U2OS and ∆ ZAK cells were embedded in agarose and subjected to cyclic compression ( 0 . 5 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies .

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

U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.


U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min . Lysates were analyzed as in ( B ) .

O O O B-CONTROLLED_VAR 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

U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).


U2OS and ∆ ZAK cells were pretreated with ZAK inhibitor ( ZAKi ) for 30 min and subjected to equibiaxial ( Equi ) or uniaxial ( Uni ) cyclic stretch ( 0 . 5 h ) as indicated . Lysates were analyzed as in ( B ) .

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

U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).


U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch ( 5 min ) as indicated . Lysates were analyzed as in ( B ) .

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

U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).


Expression levels of MAP3 kinase genes in human skeletal muscle ( GTEx Portal ) . TPM , Transcripts Per Million .

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

Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.


C2C12 cells were incubated in diff . medium for 3 or 6 days . Lysates were analyzed for ZAK isoform expression by immunoblotting .

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

C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.


C2C12 myoblasts and myotubes ( 6 d differentiation ) were pre - treated with ZAK inhibitor ( ZAKi , 0 . 5 h ) and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies .

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

C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


Skeletal muscle ( tibialis anterior , TA ) and liver from WT and ZAK - / - mice were lysed and analyzed for ZAK isoform expression by immunoblotting . Ponceau staining of the membrane indicates equal loading .

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

Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.


Mouse embryonic fibroblasts ( MEF ) isolated from WT and ZAK - / - mice were treated with 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . * , unspecific band .

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

Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.


16 - 18 - week - old WT and ZAK - / - male mice ( n = 3 biological replicates ) were subjected to the protocol in ( D ) . TA lysates were analyzed by immunoblotting with the indicated antibodies .

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

16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.


ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from ( D ) . Values indicate absolute phospho - peptide abundances and error bars represent the standard deviation ( n = 3 biological replicates ) .

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

ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).


TA muscle in an 8 - week old WT male mouse was electroporated with a ZAKβ - tdTomato construct . After 7 days the muscle was harvested , sectioned longitudinally and immunostained with an antibody against the Z - disc marker α - actinin1 . The right panel shows a higher magnification of the region highlighted in yellow . Data information : scale bars , 20 μm .

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 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

TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow. Data information: scale bars, 20 μm.


C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP - ZAKβ constructs . Cells were fixed and immunostained with an antibody against α - actinin1 . Data information : scale bars , 20 μm .

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

C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1. Data information: scale bars, 20 μm.


16 - 18 - week - old WT and ZAK - / - male mice were subjected to the experimental protocol in ( I ) . TA muscle lysates were analyzed by immunoblotting with the indicated antibodies .

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

16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.


H & E staining of soleus muscle cross sections from 8 - and 22 - week - old WT and ZAK - / - male mice . Arrows indicate the presence of centralized nuclei . Scale bars , 50 μm . Quantification of ( A ) . Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and * * * , p < 0 . 001 in t - test with Bonferroni - Dunn correction for multiple comparison .

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

H&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm. Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.


Muscles from ( A ) were immunostained for type I ( red ) and type IIa ( green ) fibers using myosin isoform - specific antibodies . Scale bars , 500 μm . Quantification of ( C ) . Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and ns , not significant in t - test with Bonferroni - Dunn correction for multiple comparison .

O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm. Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.


Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect - size heat maps of six drugs ( see titles of panels ) targeting EGFR . It is evident that the different drugs showed different profiles but also that high MAP2K1 expression ( blue / red gradient across cell lines ) was consistently associated with drug resistance ( dark blue / yellow gradient across cell lines ; AUC : area under the curve ; see main text and Appendix for details ) . See also Figure EV5 .

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

Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect-size heat maps of six drugs (see titles of panels) targeting EGFR. It is evident that the different drugs showed different profiles but also that high MAP2K1 expression (blue/red gradient across cell lines) was consistently associated with drug resistance (dark blue/yellow gradient across cell lines; AUC: area under the curve; see main text and Appendix for details). See also Figure EV5.


( A ) Effect - size heat maps of two drugs ( one from two different drug sensitivity screens ) targeting MEK1 / 2 show consistent association of high MERTK expression with drug resistance . The colour scheme is the same as in Figure 4 .

O O O O O 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

(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4.


( C ) Dose - response curves of two drugs for which high MERTK ( left panels ) or ACVR2A ( right panels ) expression was predicted to confer drug resistance . For each drug , three cell lines predicted to be sensitive ( dark blue ) and three cell lines predicted to be resistant ( yellow ) were assayed for viability . The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype ( data represents the average of three technical replicates ; see Appendix for details ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details).


( D ) Boxplots summarizing the data shown in panel ( C ) using the area under the curve ( AUC ) as a measure for drug sensitivity . High AUC values indicate drug resistance . Again , it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly ( p ≤ 0 . 05 , one - sided Mann - Whitney test ) more resistant than cell lines predicted to be more sensitive .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.


( E ) Western blot of and cells , visualising successful knockout by CRISPR / Cas9 .

(E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9.


( F ) Dose - response curve of RDEA119 ( MEK1 / 2 inhibitor ) in and cells . The knockout is more sensitive to MEK1 / 2 inhibition than the wild type . See also Figure EV6 .

O O O O O O O O O O O O O O O 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) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.


( A ) Western Blot visualising MERTK expression in two negative ( RKO , CC07 ) and two positive control cell lines ( CaCo - 2 ; HT55 ) .

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

(A) Western Blot visualising MERTK expression in two negative (RKO, CC07) and two positive control cell lines (CaCo-2; HT55).


( B ) IHC staining of MERTK expression in RKO ( negative control ) and CaCo - 2 ( positive control ) cells .

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

(B) IHC staining of MERTK expression in RKO (negative control) and CaCo-2 (positive control) cells.


( C ) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists .

O O O O 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) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists.


( D ) Kaplan - Meier plots showing worse overall , disease - free and recurrence - free survival of patients with high cytoplasmic / membranous expression of MERTK .

O O O O O O 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) Kaplan-Meier plots showing worse overall, disease-free and recurrence-free survival of patients with high cytoplasmic/membranous expression of MERTK.


( A ) Neutrophil bacterial killing were assessed at day 6 ( section iii ) . Controls were without treatment . . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve .

O O 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

(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.


( A ) Neutrophil morphologic differentiation were assessed at day 6 ( section ii ) . Controls were without treatment . White arrows indicate neutrophilnuclear segmentation . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve .

O O O O O O O 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

(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.


( A ) Neutrophil ROS production were assessed at day 6 ( iv ) . Controls were without treatment . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve .

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 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

(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.


( A ) Proliferation analysis of hematopoietic CD34 + precursors for up to 6 days ( section i ) .

(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i).


( B ) qRT - PCR assessed RA - target gene expression after culturing CD34 + cells for 1 , 2 , and 6 days .

O O 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) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days.


( C ) Flow cytometry analysis of CD66 - CD18 co - expression after culturing CD34 + cells for 6 days , using anti - human CD66 - PE and CD18 - FITC antibodies ( section i ) . Isotypes were used for controls ( section i , top panel ) . CD66 - CD18 co - expression was quantified in section ii .

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

(C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii.


( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear cells were evaluated by ROS production ( section ii ) .

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 B-MEASURED_VAR O O O O O O

(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).


( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bacterial killing was assessed in the presence or absence of PBneutrophils ( section i ) .

O O O O O O O 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) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i).


( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear cells were evaluated by bacterial killing assays ( section iii ) .

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

(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).


( E ) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies . Controls were with or without IgG . Normal IgG , φ ; Ab , antibodies ; Comb Ab , combined anti - CD18 and - CD11b antibodies .

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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O

(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.


( F ) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days . Bacterial killing ( sections i ) and neutralization of bacterial killing in the presence of IgG or anti - CD18 antibody were assessed at day 3 ( section ii ) .

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

(F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).


( A ) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia ( AML ) patient . ROS production ( section ii ) were analyzed after cells were treated for 4 days .

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

(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.


( A ) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia ( AML ) patient . Proliferation ( section i ) were analyzed after cells were treated for 4 days .

O O O O O O O O O O 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) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days.


( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . Proliferation was assessed at day 3 in parallel ( section ii ) .

O O O O O O O O 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) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii).


( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . Transcription of RA - target genes was analyzed with qRT - PCR at day 2 , 3 , and 6 ( sections i ) .

O O O O O O O O O O O O O O O O O O O O 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) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i).


( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . ROS production were assessed at day 3 in parallel ( section iii ) .

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

(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).


( A ) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF ( 25 ng / ml ) for up to 9 days . Dynamic changes in proliferation ( left sections ) and live cells on day 9 ( right sections ) were illustrated for each of specimens . Controls were without treatment .

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 O O O O O O O O O O O O O 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) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.


( B ) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . Dynamic changes in proliferation ( left sections ) and live cells on day 12 ( right sections ) were illustrated for each of specimens .

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

(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.


( C ) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF . Proliferation ( left sections ) and bacterial killing ( right sections ) were analyzed on day 12 for each of samples .

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

(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.


( B ) Fresh BM mononuclear cells collected from AML patients were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . ROS production was assessed at day 12 ( section ii ) .

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

(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).


( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Morphologic differentiation was assessed at day 12 ( section iv ) . White arrows indicate neutrophil nuclear segmentation .

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

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.


( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Proliferation was assessed for up to 12 days ( section i ) .

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

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).


( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Bacterial killing was assessed at day 12 ( sections iii ) .

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

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).


( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . RA - target gene expression was assessed at day 12 ( section v ) .

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

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).


( A ) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide ( CPA ) consists of both neutrophil decrease and neutrophil recovery stages . ANC , absolute neutrophil count .

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

(A) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide (CPA) consists of both neutrophil decrease and neutrophil recovery stages. ANC, absolute neutrophil count.


(D) Vetscan counting PBleukocytes.


( E ) Neutrophils induced by low dose treatment in PB ( section i ) and BM ( section ii ) .

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

(E) Neutrophils induced by low dose treatment in PB (section i) and BM (section ii).


( F ) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post - infection and in spleen ( sections i - iii ) , using blood agar analysis of total extracellular viable bacteria .

O O O O O O O O O O O O O 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) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post-infection and in spleen (sections i-iii), using blood agar analysis of total extracellular viable bacteria.


(H) Vetscan counting PBleukocytes.


( I ) Neutrophils induced by medium dose treatment in PB ( section i ) and BM ( section ii ) .

(I) Neutrophils induced by medium dose treatment in PB (section i) and BM (section ii).


( J ) Similar to panel F , bacterial killing by PBneutrophils was evaluated 3 and 16 hr post - infection and in heart .

O 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) Similar to panel F, bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection and in heart.


(B) Vetscan counting PBleukocytes.


( C ) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post - infection ( sections i , ii ) , using blood agar analysis of extracellular viable bacteria .

(C) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection (sections i, ii), using blood agar analysis of extracellular viable bacteria.


( D E ) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and / or GCSF in BM ( panel D ) and PB ( panel E ) . White arrows indicate neutrophilnuclear segmentation .

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

(D E) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and/or GCSF in BM (panel D) and PB (panel E). White arrows indicate neutrophilnuclear segmentation.


( B ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 3 independent experiments with similar results .

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

(B) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 3 independent experiments with similar results.


( C ) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9 . White arrows indicate phagocytosed S . aureus in neutrophils .

O O O O O O 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) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9. White arrows indicate phagocytosed S. aureus in neutrophils.


( E ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 2 independent experiments with similar results .

(E) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 2 independent experiments with similar results.


( G ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) .

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

(G) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii).


( A B ) Neutrophil recovery and morphologic differentiation were assessed in BM ( panel A ) and PB ( panel B ) of CCIN mice surviving on day 9 . White arrows indicate neutrophil nuclear segmentation .

O O O O O O O O O O O O O O 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 B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation.


( C ) PBneutrophil production and nuclear segmentation were assessed in control and Am80 - GCSF mice surviving on day 13 ( section i ) .

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

(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i).


( C ) PBneutrophil production and nuclear segmentation were assessed in control and Am80 - GCSF mice surviving on day 13 ( section ii ) .

(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii).


( 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 ) .

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

(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 ) .

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

(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 .

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

(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 .

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

(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 .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

B-MEASURED_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 B-CONTROLLED_VAR O O

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 .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

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 B-CONTROLLED_VAR O

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 ) .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

O O O O 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

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 ) .

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

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 .

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

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 )

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

D, E, 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 ) .

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 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 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 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 B-MEASURED_VAR 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

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 ) .

O O O O O O O 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 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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 .

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

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 .

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

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 )

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

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 .

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

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 ) .

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

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 .

O O O O O O O O B-MEASURED_VAR I-MEASURED_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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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 ) .

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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

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 .

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

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 .

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

B. 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 .

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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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

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 ) .

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

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 .

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

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 .

O O O O 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. 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 .

O O O O O O 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. 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 ) .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

O O O 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

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 .

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

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 .

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-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 O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) ) .

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

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 .

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

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 ) .

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

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 ) .

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

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 ) .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_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 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-MEASURED_VAR 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 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

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 ) .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_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 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-MEASURED_VAR 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 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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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).