( C ) The increased spleen weight on day 9 in Am80 - GCSF mice dropped to the levels similar to those in control mice on day 13 ( sections iii , iv ) .

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(C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv).


( C ) The increased spleen size on day 9 in Am80 - GCSF mice dropped to the levels similar to those in control mice on day 13 ( section iii ) .

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(C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii).


( D ) Significant loss of body weight occurred in GCSFmice on day 9 .

(D) Significant loss of body weight occurred in GCSFmice on day 9.


( E ) Ultrastructure analysis of neutrophils collected from mice surviving on day 11 . Neutrophil from GCSFmice showed less granules ( section i , images 1 vs . 2 or 3 ) , more intracytoplasmic vacuoles ( sections i , ii ) , and inner nuclear membrane dilation ( sections i , iii ) . Pg : primary granules ; Sg : secondary granules .

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(E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSFmice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules.


Sqr mRNA levels ( A , B , C ) , in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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Sqr mRNA levels (A, B, C), in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


SQR protein levels ( D , E , F ) in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . Note that SQRwestern - blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum . In kidneys and muscle the western - blots were performed in tissue homogenates .

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SQR protein levels (D, E, F) in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice. Note that SQRwestern-blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum. In kidneys and muscle the western-blots were performed in tissue homogenates.


SQR activity ( G , H ) in kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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SQR activity (G, H) in kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Levels of CoQ10 in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency ( A ) .

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Levels of CoQ10 in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency (A).


Levels of SQR protein in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency cultured without ( vehicle ) and with 5μM of CoQ10 ( + CoQ10 ) during 1 day or 7 days ( B ) .

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Levels of SQR protein in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency cultured without (vehicle) and with 5μM of CoQ10 (+ CoQ10) during 1 day or 7 days (B).


Total CoQ levels ( CoQ9 + CoQ10 ) in kidneys ( C ) and muscle ( D ) of Coq9 + / + , Coq9R239X and Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice .

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Total CoQ levels (CoQ9 + CoQ10) in kidneys (C) and muscle (D) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


Levels of SQR protein in kidneys ( E ) and muscle ( F ) of Coq9 + / + , Coq9R239X and Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice .

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Levels of SQR protein in kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


TST protein levels in cerebrum ( A ) , kidneys ( B ) and muscle ( C ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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TST protein levels in cerebrum (A), kidneys (B) and muscle (C) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


TST activity in cerebrum ( D ) , kidneys ( E ) and muscle ( F ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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TST activity in cerebrum (D), kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


ETHE1 ( SDO ) protein levels in cerebrum ( G ) , kidneys ( H ) and muscle ( I ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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ETHE1 (SDO) protein levels in cerebrum (G), kidneys (H) and muscle (I) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


SUOX protein levels in cerebrum ( J ) , kidneys ( K ) and muscle ( L ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

SUOX protein levels in cerebrum (J), kidneys (K) and muscle (L) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Quantification of sulfide levels in cerebrum and kidneys of Coq9 + / + , Coq9R239X and Coq9Q95X mice ( A ) .

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Quantification of sulfide levels in cerebrum and kidneys of Coq9+/+, Coq9R239X and Coq9Q95X mice (A).


Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9 + / + and Coq9R239X mice ( B ) .

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Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).


Total GSH in cytosol and mitochondria of cerebrum of Coq9 + / + and Coq9R239X mice ( A ) .

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Total GSH in cytosol and mitochondria of cerebrum of Coq9+/+ and Coq9R239X mice(A).


Cytosolic GPx and GRd activities in cerebrum of Coq9 + / + and Coq9R239X mice ( B ) .

Cytosolic GPx and GRd activities in cerebrum of Coq9+/+ and Coq9R239X mice (B).


Levels of GPx4 ( C ) and GRd ( D ) protein in cerebral homogenate of Coq9 + / + and Coq9R239X mice .

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Levels of GPx4 (C) and GRd (D) protein in cerebral homogenate of Coq9+/+ and Coq9R239X mice.


Levels of L - Glutamate ( L - Glu ) , N - Acetyl - Glutamate ( NacGlu ) , L - Tryptophan ( L - Trp ) , 5HIAA , N - Acetyl - Tryptophan ( NALT ) , L - Tyrosine ( L - Tyr ) in cerebrum of Coq9 + / + and Coq9R239X mice ( E ) .

Levels of L-Glutamate (L-Glu), N-Acetyl-Glutamate (NacGlu), L-Tryptophan (L-Trp), 5HIAA, N-Acetyl-Tryptophan (NALT), L-Tyrosine (L-Tyr) in cerebrum of Coq9+/+ and Coq9R239X mice (E).


SQR ( A ) and TST ( B ) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137 .

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SQR (A) and TST (B) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137.


TST protein level in kidneys ( C ) and cerebrum ( D ) of Coq9 + / + mice supplemented with the H2S donor GYY4137 .

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TST protein level in kidneys (C) and cerebrum (D) of Coq9+/+ mice supplemented with the H2S donor GYY4137.


Levels of neurotransmitters in cerebrum of Coq9 + / + mice supplemented with the H2S donor GYY4137 ( E ) .

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Levels of neurotransmitters in cerebrum of Coq9+/+ mice supplemented with the H2S donor GYY4137 (E).


COX activity in cerebrum , kidneys and muscle ( A ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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COX activity in cerebrum, kidneys and muscle (A) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Images COX histochemistry ( B ) in gastrocnemious of Coq9 + / + , Coq9R239X and Coq9Q95X mice ; scale bars : 100 μm .

Images COX histochemistry (B) in gastrocnemious of Coq9+/+, Coq9R239X and Coq9Q95X mice; scale bars: 100 μm.


Systolic , diastolic and mean blood pressure in Coq9 + / + and Coq9R239X mice ( A ) .

Systolic, diastolic and mean blood pressure in Coq9+/+ and Coq9R239X mice (A).


Heart rate in Coq9 + / + and Coq9R239X mice ( B ) . * * P < 0 . 01 ; Coq9R239X mice versus Coq9 + / + mice ( t test ; n = 5 for each group ) .

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Heart rate in Coq9+/+ and Coq9R239X mice (B). **P < 0.01; Coq9R239X mice versus Coq9+/+ mice (t test; n = 5 for each group).


A . Enrichment of lamin B1 in the SGZ of DG .

A. Enrichment of lamin B1 in the SGZ of DG.


B . Immunohistochemical analyses of lamin B1 - high cells . The lamin B1 - high cells express Sox2 or PSA - NCAM , but not Prox1 . Arrowheads indicate Prox1 negative lamin B1 - high cells in the SGZ .

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B. Immunohistochemical analyses of lamin B1-high cells. The lamin B1-high cells express Sox2 or PSA-NCAM, but not Prox1. Arrowheads indicate Prox1 negative lamin B1-high cells in the SGZ.


C . Immunohistochemical analyses of lamin B1 in pNestin - GFP mice . An open arrowhead indicates RGL - ANSC and a closed arrowhead indicates a non - RGL ( NR ) - NPC .

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C. Immunohistochemical analyses of lamin B1 in pNestin-GFP mice. An open arrowhead indicates RGL-ANSC and a closed arrowhead indicates a non-RGL(NR)-NPC.


E . Immunocytochemistry of lamin B1 in NPCs and Tuj1 + differentiating neurons 4 days after the induction of differentiation .

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E. Immunocytochemistry of lamin B1 in NPCs and Tuj1+ differentiating neurons 4 days after the induction of differentiation.


F . Relative expression of Lmnb1 and Tubb3 revealed by qRT - PCR from NPCs and differentiating neurons at the indicated days . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one sample t - test ( n = 4 ) .

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F. Relative expression of Lmnb1 and Tubb3 revealed by qRT-PCR from NPCs and differentiating neurons at the indicated days. ***P < 0.001, **P < 0.01, *P < 0.05, one sample t-test (n = 4).


G . Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types . F ( 3 , 82 ) = 173 . 1 , P = 1 . 2 e - 34 , n = 12 - 30 cells per cell type , one - way ANOVA followed by Tukey - Kramer test ( * * P < 0 . 01 ) .

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G. Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types. F (3,82) = 173.1, P = 1.2 e-34, n = 12-30 cells per cell type, one-way ANOVA followed by Tukey-Kramer test (**P < 0.01).


A . Age - dependent reduction of lamin B1 in the SGZ . As early as 5 . 5 months of age , the levels of lamin B1 in the SGZ were markedly reduced . Insets show higher magnification images in the SGZ .

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A. Age-dependent reduction of lamin B1 in the SGZ. As early as 5.5 months of age, the levels of lamin B1 in the SGZ were markedly reduced. Insets show higher magnification images in the SGZ.


B - D . Quantification of lamin B1 levels using immunofluorescent signals . Lamin B1 levels in Sox2 - positive , PSA - NCAM - negative - ANSPCs were selectively downregulated at 5 . 5 months of age ( ANPCs , P = 1 . 02129e - 19 ; Neuroblasts , P = 0 . 09 , DGCs , P = 6 . 4011e - 15 , ANOVA ) . * * P < 0 . 001 , ANOVA followed by Tukey - Kramer test . ( 3 animals , 30 cells per conditions )

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B-D. Quantification of lamin B1 levels using immunofluorescent signals. Lamin B1 levels in Sox2-positive, PSA-NCAM-negative-ANSPCs were selectively downregulated at 5.5 months of age (ANPCs, P = 1.02129e-19; Neuroblasts, P = 0.09, DGCs, P = 6.4011e-15, ANOVA). ** P < 0.001, ANOVA followed by Tukey-Kramer test. (3 animals, 30 cells per conditions)


E . Age - dependent reduction of lamin B1 in RGL - ANSCs ( upper ) and NR - ANPC ( lower ) from 2 to 5 . 5 months of age . Arrowheads indicate RGL - ANSCs or NR - ANPCs . F . Quantification of lamin B1 levels in RGL - ANSCs and ANPCs . * P < 0 . 05 , * * * P < 0 . 001 , ( 4 animals , 18 - 32 cells per conditions , Mann - Whitney test ) .

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E. Age-dependent reduction of lamin B1 in RGL-ANSCs (upper) and NR-ANPC (lower) from 2 to 5.5 months of age. Arrowheads indicate RGL-ANSCs or NR-ANPCs. F. Quantification of lamin B1 levels in RGL -ANSCs and ANPCs. * P < 0.05, *** P < 0.001, (4 animals, 18-32 cells per conditions, Mann-Whitney test).


Open field ( OF ) test . ( B ) Color - coded distribution of the exploratory path in the open field test . X - Y axes of squares correspond to the OF arena . Old WT mice as well as Lmnb1 - cKO mice spend more time in the periphery of the OF arena .

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Open field (OF) test. (B) Color-coded distribution of the exploratory path in the open field test. X-Y axes of squares correspond to the OF arena. Old WT mice as well as Lmnb1-cKO mice spend more time in the periphery of the OF arena.


Open field ( OF ) test . ( C ) Total distance traveled during the OF test . No significant difference among groups . F ( 2 , 25 ) = 2 . 23 , P = 0 . 13 , ANOVA . ( D ) Velocity of travel during the OF test . F ( 2 , 25 ) = 1 . 60 , P = 0 . 22 , ANOVA . N . S . ( not significant ) . ( E ) Fraction of time spent in the periphery . F ( 2 , 25 ) = 10 . 71 , P = 0 . 00044 , ANOVA followed by Tukey Kramer , * * P < 0 . 01 . ( F ) Fraction of traveled distance in the periphery , F ( 2 , 25 ) = 3 . 84 , P = 0 . 035 , ANOVA followed by Tukey Kramer , * P < 0 . 05 ( n : WT = 10 , WT - old = 7 , Lmnb1 - cKO = 11 for the OF test ) .

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Open field (OF) test. (C) Total distance traveled during the OF test. No significant difference among groups. F(2, 25) = 2.23, P = 0.13, ANOVA. (D) Velocity of travel during the OF test. F(2, 25) = 1.60, P = 0.22, ANOVA. N.S. (not significant). (E) Fraction of time spent in the periphery. F(2, 25) = 10.71, P = 0.00044, ANOVA followed by Tukey Kramer, ** P < 0.01. (F) Fraction of traveled distance in the periphery, F(2, 25) = 3.84, P = 0.035, ANOVA followed by Tukey Kramer, * P < 0.05 (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test).


H - J . Novelty suppressed feeding . ( H ) Total feeding time during the NSF test , P = 0 . 0068 , Kruskal - Wallis test followed by Dunn ' s test . * * P < 0 . 01 . ( n : WT = 6 , WT - old = 7 , Lmnb1 - cKO = 6 for novelty suppressed feeding ) . ( I ) Food consumption during the NSF test , P = 0 . 014 , Kruskal - Wallis test followed by Dunn ' s test . * P < 0 . 05 . ( J ) Total number of feeding instances during NSF test . N . S . ( not significant ) .

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H-J. Novelty suppressed feeding. (H) Total feeding time during the NSF test, P = 0.0068, Kruskal-Wallis test followed by Dunn's test. ** P < 0.01. (n: WT = 6, WT-old =7, Lmnb1-cKO = 6 for novelty suppressed feeding). (I) Food consumption during the NSF test, P = 0.014, Kruskal-Wallis test followed by Dunn's test. * P < 0.05. (J) Total number of feeding instances during NSF test. N.S. (not significant).


B , C . Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment . Note that the numbers of DCX - positive cells are increased . Boxes indicate magnified regions in C .

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B, C. Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment. Note that the numbers of DCX-positive cells are increased. Boxes indicate magnified regions in C.


D , E , F . Quantification three weeks after TAM treatment . Among EYFP - positive cells , the density of RGL - ANSCs is not different ( P = 0 . 65 , t - test ) ( D ) . The density of ANPCs is significantly decreased ( P = 0 . 0037 , t - test ) ( E ) whereas the density of DCX - positive cells is markedly increased ( P = 0 . 014 , t - test ) ( F ) . ( n = 4 for Control , n = 3 for cKO ) . Open arrowheads indicate respective cells . G , H , I . Quantification two months after the induction of KO ( n = 3 for Control , n = 4 for cKO ) . The density of RGL - ANSCs is not significantly different ( P = 0 . 29 , t - test ) ( G ) . The density of ANPCs is significantly decreased . ( P = 0 . 0092 , t - test ) ( H ) . The density of neuroblasts is not significantly different ( P = 0 . 76 , t - test ) ( I ) .

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D, E, F. Quantification three weeks after TAM treatment. Among EYFP-positive cells, the density of RGL-ANSCs is not different (P = 0.65, t-test) (D). The density of ANPCs is significantly decreased (P = 0.0037, t-test) (E) whereas the density of DCX-positive cells is markedly increased (P = 0.014, t-test) (F). (n = 4 for Control, n =3 for cKO). Open arrowheads indicate respective cells. G, H, I. Quantification two months after the induction of KO (n = 3 for Control, n = 4 for cKO). The density of RGL-ANSCs is not significantly different (P = 0.29, t-test) (G). The density of ANPCs is significantly decreased. (P = 0.0092, t-test) (H). The density of neuroblasts is not significantly different (P = 0.76, t-test) (I).


J , K , L . Quantification of proliferating cells three weeks after TAM treatment . The density of Ki67 + RGL - ANSCs is significantly decreased ( P = 0 . 027 , Mann Whitney test ) ( J ) , and that of Ki67 + ANPCs is also significantly decreased . ( P = 0 . 034 , t - test ) ( K ) . The density of Ki67 + neuroblasts is significantly increased ( P = 0 . 0011 , t - test ) ( N ) ( n = 4 for Control , n = 4 for cKO ) ( L ) . M , N , O . Quantification of proliferating cells two months after TAM treatment . The density of Ki67 + RGL - ANSCs is significantly decreased ( P = 0 . 0086 , t - test ) ( M ) , and densities of Ki67 + ANPCs and Ki67 + neuroblasts are also significantly decreased . ( P = 0 . 023 , P = 0 . 025 , t - test ) ( n = 3 for Control , n = 3 for cKO ) ( N , O ) .

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J, K, L. Quantification of proliferating cells three weeks after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.027, Mann Whitney test) (J), and that of Ki67+ANPCs is also significantly decreased. (P = 0.034, t-test) (K). The density of Ki67+ neuroblasts is significantly increased (P = 0.0011, t-test) (N) (n = 4 for Control, n = 4 for cKO) (L). M, N, O. Quantification of proliferating cells two months after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.0086, t-test) (M), and densities of Ki67+ANPCs and Ki67+neuroblasts are also significantly decreased. (P = 0.023, P = 0.025, t-test) (n = 3 for Control, n = 3 for cKO) (N, O).


P . Declined return to RGL - ANSCs . Left , experimental scheme . Analysis was performed 3 weeks after BrdU injections . Middle , a representative image of BrdU + GFAP + RGL - ANSCs . Right , Quantification of BrdU + GFAP + RGL - ANSCs ( P = 0 . 019 , n = 4 for Control , n = 4 for cKO ) .

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P. Declined return to RGL-ANSCs. Left, experimental scheme. Analysis was performed 3 weeks after BrdU injections. Middle, a representative image of BrdU+GFAP+ RGL-ANSCs. Right, Quantification of BrdU+GFAP+RGL-ANSCs (P = 0.019, n = 4 for Control, n = 4 for cKO).


Representative confocal images of the SGZ 6 . 5 months after the treatment with TAM .

Representative confocal images of the SGZ 6.5 months after the treatment with TAM.


C . Representative confocal images of the SGZ 6 . 5 months after the treatment with TAM .

C. Representative confocal images of the SGZ 6.5 months after the treatment with TAM.


Quantification of cell numbers 6 . 5 months after the induction of KO ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M , ) . ( D ) The density of RGL - ANSCs tends to be reduced in cKO . F ( 2 , 8 ) = 3 . 98 , P = 0 . 063 , One - way ANOVA . ( E ) The density of ANPCs was reduced in cKO - 8M and Cont - 15M mice . F ( 2 , 8 ) = 11 . 49 , P = 0 . 0044 , One - way ANOVA followed by Tukey ' s HSD test ( * * P < 0 . 01 ) .

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Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (D) The density of RGL-ANSCs tends to be reduced in cKO. F (2, 8) = 3.98, P = 0.063, One-way ANOVA. (E) The density of ANPCs was reduced in cKO-8M and Cont-15M mice. F (2, 8) = 11.49, P = 0.0044, One-way ANOVA followed by Tukey's HSD test (** P < 0.01).


Quantification of cell numbers 6 . 5 months after the induction of KO ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M , ) . ( F ) The density of DCX - positive cells was reduced in WT - old and cKO mice . p = 0 . 0091 , Kruskal - Wallis test followed by Dunn ' s test ( * P < 0 . 05 ) . ( G ) The density of Ki67 + RGL - ANSCs was reduced in cKO - 8M mice ( * P = 0 . 029 , Kruskal - Wallis test followed by Dunn ' s test ) . ( H ) The density of Ki67 + ANPC was not significantly different among three groups ( P = 0 . 49 , Kruskal - Wallis test ) . ( I ) The density of NeuN + EYFP + neurons was reduced in cKO - 8M mice . F ( 2 , 8 ) = 34 . 85 , P = 0 . 00011 , One - way ANOVA followed by Tukey ' s HSD test ( * * P < 0 . 001 ) . ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M ) .

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Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (F) The density of DCX-positive cells was reduced in WT-old and cKO mice. p = 0.0091, Kruskal-Wallis test followed by Dunn's test (* P < 0.05). (G) The density of Ki67+RGL-ANSCs was reduced in cKO-8M mice (* P = 0.029, Kruskal-Wallis test followed by Dunn's test). (H) The density of Ki67+ANPC was not significantly different among three groups (P = 0.49, Kruskal-Wallis test). (I) The density of NeuN+EYFP+ neurons was reduced in cKO-8M mice. F (2, 8) = 34.85, P = 0.00011, One-way ANOVA followed by Tukey's HSD test (** P < 0.001). (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M).


MA plot of differentially expressed genes between control and Lmnb1 - KO NPCs .

MA plot of differentially expressed genes between control and Lmnb1-KO NPCs.


E . Fraction of direction of gene regulation by LaminB1 . Genes were allocated dependent on where LaminB1 interacts with genes . Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout .

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E. Fraction of direction of gene regulation by LaminB1. Genes were allocated dependent on where LaminB1 interacts with genes. Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout.


Confirmation of Lamin B1 overexpression by qRT - PCR ( * P = 0 . 020 , n = 3 , one - sample t - test )

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Confirmation of Lamin B1 overexpression by qRT-PCR (*P = 0.020, n = 3, one-sample t-test)


C . Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs . Scale bar = 10 µm .

C. Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs. Scale bar = 10 µm.


D , Relative induced expression of differentiation markers upon the withdrawal of FGF2 . 0 % indicates the same expression levels with control . Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation ( Tuj1 , * P = 0 . 023 ; Prox1 , * * * P = 0 . 00012 ; NeuroD1 , * * P = 0 . 0046 ; Ascl1 , * * * P = 0 . 00064 ; S100β , * * * P = 0 . 000097 ; n = 3 , one - sample t - test ) .

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D, Relative induced expression of differentiation markers upon the withdrawal of FGF2. 0 % indicates the same expression levels with control. Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation (Tuj1, *P = 0.023; Prox1, ***P = 0.00012; NeuroD1, ** P = 0.0046; Ascl1, ***P = 0.00064; S100β, ***P = 0.000097; n = 3, one-sample t-test).


F , Confocal images after exogenous expression of lamin B1 in EGFP + cells with RV ; LaminB1 - IRES - GFP ( laminB1 - OE . ) Scale bar = 20 µm .

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F, Confocal images after exogenous expression of lamin B1 in EGFP+ cells with RV;LaminB1-IRES-GFP (laminB1-OE.) Scale bar = 20 µm.


G , H , Exogenous expression of laminB1 repressed neuronal differentiation in vivo . An arrowhead indicate DCX + GFP + cell in control ( G , left ) and open arrowheads indicate Sox2 + GFP + cells in lamin B1 - OE cells . The fraction of EGFP + DCX + cells was significantly reduced by lamin B1 overexpression ( * * * P < 0 . 0002 , n = 3 ) , whereas the fractions of EGFP + DCX + Sox2 + cells and the EGFP + Sox2 + were increased ( EGFP + DCX + Sox2 + , * * P = 0 . 0024 ; EGFP + Sox2 + , P = 0 . 058 ; others , * P = 0 . 02 ) . Scale bar = 20 µm .

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G, H, Exogenous expression of laminB1 repressed neuronal differentiation in vivo. An arrowhead indicate DCX+GFP+ cell in control (G, left) and open arrowheads indicate Sox2+GFP+ cells in lamin B1-OE cells. The fraction of EGFP+ DCX+ cells was significantly reduced by lamin B1 overexpression (***P < 0.0002, n = 3), whereas the fractions of EGFP+DCX+Sox2+ cells and the EGFP+Sox2+ were increased (EGFP+DCX+Sox2+, **P = 0.0024; EGFP+ Sox2+, P = 0.058; others, *P = 0.02). Scale bar = 20 µm.


E . As in ( D ) , except that cells were treated with Cisplatin for 24 h ( mean ± SEM ; n = 3 independent experiments ) . F . U2OS WT , U2OS / SCAI KO and U2OS / SCAI KO / Strep - HA - SCAI cells were treated or not with MMC ( 9 nM ) for 48 h , fixed and co - stained with PCNA antibody and DAPI . Cell cycle distribution was analyzed by quantitative image - based cytometry ( QIBC ) ( ≥ 2000 cells analyzed per condition ) . Data from a representative experiment are shown .

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E. As in (D), except that cells were treated with Cisplatin for 24 h (mean±SEM; n=3 independent experiments). F. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (9 nM) for 48 h, fixed and co-stained with PCNA antibody and DAPI. Cell cycle distribution was analyzed by quantitative image-based cytometry (QIBC) (≥2000 cells analyzed per condition). Data from a representative experiment are shown.


G . U2OS WT , U2OS / SCAI KO and U2OS / SCAI KO / Strep - HA - SCAI cells were treated or not with MMC ( 90 nM ) for 1 h , fixed 24 h later and co - stained with RPA2 antibody and DAPI . RPA2 foci were quantified by QIBC ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; ns , not significant , two - tailed paired t - test ) . H . As in ( G ) , except that cells were co - stained with γH2AX antibody and DAPI ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant , two - tailed paired t - test ) .

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G. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). H. As in (G), except that cells were co-stained with γH2AX antibody and DAPI (≥3000 cells analyzed per condition; mean± SD; n=3 independent experiments; *, p<0.05; **, p<0.01; ns, not significant, two-tailed paired t-test).


I . Experimental workflow for metaphase chromosome morphology analysis ( top ) and representative images of metaphase spreads from indicated cell lines treated or not with MMC ( bottom ) . DNA was stained with DAPI . Scale bars , 10 µm . J . Quantification of radial chromosomes in ( I ) ( mean ± SD ; 180 cells analyzed per condition ; n = 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 , ns , not significant , two - tailed t - test ) . K . Quantification of chromosomal breaks / gaps in ( I ) ( mean ± SEM ; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 , ns , not significant , Mann - Whitney U 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 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 O O O O O O O O O

I. Experimental workflow for metaphase chromosome morphology analysis (top) and representative images of metaphase spreads from indicated cell lines treated or not with MMC (bottom). DNA was stained with DAPI. Scale bars, 10 µm. J. Quantification of radial chromosomes in (I) (mean±SD; 180 cells analyzed per condition; n=3 independent experiments; *, p<0.05; **, p<0.01, ns, not significant, two-tailed t-test). K. Quantification of chromosomal breaks/gaps in (I) (mean±SEM; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments; **, p<0.01; ****, p<0.0001, ns, not significant, Mann-Whitney U test).


F . U2OS and U2OS / SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC ( 90 nM ) for 1 h , fixed 24 h later and co - stained with RPA2 antibody and DAPI . RPA2 foci were quantified by QIBC ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; ns , not significant , two - tailed paired t - test ) . G . As in ( F ) , except that cells were stained with γH2AX antibody ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * * , p < 0 . 01 ; ns , not significant , two - tailed paired t - test ) .

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F. U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). G. As in (F), except that cells were stained with γH2AX antibody (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; **, p<0.01; ns, not significant, two-tailed paired t-test).


I . Clonogenic survival of U2OS and U2OS / FANCA KO cells subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) . J . Clonogenic survival of U2OS , U2OS / SCAI KO , U2OS / FANCA KO , and U2OS / FANCA + SCAI DKO cells subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) .

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I. Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). J. Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI DKO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).


K . Immunoblot analysis of U2OS , U2OS / SCAI KO , U2OS / FANCA KO , and U2OS / FANCA SCAI DKO cell lines exposed or not to MMC as indicated .

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K. Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.


B . pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i ( 100 µM ) and associated proteins were analyzed by immunoblotting .

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B. pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i (100 µM) and associated proteins were analyzed by immunoblotting.


F . pICLpt was replicated in mock - or SCAI - depleted egg extract in the presence of [ α - 32P ] dATP for the indicated times , and reactions were analyzed by native agarose gel electrophoresis . ∆ SCAI - C and ∆ SCAI - N denote SCAI immunodepletion with an antibody raised against the C - or N - terminus of SCAI , respectively ; RI , replication intermediates ; OC , open circular ; SC , supercoiled .

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F. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ∆SCAI-C and ∆SCAI-N denote SCAI immunodepletion with an antibody raised against the C- or N-terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled.


A . Analysis of SCAI - interacting proteins in Xenopus egg extracts . Volcano plot shows enrichment of proteins in both SCAI - C and SCAI - N immunoprecipitates ( IPs ) compared to an IgG control , plotted against the P - value ( n = 4 independent experiments ; permutation - based FDR < 0 . 01 , s0 = 2 ) .

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A. Analysis of SCAI-interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI-C and SCAI-N immunoprecipitates (IPs) compared to an IgG control, plotted against the P-value (n=4 independent experiments; permutation-based FDR<0.01, s0=2).


D . GFP IPs from U2OS or U2OS / GFP - SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies . Note that a small fraction of REV7 is still immunoprecipitated with GFP - SCAI in REV3 knockdown cells . This could be due to incomplete knockdown , and / or additional interactions of SCAI with the REV1 - Polζ complex that are independent of REV3 .

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D. GFP IPs from U2OS or U2OS/GFP-SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP-SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1-Polζ complex that are independent of REV3.


J . pICLpt was replicated in egg extracts immunodepleted with indicated antibodies , isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting .

J. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting.


B . Immunoblot analysis of extracts immunodepleted with indicated antibodies . Asterisks denote antibody cross - reactivity .

B. Immunoblot analysis of extracts immunodepleted with indicated antibodies. Asterisks denote antibody cross-reactivity.


E . Representative images of metaphase spreads from U2OS / SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I . Scale bars , 10 µm .

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E. Representative images of metaphase spreads from U2OS/SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I. Scale bars, 10 µm.


H . Clonogenic survival of U2OS and U2OS / SCAI KO cells transfected with non - targeting control ( CTRL ) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) .

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H. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).


To verify the specificity of Cx43 - CreERT mice for astrocytes , mice were crossed to a tdTomato reporter line to induce Cre - dependent tdTomato expression after tamoxifen administration . TdTomato fluorescence was enhanced with an antibody against red fluorescent protein , and astrocytes were stained with antibodies against GFAP or S100β ( not shown ) . Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes ( arrowheads ) in 8 - month and 11 - month old animals . Expression was small in neurons ( stained with NeuN , not shown ) and negligible in NG2 cells and oligodendrocytes . Scale bars , 250 µm ( upper panel ) and 50 µm ( lower panel ) .

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To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration. TdTomato fluorescence was enhanced with an antibody against red fluorescent protein, and astrocytes were stained with antibodies against GFAP or S100β (not shown). Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes (arrowheads) in 8-month and 11-month old animals. Expression was small in neurons (stained with NeuN, not shown) and negligible in NG2 cells and oligodendrocytes. Scale bars, 250 µm (upper panel) and 50 µm (lower panel).


The majority of peri - plaque astrocytes in APP / PS1 - Stat3WT were positive for activated / phosphorylated Stat3 ( pStat3 ) in the cortex ( Cx ) and hippocampus ( HC ) , whereas the fraction of pStat3 - positive astrocytes was significantly lower in APP / PS1 - Stat3KO mice in both regions ( n = 8 mice ( 3 females and 5 males ) per group ; age , 8 months ; Mann - Whitney test ) . The occurrence of pStat3 in WT - Stat3KO and WT - Stat3WT mice was negligible ( n = 8 mice ( 4 females and 4 males ) per group ; age , 8 months ; Mann - Whitney test ) .

The majority of peri-plaque astrocytes in APP/PS1-Stat3WT were positive for activated/phosphorylated Stat3 (pStat3) in the cortex (Cx) and hippocampus (HC), whereas the fraction of pStat3-positive astrocytes was significantly lower in APP/PS1-Stat3KO mice in both regions (n = 8 mice (3 females and 5 males) per group; age, 8 months; Mann-Whitney test). The occurrence of pStat3 in WT-Stat3KO and WT-Stat3WT mice was negligible (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test).


Examples of Stat3 activation in APP / PS1 - Stat3KO and APP / PS1 - Stat3WT . D , pStat3 ( arrowheads ) was abundantly present in reactive astrocytes ( identified by GFAP ) around plaques ( identified by IC16 ) . E , only few astrocytes were positive for pStat3 in APP / PS1 - Stat3WT mice . Scale bars , 50 µm .

Examples of Stat3 activation in APP/PS1-Stat3KO and APP/PS1-Stat3WT. D, pStat3 (arrowheads) was abundantly present in reactive astrocytes (identified by GFAP) around plaques (identified by IC16). E, only few astrocytes were positive for pStat3 in APP/PS1-Stat3WT mice. Scale bars, 50 µm.


in postmortem brain sections from AD patients , we detected nuclear pStat3 ( arrows ) in the majority of peri - plaque reactive astrocytes ( identified by GFAP ) in the cortex and hippocampus ( plaques were stained with methoxy - XO4 , arrowheads ) . Scale bar , 50 µm .

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in postmortem brain sections from AD patients, we detected nuclear pStat3 (arrows) in the majority of peri-plaque reactive astrocytes (identified by GFAP) in the cortex and hippocampus (plaques were stained with methoxy-XO4, arrowheads). Scale bar, 50 µm.


Overall astrocytic and microglial coverage , as assessed by GFAP and Iba1 stainings , remained unchanged in APP / PS1 - Stat3WT vs . APP / PS1 - Stat3KO mice ( n = 8 mice ( 4 females and 4 males ) per group ; age , 8 months ; Mann - Whitney test for each comparison ) . Scale bars , 500 µm .

Overall astrocytic and microglial coverage, as assessed by GFAP and Iba1 stainings, remained unchanged in APP/PS1-Stat3WT vs. APP/PS1-Stat3KO mice (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test for each comparison). Scale bars, 500 µm.


The volume of peri - plaque reactive astrocytes was increased by the Stat3 deletion ( Mann - Whitney test ) , and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length ( Mann - Whitney test ; APP / PS1 - Stat3WT , n = 5 ( 2 female and 3 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 3 female and 4 male ) mice ; age , 8 - 10 months ) . Scale bar , 20 µm .

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The volume of peri-plaque reactive astrocytes was increased by the Stat3 deletion (Mann-Whitney test), and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length (Mann-Whitney test; APP/PS1-Stat3WT, n = 5 (2 female and 3 male) mice; APP/PS1-Stat3KO, n = 7 (3 female and 4 male) mice; age, 8-10 months). Scale bar, 20 µm.


Microglial volume was not affected by the Stat3 deletion ( Mann - Whitney test ) , but peri - plaque microglia had significantly more microglial branches and junctions per plaque , and the total process length of peri - plaque microglia was increased ( Mann - Whitney test ; same mice as in C - D ) . Scale bar , 20 µm .

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Microglial volume was not affected by the Stat3 deletion (Mann-Whitney test), but peri-plaque microglia had significantly more microglial branches and junctions per plaque, and the total process length of peri-plaque microglia was increased (Mann-Whitney test; same mice as in C-D). Scale bar, 20 µm.


Aβ plaque burden , as assessed by plaque load and size using an anti - Aβ antibody , was strongly reduced in APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ( Mann - Whitney test ; scale bar , 300 µm ) .

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Aβ plaque burden, as assessed by plaque load and size using an anti-Aβ antibody, was strongly reduced in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 300 µm).


Plaque density , assessed by staining brain sections with thioflavin ( yellow ) , remained unchanged in APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ( Mann - Whitney test ; scale bar , 250 µm ) .

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Plaque density, assessed by staining brain sections with thioflavin (yellow), remained unchanged in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 250 µm).


Electrochemiluminescence ELISA after sequential extraction from whole - brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1 - 42 and Aβ1 - 40 were significantly reduced ( Mann - Whitney test ) , whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1 - 42 and Aβ1 - 40 ( Mann - Whitney test ) .

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Electrochemiluminescence ELISA after sequential extraction from whole-brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1-42 and Aβ1-40 were significantly reduced (Mann-Whitney test), whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1-42 and Aβ1-40 (Mann-Whitney test).


Western Blot quantification of full - length amyloid precursor protein ( APP ) and its C - terminal fragments ( CTF ) showed no differences between both groups ( Mann - Whitney test ; for all datasets , n = 9 male mice for both groups ( age , 11 months ) ) .

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Western Blot quantification of full-length amyloid precursor protein (APP) and its C-terminal fragments (CTF) showed no differences between both groups (Mann-Whitney test; for all datasets, n = 9 male mice for both groups (age, 11 months)).


Internalization of Aβ ( stained with IC16 antibody or methoxy - XO4 ) was assessed using an engulfment assay , in which glial and Aβ structures were surface - rendered and Aβ volumes co - localized with glial volumes were quantified . Scale bars , 10 µm .

Internalization of Aβ (stained with IC16 antibody or methoxy-XO4) was assessed using an engulfment assay, in which glial and Aβ structures were surface-rendered and Aβ volumes co-localized with glial volumes were quantified. Scale bars, 10 µm.


Microglia ( left Y axes ) from APP / PS1 mice internalized significantly more Aβ positive for IC16 when Stat3 was deleted in astrocytes ( Mann - Whitney test ) , whereas no changes were seen in astrocytes ( right axes ; APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 11 ( 5 female and 6 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for IC16 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).


Microglia ( left Y axes ) from APP / PS1 mice internalized significantly more Aβ positive for methoxy - XO4 when Stat3 was deleted in astrocytes ( Mann - Whitney test ) , whereas no changes were seen in astrocytes ( right axes ; APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 11 ( 5 female and 6 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for methoxy-XO4 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).


Western Blot quantification of protein levels of the Aβ - degrading enzymes neprilysin / CD10 and CD36 , as well as the Aβ - binding apoliprotein E ( apoE ) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE ( APP / PS1 - Stat3WT , n = 9 ( 5 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 9 ( 5 female and 4 male ) mice ; age , 11 months ; Mann - Whitney test for all comparisons ) . In contrast , TREM2 expression remained unchanged ( APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 4 female and 3 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Western Blot quantification of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).


Western Blot of protein levels of the Aβ - degrading enzymes neprilysin / CD10 and CD36 , as well as the Aβ - binding apoliprotein E ( apoE ) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE ( APP / PS1 - Stat3WT , n = 9 ( 5 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 9 ( 5 female and 4 male ) mice ; age , 11 months ; Mann - Whitney test for all comparisons ) . In contrast , TREM2 expression remained unchanged ( APP / PS1 - Stat3WT , n = 8 ( 4 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 7 ( 4 female and 3 male ) mice ; age , 11 months ; Mann - Whitney test ) .

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Western Blot of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).


Quantitative PCR from cortex of APP / PS1 - Stat3KO compared to APP / PS1 - Stat3WT mice revealed lower expression of ' A1 ' markers Amigo2 and C3 , whereas Ggta1 remained unchanged . In turn , the ' A2 ' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 ( n = 6 mice ( 3 females and 3 males ) per group ; age , 8 months ; Mann - Whitney test ) .

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Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of 'A1' markers Amigo2 and C3, whereas Ggta1 remained unchanged. In turn, the 'A2' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 (n = 6 mice (3 females and 3 males) per group; age, 8 months; Mann-Whitney test).


Confirming lower expression of the ' A1 ' marker C3d , western blot analysis indicated lower protein levels of C3d in APP / PS1 - Stat3KO . APP / PS1 - Stat3WT , n = 6 ( 2 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 3 female and 3 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Confirming lower expression of the 'A1' marker C3d, western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).


Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri - plaque reactive astrocytes ( arrows ; plaques were visualized with methoxy - XO4 , arrowheads ; scale bars , 50 µm ; APP / PS1 - Stat3WT , n = 6 ( 2 female and 4 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 3 female and 3 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri-plaque reactive astrocytes (arrows; plaques were visualized with methoxy-XO4, arrowheads; scale bars, 50 µm; APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).


Whole - brain levels of the pro - inflammatory cytokines IL - 1β and TNF - α were significantly reduced in APP / PS1 - Stat3KO mice ( Mann - Whitney test ) , whereas no changes were seen for IL - 10 ( APP / PS1 - Stat3WT , n = 13 ( 6 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 13 ( 8 female and 5 male ) mice ; age , 11 months ; Mann - Whitney test ) .

Whole-brain levels of the pro-inflammatory cytokines IL-1β and TNF-α were significantly reduced in APP/PS1-Stat3KO mice (Mann-Whitney test), whereas no changes were seen for IL-10 (APP/PS1-Stat3WT, n = 13 (6 female and 7 male) mice; APP/PS1-Stat3KO, n = 13 (8 female and 5 male) mice; age, 11 months; Mann-Whitney test).


These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP / PS1 - Stat3KO compared to APP / PS1 - Stat3WT mice , as assessed by LAMP1 staining ( Mann - Whitney test ; n = 10 male mice for both groups ; age , 8 months ; scale bars , 300 µm ) .

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These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice, as assessed by LAMP1 staining (Mann-Whitney test; n = 10 male mice for both groups; age, 8 months; scale bars, 300 µm).


For in vivo two - photon imaging , astrocytes ( arrows ) and neurons ( arrowheads ) were labeled with the calcium indicator OGB - 1 , and astrocytes were co - labeled with sulforhodamine 101 ( SR101 ; arrows ) . Aβ plaques were labeled with the intravital dye methoxy - XO4 ( open arrowheads ) . Scale bar , 50 µm .

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For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows). Aβ plaques were labeled with the intravital dye methoxy-XO4 (open arrowheads). Scale bar, 50 µm.


Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP / PS1 - Stat3KO mice was reduced to levels comparable to WT - Stat3WT mice , but significantly increased in APP / PS1 - Stat3WT mice ( One - Way ANOVA followed by Bonferroni ' s multiple comparison test ; n = 6 mice ( 3 female and 3 male ) for each group ; age , 8 months ) .

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Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP/PS1-Stat3KO mice was reduced to levels comparable to WT-Stat3WT mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; n = 6 mice (3 female and 3 male) for each group; age, 8 months).


Similarly , neuronal activity was also reduced to levels comparable to WT - Stat3WT mice in APP / PS1 - Stat3KO mice , but significantly increased in APP / PS1 - Stat3WT mice ( One - Way ANOVA followed by Bonferroni ' s multiple comparison test ; same mice as in B ) . The cumulative distributions of neuronal calcium transients in APP / PS1 - Stat3KO mice were not different from those of WT - Stat3WT mice ( p = 0 . 31 , Kolmogorov - Smirnov test ) , but significantly different from those of APP / PS1 - Stat3WT mice ( p = 0 . 001 , Kolmogorov - Smirnov test ) .

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Similarly, neuronal activity was also reduced to levels comparable to WT-Stat3WT mice in APP/PS1-Stat3KO mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; same mice as in B). The cumulative distributions of neuronal calcium transients in APP/PS1-Stat3KO mice were not different from those of WT-Stat3WT mice (p = 0.31, Kolmogorov-Smirnov test), but significantly different from those of APP/PS1-Stat3WT mice (p = 0.001, Kolmogorov-Smirnov test).


Two - photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Two-photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).


Two - photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) .

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Two-photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).


MRS2179 ( APP / PS1 - Stat3WT , n = 6 ( 4 female and 2 male ) mice ; APP / PS1 - Stat3KO , n = 6 ( 2 female and 4 male ) mice ; age , 8 months ; Mann - Whitney test ) . This network normalization induced a reduction of activated Stat3 ( pStat3 ) in astrocytes assessed in fixed brain sections of the same mice ( Mann - Whitney test ) . Scale bars , 50 µm .

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MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). This network normalization induced a reduction of activated Stat3 (pStat3) in astrocytes assessed in fixed brain sections of the same mice (Mann-Whitney test). Scale bars, 50 µm.


Spatial learning and memory were assessed in the Morris Water Maze paradigm . APP / PS1 - Stat3KO mice showed faster latencies to reach the hidden platform compared with APP / PS1 - Stat3WT on days 4 and 5 , but were similar to WT - Stat3WT and WT - Stat3KO mice ( Two - way repeated - measures ANOVA followed by Bonferroni post - hoc test ; P values are for APP / PS1 - Stat3KO vs . APP / PS1 - Stat3WT mice ) .

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Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test; P values are for APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice).


The area under the curve ( AUC ) for the latency to reach the hidden platform was similar in APP / PS1 - Stat3WT compared to WT - Stat3WT and WT - Stat3KO mice , but significantly higher in APP / PS1 - Stat3KO mice ( Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ) .

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The area under the curve (AUC) for the latency to reach the hidden platform was similar in APP/PS1-Stat3WT compared to WT-Stat3WT and WT-Stat3KO mice, but significantly higher in APP/PS1-Stat3KO mice (Kruskal-Wallis test followed by Dunn's multiple comparisons test).


The swimming velocity was similar in all groups ( Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ) .

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The swimming velocity was similar in all groups (Kruskal-Wallis test followed by Dunn's multiple comparisons test).


In the probe trial 24 h after the last training day , the time mice spent in the target quadrant ( TQ ) was different from chance in all groups except for APP / PS1 - Stat3WT mice ( p < 0 . 05 , one - tailed one - sample t test ) . APP / PS1 - Stat3KO , WT - Stat3WT and WT - Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants ( AO ) , whereas APP / PS1 - Stat3WT spent equal times in the target and all other quadrants ( Wilcoxon matched - pairs signed rank test for each comparison ; APP / PS1 - Stat3WT , n = 15 ( 8 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 15 ( 9 female and 6 male ) mice ; age , 8 - 9 months ) .

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In the probe trial 24 h after the last training day, the time mice spent in the target quadrant (TQ) was different from chance in all groups except for APP/PS1-Stat3WT mice (p < 0.05, one-tailed one-sample t test). APP/PS1-Stat3KO, WT-Stat3WT and WT-Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants (AO), whereas APP/PS1-Stat3WT spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for each comparison; APP/PS1-Stat3WT, n = 15 (8 female and 7 male) mice; APP/PS1-Stat3KO, n = 15 (9 female and 6 male) mice; age, 8-9 months).


APP / PS1 mice were systemically treated with SH - 4 - 54 for 6 weeks and compared to age - matched APP / PS1 mice treated with vehicle . In the Morris Water Maze test , treatment with SH - 4 - 54 led to significantly faster latencies to reach the hidden platform on the last training day ( Two - way repeated - measures ANOVA followed by Bonferroni post - hoc test ) .

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APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test).


The area under the curve ( AUC ) for the latency to reach the hidden platform was also smaller in APP / PS1 mice treated with the Stat3 inhibitor ( Mann - Whitney test ) .

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The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (Mann-Whitney test).


The swimming velocity was not affected by the treatment ( Mann - Whitney test ) .

The swimming velocity was not affected by the treatment (Mann-Whitney test).


In the probe trial test , APP / PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant ( TQ ) compared to the mean of all other quadrants ( AO ) , whereas APP / PS1 mice treated with vehicle spent equal times in the target and all other quadrants ( Wilcoxon matched - pairs signed rank test for all comparisons ; APP / PS1 - Stat3WT , n = 12 ( 5 female and 7 male ) mice ; APP / PS1 - Stat3KO , n = 12 ( 4 female and 8 male ) mice ; age , 8 months ) .

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In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO), whereas APP/PS1 mice treated with vehicle spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for all comparisons; APP/PS1-Stat3WT, n = 12 (5 female and 7 male) mice; APP/PS1-Stat3KO, n = 12 (4 female and 8 male) mice; age, 8 months).


( E ) To verify target engagement in the brain , the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two - photon microscopy of calcium activity . Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons ( Mann - Whitney test ) .

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(E) To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (Mann-Whitney test).


SH - 4 - 54 significantly decreased plaque growth , as assessed by IC16 immunohistochemistry , while plaque load remained unchanged .

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SH-4-54 significantly decreased plaque growth, as assessed by IC16 immunohistochemistry, while plaque load remained unchanged.


SH - 4 - 54 significantly There was also no significant change in dystrophic neurite area during the treatment time ( Mann - Whitney test for all comparisons ; scale bars , 500 µm ) .

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SH-4-54 significantly There was also no significant change in dystrophic neurite area during the treatment time (Mann-Whitney test for all comparisons; scale bars, 500 µm).


The fraction of pStat3 - positive astrocytes in the peri - plaque region was strongly reduced by the treatment with the Stat3 inhibitor ( arrowheads indicate pStat3 signals ; scale bars , 100 µm ; Mann - Whitney test ) .

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The fraction of pStat3-positive astrocytes in the peri-plaque region was strongly reduced by the treatment with the Stat3 inhibitor (arrowheads indicate pStat3 signals; scale bars, 100 µm; Mann-Whitney test).


While no changes were seen in morphological parameters of peri - plaque astrocytes , there was a significant increase in the process length of near - plaque microglia , indicating higher microglial complexity ( Mann - Whitney test for all comparisons ; APP / PS1 - Stat3WT , n = 12 ( 6 female and 6 male ) mice ; APP / PS1 - Stat3KO , n = 12 ( 7 female and 5 male ) mice ; age , 8 months ) .

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While no changes were seen in morphological parameters of peri-plaque astrocytes, there was a significant increase in the process length of near-plaque microglia, indicating higher microglial complexity (Mann-Whitney test for all comparisons; APP/PS1-Stat3WT, n = 12 (6 female and 6 male) mice; APP/PS1-Stat3KO, n = 12 (7 female and 5 male) mice; age, 8 months).


Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin .

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

Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.


Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin .

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

Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.


Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPO , erythropoietin .

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

Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPO, erythropoietin.


Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice .

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

Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting B , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA ,

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting B, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting E ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA ,

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

Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting E). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290


APP / PS1 - 21 mice ( 5 m ) treated with rhEPO ( 10000 IU / kg / d ) or ARA290 ( 0 . 03 mg / kg / d ) ( i . p . , once a day , for 14 days ) ( n = 10 ) , and Aβ plaques in cortex and hippocampus ( n = 5 ) were detected by IHC ( I ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290 ; IHC , Immunohistochemical staining .

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 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 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

APP/PS1-21 mice (5 m) treated with rhEPO (10000 IU/kg/d) or ARA290 (0.03 mg/kg/d) (i.p., once a day, for 14 days) (n=10), and Aβ plaques in cortex and hippocampus (n = 5) were detected by IHC (I). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290; IHC, Immunohistochemical staining.


Proteins were extracted from spleens of 5 - or 11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , and soluble Aβ levels were measured by ELISA ( n = 5 ) ( J Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ELISA ,

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

Proteins were extracted from spleens of 5- or 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), and soluble Aβ levels were measured by ELISA (n = 5) (J Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ELISA,


11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , Aβ plaques in the cortex and hippocampus of mice of 11 - month - old ( M ) ( n = 5 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining .

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

11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), Aβ plaques in the cortex and hippocampus of mice of 11-month-old (M) (n = 5) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.


R Aβ plaques in the cortex and hippocampus ( n = 4 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining .

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

R Aβ plaques in the cortex and hippocampus (n = 4) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.


A , Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


B Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Both non - cognitive and cognitive behavioral impairments of APP / PS1 - 21 mice ( 11 m ) were analyzed by nest construction assay , social interaction assay , NORT , FCT ( n = 10 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . NORT , novel object recognition test ; FCT , fear conditioning 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Both non-cognitive and cognitive behavioral impairments of APP/PS1-21 mice (11 m) were analyzed by nest construction assay, social interaction assay, NORT, FCT (n = 10). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. NORT, novel object recognition test; FCT, fear conditioning test.


Splenic macrophages from APP / PS1 - 21 ( 5 - or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Splenic macrophages from APP/PS1-21 (5- or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Splenic macrophages from APP / PS1 - 21 11 - month - old ) or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) B ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Splenic macrophages from APP/PS1-21 11-month-old) or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) B). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies ( n = 3 ) ( G ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies (n = 3) (G). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( I ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin .

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

Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (I). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.


PMs were treated with Aβ42 ( 0 , 5 , 7 . 5 or 10 μM ) for 12 hr , and cell lysates were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( K ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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

PMs were treated with Aβ42 (0, 5, 7.5 or 10 μM) for 12 hr, and cell lysates were detected by immunoblotting with the indicated antibodies (n = 3) (K). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.


PMs were treated with Aβ42 ( 0 , 5 , 10 μM ) for 12 hr , and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( M ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin .

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

PMs were treated with Aβ42 (0, 5, 10 μM) for 12 hr, and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (M). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.


E Aβ plaques in the cortex and hippocampus ( n = 5 ) were detected by IHC . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance .

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 O O 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 Aβ plaques in the cortex and hippocampus (n = 5) were detected by IHC. Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.