middle), and quantification of the ER by stereology (right). Scale bars (left and middle), 1 μm.


( b ) G6Pase electron - microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS ( left ) , and quantification of G6Pase + ( ER ) area , relative to total cell area ( right ) . Scale bars ( left ) , 1 μm .

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

(b) G6Pase electron-microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS (left), and quantification of G6Pase+ (ER) area, relative to total cell area (right). Scale bars (left), 1 μm.


( c ) Quantitative RT - PCR analysis of transcripts encoding spliced XBP - 1 ( sXBP - 1 ) , total XBP - 1 ( tXBP - 1 ) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS , presented relative to that of unstimulated Atg5f / f B cells at day 3 .

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

(c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3.


( d ) Quantitative RT - PCR analysis of CHOP mRNA in Atg5f / f and Atg5f / fCD19 - Cre B cells stimulated for 3 d with LPS ( left and middle ) , and in Atg5f / f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin ( Atg5f / f 24 h tun ; 0 . 5 μg / ml ; positive control ) , presented relative to that in Atg5f / f B cells stimulated for 3 d with LPS .

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

(d) Quantitative RT-PCR analysis of CHOP mRNA in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS (left and middle), and in Atg5f/f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin (Atg5f/f 24 h tun; 0.5 μg/ml; positive control), presented relative to that in Atg5f/f B cells stimulated for 3 d with LPS.


( e ) Immunoblot analysis ( left ) of ER proteins in Atg5f / f B cells left untreated ( UT ) or treated with lysosomal protease inhibitors ( 10 μM leupeptin and E - 64d ) for the final 8 h of the third day of LPS - induced differentiation ( Leu + E - 64d ( 8 h ) ) . Right , quantification of band intensity ( presented as in Fig . 1c ) . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(e) Immunoblot analysis (left) of ER proteins in Atg5f/f B cells left untreated (UT) or treated with lysosomal protease inhibitors (10 μM leupeptin and E-64d) for the final 8 h of the third day of LPS-induced differentiation (Leu + E-64d (8 h)). Right, quantification of band intensity (presented as in Fig. 1c). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Enzyme - linked immunosorbent assay ( ELISA ) of IgM in supernatants of Atg5f / fCD19 - Cre and Atg5f / f B cells stimulated for 3 d with LPS and plated for 4 h ( as trypan blue - negative cells at a density of 1 × 106 cells per ml in dedicated media ) , presented relative to results obtained for Atg5f / f cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells.


( b , c ) Immunoglobulin assembly ( b ) and total intracellular ( IC ) and secreted ( Sec ) IgM heavy chain ( IgM - H , c ) in Atg5f / f and Atg5f / fCD19 - CreB cells stimulated for 3 d with LPS , then pulse - labeled and chased ( time , above lanes ) , followed by immunoprecipitation of the μ - chain from lysates and SDS - PAGE ( left ) in nonreducing conditions ( b ) or reducing conditions ( c ) . Right , quantification of intracellular IgM polymers by densitometry ( b ) and of secretion efficiency , estimated as the amount of radioactive secreted μ - chain at the end of the chase relative to intracellular μ - chain at the end of the pulse ( c ) . AU , arbitrary units .

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

(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.


( d ) Quantitative RT - PCR analysis of mRNA encoding Blimp - 1 and the secreted μ - chain in Atg5f / f and Atg5f / fCD19 - Cre B cells stimulated for 3 d with LPS , presented relative to that of Atg5f / f cells .

(d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells.


( e ) Quantitative RT - PCR analysis of transcripts encoding spliced XBP - 1 , BiP , Blimp - 1 and secreted μ - chain in Atg5f / f B cells stimulated for 3 d with LPS and given no further treatment ( − tun ) or treated for the final 24 h with tunicamycin ( tun 24 h ; 0 . 5 μg / ml ) , presented relative to results obtained with LPS - stimulated cells given no further treatment . * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ( Student ' s t - test ) .

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

(e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).


( a ) Intracellular ATP in fresh lysates of Atg5f / f and Atg5f / fCD19 - Cre splenic B cells activated for 3 d with LPS ( 3 ) or left untreated ( 0 ) .

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(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0).