( C ) To assess in vivo , bronchoalveolar lavage fluid from Mtb - WT - , Mtb - Δeis - , Mtb - c - eis - infected mice were subjected to PI , analyzed by flow cytometry . Data are presented as mean ± ( n = 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 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

(C) To assess in vivo cell death, bronchoalveolar lavage fluid cells from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice were subjected to PI staining, and analyzed by flow cytometry. Data are presented as the mean±SEM (n = 4).


( D ) Numbers of CFUs in lung spleen 4 after infection with Mtb - WT , Mtb - Δeis , or Mtb - c - eis . Data are presented as log10 CFU ± ( n = 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(D) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis. Data are presented as log10 CFU±SEM (n = 4).


( E ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis then analyzed by CFU . CFU data represent mean ± SD of four experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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

(E) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis and then analyzed by CFU assay. CFU data represent the mean±SD of four individual experiments. **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A ) Intracellular superoxide production was analyzed by flow cytometric . BMDMs were infected with Mtb - Δeis ( MOI = 10 ) in presence or absence of recombinant Eis ( rEis ; 5 , 10 , 20 µg / mL ) or 30 kDa Mtb antigen ( 30 k ; 5 , 10 , 20 µg / mL ) . Upper , representative flow cytometric ; lower , quantitation of superoxide generation .

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

(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation.


( B ) THP - 1 were transfected with mock , Eis - WT , or Eis - ΔAT constructs , infected with Mtb - Δeis for 30 min . were stained with DHE ( for superoxide ) or DCFH - DA ( for H2O2 ) subjected to flow cytometric . Inset , transfection efficiency .

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

(B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency.


( C D ) Experimental conditions were identical to those outlined in panels A B , respectively . Supernatants were collected 18 h after infection assessed by ELISA for of TNF - α IL - 6 . Data are presented as mean ± SD of five experiments .

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

(C and D) Experimental conditions were identical to those outlined in panels A and B, respectively. Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments.


( E F ) THP - 1 transfected with mock , Eis - WT , or Eis - ΔAT constructs were pretreated with SP600125 ( SP ; 20 µM ) or SB203580 ( SB ; 5 µM ) for 45 min before infection with Mtb - Δeis for 30 min ( E ) or 18 h ( F ) . E , Intracellular superoxide production was analyzed by flow cytometric . F , ELISA for TNF - α IL - 6 . Data are presented as mean ± SD of three experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . SC ( A , C , E , F ) ; mock control ( B D ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 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 and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).


DFL inhibits HMGB1 - induced , but not fMLP - induced migration . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers , 1 nM HMGB1 or no chemoattractant was added in lower chamber , together with indicated concentrations of DFL . Data points with average ± standard deviation ( avg ± sd ; n = 3 , each point represents a biological replicate ) in a representative experiment .

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

DFL inhibits HMGB1-induced, but not fMLP-induced cell migration. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers, 1 nM HMGB1 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points with average ± standard deviation (avg ± sd; n=3, each point represents a biological replicate) in a representative experiment.


Histogram showing residue - specific CSPs of 15N - labelled HMGB1 ( ~ 0 . 1 mM ) upon addition of ten - fold excess of DFL ( helices are schematically represented on top ) . Missing residues are prolines or are absent because of exchange with solvent . Box A Box B residues with CSP > avg + sd are represented in magenta blue , respectively .

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

Histogram showing residue-specific CSPs of 15N-labelled HMGB1 (~0.1 mM) upon addition of ten-fold excess of DFL (helices are schematically represented on the top). Missing residues are prolines or are absent because of exchange with the solvent. Box A and Box B residues with CSP > avg + sd are represented in magenta and blue, respectively.


R23A / R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL ( experiment was carried out as described in panel B ) . data points represent three biological replicates in one experiment ( of 2 performed in different days ) ; avg ± sd is shown .

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

The R23A/R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL (the experiment was carried out as described in panel B). The data points represent three biological replicates in one experiment (of 2 performed in different days); the avg ± sd is shown.


Histogram showing CSPs of 15N - labelled CXCL12 amides ( ~ 0 . 1 mM ) upon addition of ten - fold excess of DFL . Missing residues are prolines . Elements of secondary structure are depicted on top .

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

Histogram showing the CSPs of 15N-labelled CXCL12 amides (~0.1 mM) upon addition of ten-fold excess of DFL. Missing residues are prolines. Elements of secondary structure are depicted on top.


Top : 1H spectrum of DFL , where numbered peaks correspond to DFL protons ( left ) . Middle : superimposition of STD spectra obtained for 0 . 5 mM DFL with 0 . 05 mM CXCL12 ( black ) upon addition of 0 . 2 mM of CXCR41 - 38sY21 ( red ) . Bottom : waterLOGSY spectra obtained for 0 . 5 mM DFL with 0 . 05 mM CXCL12 ( black ) upon addition of 0 . 2 mM of CXCR41 - 38sY21 ( red ) .

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

Top: 1H spectrum of DFL, where the numbered peaks correspond to DFL protons (left). Middle: superimposition of STD spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) and upon addition of 0.2 mM of CXCR41-38sY21 (red line). Bottom: waterLOGSY spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) upon addition of 0.2 mM of CXCR41-38sY21 (red line).


DFL does not inhibit CXCL12 induced chemotaxis . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers ; 1 nM CXCL12 or no chemoattractant was added in lower chamber , together with indicated concentrations of DFL . Data points ( n = 3 ) with avg ± sd in one representative experiment ( of 2 performed in different days ) ; migration in presence of increasing concentrations of

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

DFL does not inhibit CXCL12 induced chemotaxis. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM CXCL12 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points (n=3) with avg ± sd in one representative experiment (of 2 performed in different days); the migration in the presence of increasing concentrations of DFL


DFL inhibits chemotaxis toward HMGB1 / CXCL12 heterocomplex . Data points ( n = 3 ) with avg ± sd in one representative experiment ( of 3 performed in different days ) . Migration in presence or absence of DFL is significantly different ( p = 0 . 0037 , one - way ANOVA plus Dunnett ' s post ; * * p < 0 . 01 relative to no DFL addition ) .

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

DFL inhibits chemotaxis toward the HMGB1/CXCL12 heterocomplex. Data points (n=3) with avg ± sd in one representative experiment (of 3 performed in different days). Migration in the presence or absence of DFL is significantly different (p=0.0037, one-way ANOVA plus Dunnett's post test; ** p<0.01 relative to no DFL addition).


1H-15N HSQC HMGB1 (0.1 mM) spectrum


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

1H-15N HSQC HMGB1 (0.1 mM) spectrum with 0.2 mM CXCL12.


MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1 ; signal increases from ∼ 930 ( free ) to ∼ 950 a . u . ( bound ) yielding an apparent Kd of 4 ± 0 . 4 μM . n = 3 ; error bars represent avg ± sd .

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

MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1; the signal increases from ∼930 (free) to ∼950 a.u. (bound) yielding an apparent Kd of 4 ± 0.4 μM. n=3; error bars represent avg ± sd.


H - 15N HSQC of HMGB1 ( 0 . 1 mM ) with CXCL12 ( 0 . 2 mM ) upon addition of ( D ) 0 . 2 mM ( E ) 1 mM DFL .

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 O O

H-15N HSQC of HMGB1 (0.1 mM) with CXCL12 (0.2 mM) upon addition of (D) 0.2 mM and (E) 1 mM DFL.


MST measurements of heterocomplex ( preformed using 50 nM HMGB1 50 μM CXCL12 ) in presence of increasing concentrations of DFL . Decreasing MST signal ( from ∼ 950 to ∼ 930 a . u . ) upon DFL titration indicates that CXCL12 has been displaced from HMGB1 . n = 3 ; error bars represent avg ± sd .

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

MST measurements of the heterocomplex (preformed using 50 nM HMGB1 and 50 μM CXCL12) in the presence of increasing concentrations of DFL. Decreasing MST signal (from ∼950 to ∼930 a.u.) upon DFL titration indicates that CXCL12 has been displaced from HMGB1. n=3; error bars represent avg ± sd.


DFL does not affect cytokine - inducing activities of disulfide HMGB1 ( ds - HMGB1 ) . Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg / ml ds - HMGB1 ( ~ 100 nM ) or 10 ng / ml LPS , in presence of indicated concentrations of DFL . data points represent n = 3 biological replicates , with avg ± sd , in one representative experiment ( of 2 performed in different days ) . of TNFα mRNA are not significantly different in presence of increasing concentrations of DFL ( one - way ANOVA plus post ) .

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

DFL does not affect the cytokine-inducing activities of disulfide HMGB1 (ds-HMGB1). Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg/ml ds-HMGB1 (~100 nM) or 10 ng/ml LPS, in the presence of the indicated concentrations of DFL. The data points represent n=3 biological replicates, with avg ± sd, in one representative experiment (of 2 performed in different days). The levels of TNFα mRNA are not significantly different in the presence of increasing concentrations of DFL (one-way ANOVA plus post tests).


DFL does not inhibit chemotaxis towards 3S . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers ; 1 nM fully reduced HMGB1 ( fr - HMGB1 ) ( green ) or 3S ( red ) , or no chemoattractant ( black ) , were added in lower chamber , together with 1 µg / ml of anti - CXCL12 monoclonal antibody or 30 nM DFL . Data points ( n = 4 ) with avg ± sd in one representative experiment ( of two performed in different days ) . Statistics : one - way ANOVA ( p < 0 . 0001 ) , followed by Dunnett ' s post - . * * * , p < 0 . 001 relative to migration towards HMGB1 , migrations towards 3S are not significantly different among each other .

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

DFL does not inhibit chemotaxis towards 3S. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM fully reduced HMGB1 (fr-HMGB1) (green) or 3S (red), or no chemoattractant (black), were added in the lower chamber, together with 1 µg/ml of anti-CXCL12 monoclonal antibody or 30 nM DFL. Data points (n=4) with avg ± sd in one representative experiment (of two performed in different days). Statistics: one-way ANOVA (p<0.0001), followed by Dunnett's post-test. ***, p<0.001 relative to migration towards HMGB1, the migrations towards 3S are not significantly different among each other.


of CD45 + isolated with immunobeads from injured 6 hours after damage . Before damage mice were treated either with Phosphate Buffered Saline vehicle , Glycyrrhizin or DFL . Two independent experiments . Each point represents a different mouse , avg ± sd are shown ( statistics : Kruskal - Willis plus post - ) .

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

Quantification of CD45+ cells isolated with immunobeads from injured muscles 6 hours after damage. Before damage mice were treated either with Phosphate Buffered Saline vehicle, Glycyrrhizin or DFL. Two independent experiments. Each point represents a different mouse, avg ± sd are shown (statistics: Kruskal-Willis plus post-tests).


( A ) Schematic representation of study design Western blot for TIP30 GAPDH in hearts from TIP30 wild - type ( WT ) , heterozygous ( Het ) homozygous knock - out ( KO ) mice under basal conditions .

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

(A) Schematic representation of the study design and Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT), heterozygous (Het) and homozygous knock-out (KO) mice under basal conditions.


of heart ( HW ) / tibia ( TL ) ( B ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

O O O O O 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 heart weight (HW)/tibia length (TL) ratio (B) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of lung ( LuW / TL ) ( C ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

O O 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 lung weight (LuW/TL) ratio (C) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of , echocardiographic fractional change ( D ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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 , echocardiographic fractional area change (D) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of left ventricular enddiastolic ( LVEDA ; E ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

O 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 left ventricular enddiastolic area (LVEDA; E) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of dP / dt max dP / dt min ( Millar catheter ; F , G ) N = 4 to 18 mice / group , 6 after TAC or sham surgery .

O O O O O O O O O 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 dP/dt max and dP/dt min (Millar catheter; F, G) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of α - MHC transcript abundance N = 4 to 18 mice / group , 6 after TAC or sham surgery .

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

Quantification of α-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


of β - MHC transcript abundance N = 4 to 18 mice / group , 6 after TAC or sham surgery .

Quantification of β-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.


Representative of Sirius - red - stained heart sections ( scale bar : 1mm ) ( J )

Representative images of Sirius-red-stained heart sections (scale bar: 1mm) (J)


fibrosis ( K ) of indicated mice 6 after TAC or sham surgery .

fibrosis quantification (K) of indicated mice 6 weeks after TAC or sham surgery.


of cardiomyocyte of isolated adult cardiac myocytes ( N = 4 to 11 mice / group ) of indicated mice 6 after TAC or sham surgery . * P < 0 . 05 . One - way ANOVA with Sidak ´ s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O 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 cardiomyocyte area of isolated adult cardiac myocytes (N=4 to 11 mice/group) of indicated mice 6 weeks after TAC or sham surgery. *P<0.05. One-way ANOVA with Sidak´s multiple comparisons test.


Microscopy of heart sections of indicated mice 6 after TAC surgery stained for Isolectin B4 ( green ) WGA ( red , M )

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

Microscopy images of heart sections of indicated mice 6 weeks after TAC surgery stained for Isolectin B4 (green) and WGA (red, M)


of capillaries per myocyte ( N ) . ( N = 5 to 7 mice / group , scale bar : 50µm ) . * * P < 0 . 01 . One - way ANOVA with Sidak ´ s multiple comparisons .

O O O O O O O O O O O O O O O O O O O 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 capillaries per myocyte (N). (N=5 to 7 mice/group, scale bar: 50µm). **P<0.01. One-way ANOVA with Sidak´s multiple comparisons test.


( P ) Western blot for TIP30 GAPDH in hearts from TIP30 wild - type ( WT ) heterozygous ( Het ) after AAV - TropT - TIP30 or AAV - Control ( AAV - Con ) injection followed by 6 of TAC surgery .

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

(P) Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT) and heterozygous (Het) after AAV-TropT-TIP30 or AAV-Control (AAV-Con) injection followed by 6 weeks of TAC surgery.


of HW / TL ( Q ) in AAV - Con or AAV - TropT - TIP30 treated Tip30 heterozygous ( Het ) or WT mice 6 after TAC or sham surgery ( N = 5 to 11 mice / group ) .

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

Quantification of HW/TL ratio (Q) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N=5 to 11 mice/group).


of LuW / TL ( R ) in AAV - Con or AAV - TropT - TIP30 treated Tip30 heterozygous ( Het ) or WT mice 6 after TAC or sham surgery ( N = 5 to 11 mice / group ) .

Quantification of LuW/TL (R) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N=5 to 11 mice/group).


( A ) Western blot for TIP30 GAPDH in neonatal rat cardiomyocytes ( NRCM ) after adenoviral transduction with Ad . Control ( Ad . Con ) or Ad . TIP30 .

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 B-CONTROLLED_VAR O

(A) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes (NRCM) after adenoviral transduction with Ad.Control (Ad.Con) or Ad.TIP30.


of cardiomyocyte ( B ) , N = 6 to 8 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . ET - 1 : endothelin - 1 , FBS : bovine serum , PE : phenylephrine .

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

Quantification of cardiomyocyte area (B), N=6 to 8 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1, FBS: fetal bovine serum, PE: phenylephrine.


of / ( C ) , N = 9 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . PE : phenylephrine .

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

Quantification of protein/DNA ratio (C), N=9 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.


of Acta1 mRNA transcript abundance ( D ) , N = 3 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . ET - 1 : endothelin - 1 PE : phenylephrine .

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

Quantification of Acta1 mRNA transcript abundance (D), N=3 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1 PE: phenylephrine.


of with a 7 - AAD ( E ) , N = 7 samples / group in NRCM transduced with Ad . Con or Ad . TIP30 stimulated as indicated . PE : phenylephrine .

O O 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

Quantification of cell death with a 7-AAD assay (E), N=7 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.


( G ) Western blot for TIP30 Actin in mouse hearts with AAV9 mediated overexpression of TIP30 ( AAV - TIP30 ) or from mice treated with a control AAV9 construct ( AAV - Con ) followed by 2 of TAC surgery .

O O O O O O B-MEASURED_VAR 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

(G) Western blot for TIP30 and Actin in mouse hearts with AAV9 mediated overexpression of TIP30 (AAV-TIP30) or from mice treated with a control AAV9 construct (AAV-Con) followed by 2 weeks of TAC surgery.


of HW / TL ( H ) , N = 8 to 13 mice / group 2 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice . *

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

Quantification of HW/TL ratio (H), N=8 to 13 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice. *


of cardiomyocyte ( I ) , N = 4 to 5 mice / group 2 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice .

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

Quantification of cardiomyocyte area (I), N=4 to 5 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice.


( J ) Serial echocardiography with of echocardiographic fractional change 2 , 4 6 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice ( N = 10 to 14 mice / group time point ) .

O O 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

(J) Serial echocardiography with quantification of echocardiographic fractional area change 2, 4 and 6 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice (N=10 to 14 mice/group and time point).


( A ) Western blot of GST - pulldown assays with GST - TIP30 fragments of indicated ( as : from N - terminus ) with Myc - tagged binding partners .

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

(A) Western blot analysis of GST-pulldown assays with GST-TIP30 fragments of the indicated length (as: amino acids from N-terminus) with Myc-tagged binding partners.


( B ) Western blot of co - immunoprecipitation ( IP ) from HEK transfected with GST - TIP30 eEF1A1 - Myc .

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

(B) Western blot analysis of co-immunoprecipitation (IP) from HEK cells transfected with GST-TIP30 and eEF1A1-Myc.


( C ) Confocal microscopy of neonatal rat cardiomyocytes ( NRCM ) stained with antibodies for endogenous TIP30 ( green ) endogenous eEF1A1 ( red ) . DAPI : blue . ( Scale bar : 20µm ) .

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

(C) Confocal microscopy images of neonatal rat cardiomyocytes (NRCM) stained with antibodies for endogenous TIP30 (green) and endogenous eEF1A1 (red). DAPI: blue. (Scale bar: 20µm).


( D ) Microscopy of isolated neonatal rat cardiomyocytes subsequent Proximity Ligation ( PLA ) . Red : endogenous TIP30 / eEF1A1 interaction ; Blue : DAPI . Control were stained for TIP30 / Myc - Tag interaction ( scale bar : 50µm ) .

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

(D) Microscopy images of isolated neonatal rat cardiomyocytes and subsequent Proximity Ligation Assay (PLA). Red: endogenous TIP30/eEF1A1 interaction; Blue: DAPI. Control cells were stained for TIP30/Myc-Tag interaction (scale bar: 50µm).


( E ) Western blot of co - immunoprecipitation ( IP ) for endogenous TIP30 eEF1A1 in NRCM after stimulation with phenylephrine for 24 h . IgG HC - High chain of IgG molecule .

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

(E) Western blot analysis of co-immunoprecipitation (IP) for endogenous TIP30 and eEF1A1 protein in NRCM after stimulation with phenylephrine for 24 h. IgG HC - High chain of IgG molecule.


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

Western blot analysis of GST-pulldown assays with GST or GST-tagged eEF1A1 (GST-eEF1A1) and TIP30-His full length protein (TIP30-His) and TIP30 deletion mutants ΔN25 (TIP30-ΔN25-His), ΔN52 (TIP30-ΔN52-His), ΔC15 (TIP30-ΔC15-His) and Δ102-107 (TIP30-ΔN102-104-His, G)


Western blot of GST - tagged eEF1A1 GST - Control ( H ) .

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

Western blot analysis of GST-tagged eEF1A1 and GST-Control (H).


( J ) Quantitative Real - Time PCR of eEF1A1 eEF1A2 mRNA abundance in hearts from neonatal mice , adult wild - type mice ( Adult ) adult wild - type mice 2 after TAC surgery ( Adult TAC , N = 3 to 4 mice / group ) . * P < 0 . 05 , * * * P < 0 . 001 . One - way ANOVA with Sidak ´ s multiple comparisons .

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 O 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) Quantitative Real-Time PCR analysis of eEF1A1 and eEF1A2 mRNA abundance in hearts from neonatal mice, adult wild-type mice (Adult) and adult wild-type mice 2 weeks after TAC surgery (Adult TAC, N=3 to 4 mice/group). *P<0.05, ***P<0.001. One-way ANOVA with Sidak´s multiple comparisons test.


( K ) Western blot for eEF1A1 , eEF1A2 GAPDH in hearts from wild - type mice at of 7 days ( 7d ) 8 ( 8w ) as well as 2 ( TAC 2w ) after TAC surgery .

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

(K) Western blot analysis for eEF1A1, eEF1A2 and GAPDH in hearts from wild-type mice at the age of 7 days (7d) and 8 weeks (8w) as well as 2 weeks (TAC 2w) after TAC surgery.


( L ) Western blot of co - immunoprecipitation ( IP ) with GST - TIP30 eEF1A2 - His .

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

(L) Western blot analysis of co-immunoprecipitation (IP) with GST-TIP30 and eEF1A2-His.


( A ) Western blot of GST - pulldown assays with GST or GST - eEF1A1 purified eEF1B2 - His . Purified TIP30 - His was added in indicated amounts .

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 B-CONTROLLED_VAR O O O O O O O O

(A) Western blot analysis of GST-pulldown assays with GST or GST-eEF1A1 and purified eEF1B2-His. Purified TIP30-His was added in indicated amounts.


( B ) Western blot of anti - Myc immunoprecipitation ( IP ) in NRCM co - transduced with Ad . eEF1A1 - Myc either control ( Ad . con ) or Ad . TIP30 . Endogenous eEF1B2 was detected . IP input for eEF1B2 , eEF1A1 - myc TIP30 is shown below . ( C ) of eEF1B2 abundance after eEF1A1 - myc IP under conditions shown in ( B )

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

(B) Western blot analysis of anti-Myc immunoprecipitation (IP) in NRCM co-transduced with Ad.eEF1A1-Myc and either control virus (Ad.con) or Ad.TIP30. Endogenous eEF1B2 was detected. The IP input for eEF1B2, eEF1A1-myc and TIP30 is shown below. (C) Quantification of eEF1B2 abundance after eEF1A1-myc IP under conditions shown in (B)


( D ) Microscopy of NRCM after adenoviral transduction with Ad . TIP30 or control ( Ad . Con ) stimulation with phenylephrine ( PE ) subsequent Proximity Ligation ( PLA ) . Red : eEF1A1 - eEF1B2 interaction ; Blue : DAPI . ( scale bar : 50µm ) . ( E ) of eEF1A1 - eEF1B2 interaction in conditions described in ( D )

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-MEASURED_VAR 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 O O O O

(D) Microscopy images of NRCM after adenoviral transduction with Ad.TIP30 or control virus (Ad.Con) and stimulation with phenylephrine (PE) and subsequent Proximity Ligation Assay (PLA). Red: eEF1A1-eEF1B2 interaction; Blue: DAPI. (scale bar: 50µm). (E) Quantification of eEF1A1-eEF1B2 interaction in conditions described in (D)


( F ) Microscopy of adult mouse cardiomyocytes isolated from hearts 3 days ( d ) after TAC or sham surgery subsequent PLA . Red : eEF1A1 - eEF1B2 interaction ; Blue : DAPI ( scale bar : 100µm ) . ( G ) of eEF1A1 - eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery subsequent PLA ( N = 4 to 6 mice / group ) . *

O O 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 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

(F) Microscopy images of adult mouse cardiomyocytes isolated from hearts 3 days (d) after TAC or sham surgery and subsequent PLA. Red: eEF1A1-eEF1B2 interaction; Blue: DAPI (scale bar: 100µm). (G) Quantification of eEF1A1-eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery and subsequent PLA (N=4 to 6 mice/group). *


Western blot for TIP30 , eEF1A1 GAPDH in C57BL / 6 WT mice 3 days ( A , B ) , N = 4 mice / group , 2 ( C , D ) , N = 4 to 8 mice / group 6 ( E , F ) , N = 4 to 8 mice / group after TAC or sham surgery their . KO denotes TIP30 homozygous knock - out .

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

Western blot analysis for TIP30, eEF1A1 and GAPDH in C57BL/6 WT mice 3 days (A, B), N=4 mice/group, 2 weeks (C, D), N=4 to 8 mice/group and 6 weeks (E, F), N=4 to 8 mice/group after TAC or sham surgery and their quantification. KO denotes TIP30 homozygous knock-out.


( G , H ) Western blot for TIP30 , eEF1A1 Actin in TIP30 Het mice 6 after TAC surgery their ( N = 4 mice / group ) .

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

(G, H) Western blot for TIP30, eEF1A1 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification (N=4 mice/group).


( I , J ) Western blot for TIP30 , eEF1A1 Actin 2 after sham or TAC surgery in AAV - Con or AAV - TIP30 treated C57BL / 6 WT mice their ( N = 4 mice / group ) . A of TIP30 eEF1A1 expression was calculated for each condition .

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 B-CONTROLLED_VAR 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

(I, J) Western blot for TIP30, eEF1A1 and Actin 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice and their quantification (N=4 mice/group). A ratio of TIP30 and eEF1A1 expression was calculated for each condition.


of Tip30 eEF1A1 mRNA transcript abundance in human failing hearts ( A ) , N = 6 to 8 hearts / group A of Tip30 eEF1A1 expression was

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 B-MEASURED_VAR O O O

Quantification of Tip30 and eEF1A1 mRNA transcript abundance in human failing hearts (A), N=6 to 8 hearts/group A ratio of Tip30 and eEF1A1 expression was calculated


of Tip30 eEF1A1 mRNA transcript abundance in with Hypertrophic Cardiomyopathy ( HCM ; B , N = 4 to 8 hearts / group ) A of Tip30 eEF1A1 expression was

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 B-MEASURED_VAR I-MEASURED_VAR O O

Quantification of Tip30 and eEF1A1 mRNA transcript abundance in patients with Hypertrophic Cardiomyopathy (HCM; B, N=4 to 8 hearts/group) A ratio of Tip30 and eEF1A1 expression was calculated


of Tip30 eEF1A1 mRNA transcript abundance in 6 months old mdx mice or WT mice ( C ) , N = 4 mice / group . A of Tip30 eEF1A1 expression was

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

Quantification of Tip30 and eEF1A1 mRNA transcript abundance in 6 months old mdx mice or WT mice (C), N=4 mice/group. A ratio of Tip30 and eEF1A1 expression was calculated


( E ) Echocardiographic diastolic left - ventricular wall - thickness in MDX mice at of 2 months at baseline , 1 - 9 months ( mo ) after injection of AAV9 - Con or AAV9 - TIP30 ( TIP ; N = 6 to 7 mice / group ) .

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

(E) Echocardiographic diastolic left-ventricular wall-thickness in MDX mice at the age of 2 months at baseline, and 1-9 months (mo) after injection of AAV9-Con or AAV9-TIP30 (TIP; N=6 to 7 mice/group).


( F ) of HW / TL of 9mo old MDX mice , N = 5 to 6 mice / group .

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

(F) Quantification of HW/TL ratio of 9mo old MDX mice, N=5 to 6 mice/group.


( A ) Western blot of isolated neonatal rat cardiomyocytes ( NRCM ) after adenoviral transduction either with control adenovirus ( Con ) or Ad . TIP30 ( TIP30 ) followed by stimulation with phenylephrine ( PE , for 3 hours ) puromycin incorporation ( for 30 min ) . ( B ) of Western blot shown in ( A ) ( N = 3 samples / group ) .

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

(A) Western blot analysis of isolated neonatal rat cardiomyocytes (NRCM) after adenoviral transduction either with control adenovirus (Con) or Ad.TIP30 (TIP30) followed by stimulation with phenylephrine (PE, for 3 hours) and puromycin incorporation (for 30 min). (B) Quantification of the Western blot shown in (A) (N=3 samples/group).


( C ) of surface of isolated neonatal mouse cardiomyocyte of Tip30 Het WT mice treated with endothelin - 1 ( ET - 1 ) Narciclasine ( Narci ) or without stimulation as indicated ( N = 6 samples / group ) .

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

(C) Quantification of cell surface area of isolated neonatal mouse cardiomyocyte of Tip30 Het and WT mice treated with endothelin-1 (ET-1) and Narciclasine (Narci) or without stimulation as indicated (N=6 samples/group).


of HW / TL ( E ) , N = 6 to 10 mice / group , in Tip30 Het or WT mice 2 after TAC . were treated with Narciclasine daily for 14 days after TAC as indicated .

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

Quantification of HW/TL ratio (E), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.


of , echocardiographic wall thickness ( F ) , N = 6 to 10 mice / group , in Tip30 Het or WT mice 2 after TAC . were treated with Narciclasine daily for 14 days after TAC as indicated .

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

Quantification of , echocardiographic wall thickness (F), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.


of cardiomyocyte ( G ) , N = 5 mice / group in Tip30 Het or WT mice 2 after TAC . were treated with Narciclasine daily for 14 days after TAC as indicated .

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

Quantification of cardiomyocyte area (G), N=5 mice/group in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.


of fractional change ( FAC , H , N = 6 to 10 mice / group ) in Tip30 Het or WT mice 2 after TAC . were treated with Narciclasine daily for 14 days after TAC as indicated .

Quantification of fractional area change (FAC, H, N=6 to 10 mice/group) in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.


( I ) Western blot of puromycin incorporation in hearts of Tip30 Het WT mice 3 days after TAC or sham surgery daily Narciclasine ( Narci ) injection their ( N = 2 to 4 mice / group ) . Puromycin was injected 3 hours prior to sacrifice .

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

(I) Western blot analysis of puromycin incorporation in hearts of Tip30 Het and WT mice 3 days after TAC or sham surgery and daily Narciclasine (Narci) injection and their quantification (N=2 to 4 mice/group). Puromycin was injected 3 hours prior to sacrifice.


( C ) suffering from major depressive disorder ( MDD ) show a blunted ACTH response to CRH ( black , N = 10 ) , compared with control ( gray , N = 10 ) - data from ( von Bardeleben et al , 1988 ) , shown are mean ± .

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 O O O O 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) Patients suffering from major depressive disorder (MDD) show a blunted ACTH response to CRH (black line, N=10), compared with control (gray line, N=10) - data from (von Bardeleben et al, 1988), shown are mean ± SEM.


( D ) suffering from anorexia admitted to treatment show a blunted ACTH response hypercortisolemia , which resolves within 6 - 24 months after normalization - data from ( Gold et al , 1986a ) . However , 3 - 4 after normalization , cortisol dynamics are normal whereas ACTH dynamics are blunted . Pregnancy is associated with elevated cortisol due to CRH secretion by placenta . 3 after delivery , cortisol dynamics return to normal , whereas ACTH dynamics are blunted - data from ( Magiakou et al , 1996 ) . After 12 , ACTH dynamics normalize as well , while cortisol dynamics remain normal . Individuals recovering from alcohol abuse show hypercortisolemia blunted ACTH response after admission - data from ( von Bardeleben et al , 1989 ) . After 2 - 6 , these individuals show normal cortisol dynamics , but blunted ACTH responses persist . In panels , control data are denoted by thin gray ( Anorexia : N = 13 . Pregnancy : N was unspecified . Alcohol abuse disorder : N = 11 ) , case data by a thicker black ( Anorexia : left panel , N = 9 , center panel , N = 5 , right panel , N = 6 . Pregnancy : N = 17 . Alcohol abuse disorder : N = 20 ) . Shown are mean ± for panels .

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 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 B-MEASURED_VAR 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 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

(D) Patients suffering from anorexia and admitted to treatment show a blunted ACTH response and hypercortisolemia, which resolves within 6-24 months after weight normalization- data from (Gold et al, 1986a). However, 3-4 weeks after weight normalization, cortisol dynamics are normal whereas ACTH dynamics are blunted. Pregnancy is associated with elevated cortisol levels due to CRH secretion by the placenta. 3 weeks after delivery, cortisol levels and dynamics return to normal, whereas ACTH dynamics are blunted- data from (Magiakou et al, 1996). After 12 weeks, ACTH dynamics normalize as well, while cortisol dynamics remain normal. Individuals recovering from alcohol abuse show hypercortisolemia and blunted ACTH response after admission - data from (von Bardeleben et al, 1989). After 2-6 weeks, these individuals show normal cortisol dynamics, but blunted ACTH responses persist. In all panels, control patient data are denoted by thin gray line (Anorexia: N=13. Pregnancy: N was unspecified. Alcohol abuse disorder: N=11), and case data by a thicker black line (Anorexia: left panel, N=9, center panel, N=5, right panel, N=6. Pregnancy: N=17. Alcohol abuse disorder: N=20). Shown are mean ± SEM for all panels.


WT CKO mice aged 4 were selected divided into 4 groups , fed a normal diet until to 3 months or 18 months , respectively ( n = 10 ， there were 10 mice in each group , 6 mice in each group were selected for detection at end of experiment ) . H Representative of H & E in distal femora , scale bar , 50µm . I Representative of TRAP in distal femora , scale bar , 50µm . J , K of number of osteoblasts ( J ) number of osteoclasts ( K ) in distal femora ( n = 6 ) . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 * * * P ＜ 0 . 001 ( Student ' s t ) .

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

WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H Representative images of H&E staining in distal femora, scale bar, 50µm. I Representative images of TRAP staining in distal femora, scale bar, 50µm. J, K Quantification of number of osteoblasts (J) and number of osteoclasts (K) in distal femora (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).


WT CKO mice aged 4 were selected divided into 4 groups , fed a normal diet until to 3 months or 18 months , respectively ( n = 10 ， there were 10 mice in each group , 6 mice in each group were selected for detection at end of experiment ) . L , M Representative of Ocn + ( red ) DAPI ( blue ) immunostaining ( L ) of Ocn + on trabecular ( M ) , scale bar , 50µm ( n = 6 ) . N , O Representative of TRAP ( red ) DAPI ( blue ) immunostaining ( N ) of TRAP on trabecular ( O ) , scale bar , 50µm ( n = 6 ) . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 * * * P ＜ 0 . 001 ( Student ' s t ) .

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

WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). L, M Representative images of Ocn+ (red) and DAPI (blue) immunostaining (L) and quantification of Ocn+ cells on trabecular (M), scale bar, 50µm (n=6). N, O Representative images of TRAP (red) and DAPI (blue) immunostaining (N) and quantification of TRAP cells on trabecular (O), scale bar, 50µm (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).


WT CKO mice aged 4 were selected divided into 4 groups , fed a normal diet until to 3 months or 18 months , respectively ( n = 10 ， there were 10 mice in each group , 6 mice in each group were selected for detection at end of experiment ) . R , S serum of CTX ( R ) PINP ( S ) ( n = 6 ) . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 * * * P ＜ 0 . 001 ( Student ' s t ) .

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

WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). R, S The serum levels of CTX (R) and PINP (S) (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).


BMT was conducted on 12 - month - old CKO WT mice with normal diet , then recipients from each group were performed for 3 months after interventions ( n = 10 ， there were 10 mice in each group , 6 mice in each group were selected for detection at end of experiment ) . H Representative of H & E ( upper panel ) , TRAP ( middle panel ) calcein double - labeling ( lower panel ) in distal femora , scale bar , 100 µm for H & E . 50 µm for TRAP , 50μm for calcein double - labeling . I , J of number for osteoblasts ( I ) number of osteoclasts ( J ) in distal femora ( n = 6 ) . K of bone formation rate per bone surface ( BFR / BS ) ( n = 6 ) . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 * * * P ＜ 0 . 001 ( Student ' s t ) .

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

BMT was conducted on 12-month-old CKO and WT mice with normal diet, and then the recipients from each group were performed for analysis 3 months after interventions (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H Representative images of H&E staining (upper panel), TRAP staining (middle panel) and calcein double-labeling (lower panel) in distal femora, scale bar, 100 µm for H&E staining. 50 µm for TRAP staining, 50μm for calcein double-labeling. I, J Quantification of number for osteoblasts (I) and number of osteoclasts (J) in distal femora (n=6). K Quantification of bone formation rate per bone surface (BFR/BS) (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).


in situ myeloid specific MYDGF overexpression in bone marrow was performed on CKO mice aged 9 months . Micro - CT histomorphometric of distal femora were performed after 3 months intervention . H Representative of H & E in distal femora , scale bar , 100 µm . I of number for osteoblasts in distal femora ( n = 6 ) . J Representative of TRAP in distal femora , scale bar , 50µm K of number for osteoclasts in distal femora ( n = 6 ) . L Representative of calcein double - labeling , scale bar , 50μm . M of bone formation rate per bone surface ( BFR / BS ) ( n = 6 ) . Data information : Results are shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) .

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 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

in situ myeloid specific MYDGF overexpression in bone marrow was performed on CKO mice aged 9 months. Micro-CT analysis and histomorphometric analysis of distal femora were performed after 3 months intervention. H Representative images of H&E in distal femora, scale bar, 100 µm. I Quantification of number for osteoblasts in distal femora (n=6). J Representative images of TRAP staining in distal femora, scale bar, 50µm K Quantification of number for osteoclasts in distal femora (n=6). L Representative images of calcein double-labeling, scale bar, 50μm. M Quantification of bone formation rate per bone surface (BFR/BS) (n=6). Data information: Results are shown as mean ±SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t test).


A - C BMMs from WT CKO mice were induced with M - CSF ( 100 ng / ml ) RANKL ( 50 ng / ml ) for osteoclast differentiation . Representative TRAP ( A ) , scale bar , 200μm . of osteoclast number ( B ) of osteoclast ( C ) . Results are shown as mean ± , * * * P < 0 . 001 ( Student ' s t ) . Data information : Each experiment was repeated 5 times .

O O O O O O O O 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

A-C Primary BMMs from WT and CKO mice were induced with M-CSF (100 ng/ml) and RANKL (50 ng/ml) for osteoclast differentiation. Representative TRAP staining images (A), scale bar, 200μm. Quantification of osteoclast number (B) percentage of osteoclast area (C). Results are shown as mean ± SEM, ***P<0.001 (Student's t test). Data information: Each experiment was repeated 5 times.


D - F BMMs with treatment of conditioned medium of BMCs from WT or CKO mice for 7 days of osteoclast differentiation with or without RANKL ( 50 ng / ml ) . M - CSF ( 100 ng / ml ) was present in settings . Representative TRAP , upper panel represent cultured without RANKL , lower panel represent cultured with RANKL ( 50 ng / ml ; D ) , scale bar , 200μm . of osteoclast number of TRAP - positive ( E ) of osteoclast ( F ) . Results are shown as mean ± , * P < 0 . 05 ( Student ' s t ) . Data information : Each experiment was repeated 5 times .

O 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 B-MEASURED_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 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

D-F BMMs with treatment of conditioned medium of BMCs from WT or CKO mice for 7 days of osteoclast differentiation with or without RANKL (50 ng/ml). M-CSF (100 ng/ml) was present in all settings. Representative TRAP staining images, upper panel represent cultured without RANKL, lower panel represent cultured with RANKL (50 ng/ml; D), scale bar, 200μm. Quantification of osteoclast number of TRAP-positive cells (E) and percentage of osteoclast area (F). Results are shown as mean ± SEM, *P<0.05 (Student's t test). Data information: Each experiment was repeated 5 times.


N - P BMMs were co - cultured with osteoclasts in presence of 1 , 25 ( OH ) 2D3 PGE2 with conditioned medium of BMCs from WT or CKO mice for 5 days . Representative of osteoclasts co - cultured with osteoblasts , scale bar , 200μm ( N ) . of osteoclast number ( O ) of osteoclast ( P ) . Results are shown as mean ± , * P < 0 . 05 ( Student ' s t ) . Data information : Each experiment was repeated 5 times .

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

N-P BMMs were co-cultured with osteoclasts in presence of 1,25(OH)2D3 and PGE2 with conditioned medium of BMCs from WT or CKO mice for 5 days. Representative images of osteoclasts co-cultured with osteoblasts, scale bar, 200μm (N). Quantification of osteoclast number (O) and percentage of osteoclast area (P). Results are shown as mean ± SEM, *P < 0.05 (Student's t test). Data information: Each experiment was repeated 5 times.


G - I calvarial osteoblasts cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP at 7 days or ARS at 14 days . Representative of ALP ARS of osteoblasts , scale bar , 100μm ( G ) . Quantitative of ALP activity ( H ) calcium mineralization ( I ) . Data information : Results are shown as mean ± , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) . Each experiment was repeated 5 times .

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 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

G-I Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP staining assay at 7 days or ARS staining assay at 14 days. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (G). Quantitative analysis of ALP activity (H) and calcium mineralization (I). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.


J - L calvarial osteoblasts cultured in osteogenesis induction medium with treatment of 0ng / ml ( vehicle ) or 100ng / ml rMYDGF . Representative of ALP ARS of osteoblasts , scale bar , 100μm ( J ) . Quantitative of ALP activity ( K ) calcium mineralization ( L ) . Data information : Results are shown as mean ± , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) . Each experiment was repeated 5 times .

O O O 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 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

J-L Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of 0ng/ml (vehicle) or 100ng/ml rMYDGF. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (J). Quantitative analysis of ALP activity (K) and calcium mineralization (L). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.


Bone defects models were conducted on young ( 3 - month - old ) aged ( 18 - month - old ) mice , AAV - GFP or AAV - MYDGF intra - marrow injection once 2 before bone defects surgery on young aged mice , defects was analyzed after 2 ( n = 10 , there were 10 mice in each group , 5 mice in each group were selected for detection at end of experiment ) . C , D Quantitative micro - CT of BMD ( C ) BV / TV ( D ) of regenerated bone in calvarial defects ( n = 5 ) . Data information : Results are shown as mean ± , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) .

O O O O O O 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

Bone defects models were conducted on young (3-month-old) and aged (18-month-old) mice, AAV-GFP or AAV-MYDGF intra-marrow injection once 2 weeks before bone defects surgery on young and aged mice, and the defects area was analyzed after 2 weeks (n=10, there were 10 mice in each group, and 5 mice in each group were selected for detection at the end of the experiment). C, D Quantitative micro-CT analysis of BMD (C) and BV/TV (D) of the regenerated bone in calvarial defects (n=5). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 (Student's t test).


Bone defects models were conducted on young ( 3 - month - old ) aged ( 18 - month - old ) mice , AAV - GFP or AAV - MYDGF intra - marrow injection once 2 before bone defects surgery on young aged mice , defects was analyzed after 2 ( n = 10 , there were 10 mice in each group , 5 mice in each group were selected for detection at end of experiment ) . I , J Quantitative micro - CT of BMD ( I ) BV / TV ( J ) of regenerated bone in calvarial defects ( n = 5 ) . Data information : Results are shown as mean ± , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) .

Bone defects models were conducted on young (3-month-old) and aged (18-month-old) mice, AAV-GFP or AAV-MYDGF intra-marrow injection once 2 weeks before bone defects surgery on young and aged mice, and the defects area was analyzed after 2 weeks (n=10, there were 10 mice in each group, and 5 mice in each group were selected for detection at the end of the experiment). I, J Quantitative micro-CT analysis of BMD (I) and BV/TV (J) of the regenerated bone in calvarial defects (n=5). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 (Student's t test).


OVX mice 18 - month - old mice serve as model of osteoporosis , AAV - MYDGF or AAV - GFP were injected into bone marrow cavity every 3 for 12 ( n = 10 , there were 10 mice in each group , 6 mice in each group were selected for detection at end of experiment ) . Micro - CT reconstruction , H & E of trabecular bone from distal femurs in Sham + GFP , OVX + GFP OVX + MYDGF groups ( n = 6 ) . Representative of micro - CT reconstruction , scale bar , 2mm ( A ) . Representative of H & E in distal femora , scale bar , 500μm for upper panel , 100μm for lower panel ( D ) .

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 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

OVX mice and 18-month-old male mice serve as the model of osteoporosis, AAV-MYDGF or AAV-GFP were injected into bone marrow cavity every 3 weeks for 12 weeks (n=10, there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). Micro-CT reconstruction, H&E staining of trabecular bone from distal femurs in Sham +GFP, OVX +GFP and OVX + MYDGF groups (n=6). Representative images of micro-CT reconstruction, scale bar, 2mm (A). Representative images of H&E staining in distal femora, scale bar, 500μm for upper panel,100μm for lower panel (D).


OVX mice 18 - month - old mice serve as model of osteoporosis , AAV - MYDGF or AAV - GFP were injected into bone marrow cavity every 3 for 12 ( n = 10 , there were 10 mice in each group , 6 mice in each group were selected for detection at end of experiment ) . H & E of trabecular bone from distal femurs in aged mice with AAV - GFP or AAV - MYDGF ( n = 6 ) . Representative of H & E in distal femora , scale bar , 500μm for upper panel , 100μm for lower panel ( J ) .

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 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

OVX mice and 18-month-old male mice serve as the model of osteoporosis, AAV-MYDGF or AAV-GFP were injected into bone marrow cavity every 3 weeks for 12 weeks (n=10, there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H&E staining of trabecular bone from distal femurs in aged mice with AAV-GFP or AAV-MYDGF (n=6). Representative images of H&E staining in distal femora, scale bar, 500μm for upper panel,100μm for lower panel (J).


A qRT - PCR for Nfatc1 , Ctsk , c - Fos , Mmp9 Acp5 mRNA expression in BMMs cultured in osteoclastogenesis induction medium for 48 hours . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 ( Student ' s t ) .

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

A qRT-PCR analysis for Nfatc1, Ctsk, c-Fos, Mmp9 and Acp5 mRNA expression in BMMs cultured in osteoclastogenesis induction medium for 48 hours. Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).


Representative WB of IKBα , P65 , PKCβ treated with or without rMYDGF for RANKL - induced

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

Representative WB images of IKBα, P65, PKCβ treated with or without rMYDGF for RANKL -induced osteoclastogenesis


M BMMs were treated with control , RANKL or MG132 for osteoclastogenesis , then with or without rMYDGF for 24 hours . IgG , P65 histone antibodies were used to ChIP RT - PCR was performed to determine Nfatc1 promoters . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 ( Student ' s t ) .

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

M BMMs were treated with control, RANKL or MG132 for osteoclastogenesis, then with or without rMYDGF for 24 hours. IgG, P65 and histone antibodies were used to ChIP and RT-PCR was performed to determine Nfatc1 promoters. Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).


O , P of osteoclast number ( O ) of osteoclast ( P ) . Data information : Results are shown as mean ± . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 ( Student ' s t ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of osteoclast number (O) and percentage of osteoclast area (P). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).


Representative WB of Smad2 / 3 , mTOR , STAT3 ( S ) , STAT3 ( Y ) , MAPK1 / 3 JAK1 / 2

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

Representative WB images of Smad2/3, mTOR, STAT3(S), STAT3(Y), MAPK1/3 and JAK1/2 proteins expression


H mRNA of Runx2 in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated interventions . I , J Quantitative of ALP activity ( I ) calcium mineralization ( J ) in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated intervention . Data information : Results are shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) .

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-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

H The mRNA levels of Runx2 in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated interventions. I, J Quantitative analysis of ALP activity (I) and calcium mineralization (J) in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated intervention. Data information: Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001(Student's t test).


O , P Representative WB diagram ( O ) of STAT3 ( S ) MAPK1 / 3 expression ( P ) . Data information : Results are shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t ) .

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 O O O O

O, P Representative WB diagram (O) and the protein levels of STAT3(S) and MAPK1/3 expression (P). Data information: Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001(Student's t test).


Metaphysis region in BM is rich in endomucin + capillaries expressing HO - 1 . mp - metaphysis ; gp - growth plate ; scale bar 100 μm . HO - 1 positive small capillaries in metaphysis express endomucin CD31 . Shown maximum projection , scale bar 20 μm . HO - 1 is expressed by smaller endomucin + CD31 + capillaries ( # ) as well as in bigger endomucin - / lowCD31 + arteries ( * ) . CD31 - pericytes wrapping artery also express HO - 1 ( * ) ; scale bar 20 μm . HO - 1 positive capillaries in metaphysis expressed CD31 Sca - 1 . capillaries are enveloped by HO - 1 expressing pericytes . Part of HO - 1 + pericytes express Sca - 1 ( # ) while others show no or low Sca - 1 signal ( * ) ; scale bar 20 μm .

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

Metaphysis region in the BM is rich in endomucin+ capillaries expressing HO-1. mp - metaphysis; gp - growth plate; scale bar 100 μm. The HO-1 positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm. HO-1 is expressed by smaller endomucin+CD31+ capillaries (#) as well as in bigger endomucin-/lowCD31+ arteries (*). CD31- pericytes wrapping the artery also express HO-1 (*); scale bar 20 μm. HO-1 positive capillaries in the metaphysis expressed CD31 and Sca-1. The capillaries are enveloped by HO-1 expressing pericytes. Part of the HO-1+ pericytes express Sca-1 (#) while others show no or low Sca-1 signal (*); scale bar 20 μm.


Flow cytometry revealed highest expression of HO - 1 in CD31 + Sca - 1 + ECs . CAR PαS populations also express HO - 1 , while most of non - hematopoietic CD45 - Ter119 - are HO - 1 - negative in steady - state conditions .

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

Flow cytometry analysis revealed the highest expression of HO-1 in CD31+Sca-1+ ECs. CAR and PαS populations also express HO-1, while most of non-hematopoietic CD45-Ter119- are HO-1-negative in steady-state conditions.


BM macrophages ( MQ ) express HO - 1 . MHCIIhigh MQ express higher of HO - 1 than MHCIIlow MQ . within whole HSPC compartment ( LKS ) express no or low of HO - 1 in comparison to MQ .

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

BM macrophages (MQ) express HO-1. The MHCIIhigh MQ express higher levels of HO-1 than MHCIIlow MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO-1 in comparison to MQ.


HO - 1 expression on mRNA level quantified by qPCR qPCR on two independent experiments n = 10 - 11 /

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

HO-1 expression on mRNA level quantified by qPCR qPCR analysis based on two independent experiments n = 10-11/group


HO - 1 expression on mRNA level quantified by - seq . RNAseq has n = 3 - 4 / group , two - t - for two groups comparison , one way ANOVA with Bonferroni post - for multiple group comparison . * p < 0 . 05 , * * * * p < 0 . 0001 . Data are shown as mean ± .

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

HO-1 expression on mRNA level quantified by RNA-seq. RNAseq analysis has n = 3-4/group, two-tailed t-test for two groups comparison, and one way ANOVA with Bonferroni post-test for multiple group comparison. *p< 0.05, ****p< 0.0001. Data are shown as mean ± SEM.


ECs CARs from middle - aged ( 11 - 12 months ) express lower of HO - 1 . Two independent experiments , n = 5 - 10 / group . Data are shown as mean ± . * p < 0 . 05 , * * * p < 0 . 001 , two - unpaired t - .

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 O

ECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean ± SEM. *p< 0.05, ***p<0.001, two-tailed unpaired t-test.


Middle - aged have lower frequency of ECs . Two independent experiments , n = 10 - 11 / group . Data are shown as mean ± . * * p < 0 . 01 , two - unpaired t - . control of HO - 1 on HO - 1 - / - bone marrow section is provided in Appendix Fig S1 .

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

Middle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean ± SEM. **p<0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1-/- bone marrow section is provided in Appendix Fig S1.


- seq revealed 111 DEGs in HO - 1 - / - ECs .

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

RNA-seq revealed 111 DEGs in HO-1-/- ECs.


identified DEGs separated HO - 1 - / - HO - 1 + / + ECs by ( B ) hierarchical clustering ( C ) PCA .

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

The identified DEGs separated HO-1-/- and HO-1+/+ ECs by (B) hierarchical clustering and (C) PCA.


of selected ( E ) hematopoietic factors ( F ) adhesion molecules expression in HO - 1 - / - HO - 1 + / + ECs .

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

Heatmap of selected (E) hematopoietic factors and (F) adhesion molecules expression in HO-1-/- and HO-1+/+ ECs.


- seq revealed 100 DEGs in HO - 1 - / - CARs .

RNA-seq revealed 100 DEGs in HO-1-/- CARs.


identified DEGs separated HO - 1 - / - HO - 1 + / + CARs by ( H ) hierarchical clustering ( I ) PCA .

The identified DEGs separated HO-1-/- and HO-1+/+ CARs by (H) hierarchical clustering and (I) PCA.


of selected ( K ) skeletal biology ( L ) hematopoietic factors expression in HO - 1 - / - HO - 1 + / + CARs . Correlation similarity metric average linkage clustering were used in presented heatmaps .

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

Heatmap of selected (K) skeletal biology and (L) hematopoietic factors expression in HO-1-/- and HO-1+/+ CARs. Correlation similarity metric and average linkage clustering were used in the presented heatmaps.


HO - 1 - / - mice have more ECs less CARs ( p = 0 . 053 ) in BM , while frequency of PaS does not differ .

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

HO-1-/- mice have more ECs and less CARs (p = 0.053) in BM, while frequency of PaS does not differ.


Young HO - 1 - / - mice have lower frequency of Sca - 1high fraction among BM ECs , what resembles phenotype of BM ECs in old mice .

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Young HO-1-/- mice have lower frequency of Sca-1high fraction among BM ECs, what resembles phenotype of BM ECs in old mice.


Intracellular flow cytometry reveals ( C ) lower of SDF - 1α in HO - 1 - / - CARs ( D ) lower of LAP in HO - 1 - / - ECs .

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

Intracellular flow cytometry reveals (C) lower levels of SDF-1α in HO-1-/- CARs and (D) lower levels of LAP protein in HO-1-/- ECs.


of surface SCF revealed lower frequency of SCF + ECs in HO - 1 - / - while no differences among CARs .

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

Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs.


frequency of ( F ) CD163 + MQs ( G ) CD163 - MQs in BM of HO - 1 - / - mice is altered .

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

The frequency of (F) CD163+ MQs and (G) CD163- MQs in BM of HO-1-/- mice is altered.


Gating strategy of hematopoietic progenitor .

Gating strategy of hematopoietic stem and progenitor cells.


Young HO - 1 - / - mice possess higher number of ( B ) LT - HSCs , ( C ) ST - HSCs ( D ) MPPs .

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

Young HO-1-/- mice possess higher number of (B) LT-HSCs, (C) ST-HSCs and (D) MPPs.


Exemplary analysis of cell cycle with Ki67 and nuclear dye.


More young HO - 1 - / - LT - HSCs are in G1 S / G2 / M cycle phases . presented cycle is from two independent experiments . Young HO - 1 - / - MPPs do not differ in cycling from young HO - 1 + / + MPPs . presented cycle is from two independent experiments . More old HO - 1 - / - LT - HSCs are in G1 phase in comparison to old HO - 1 + / + LT - HSCs , but not in S / G2 / M phase . presented cycle is from two independent experiments . Old MPPs do not differ in cycling between genotypes . presented cycle is from two independent experiments .

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

More young HO-1-/- LT-HSCs are in G1 and S/G2/M cell cycle phases. The presented cell cycle analysis is from two independent experiments. Young HO-1-/- MPPs do not differ in cell cycling from young HO-1+/+ MPPs. The presented cell cycle analysis is from two independent experiments. More old HO-1-/- LT-HSCs are in G1 phase in comparison to old HO-1+/+ LT-HSCs, but not in S/G2/M phase. The presented cell cycle analysis is from two independent experiments. Old MPPs do not differ in cell cycling between genotypes. The presented cell cycle analysis is from two independent experiments.


Young HO - 1 - / - LT - HSCs contain more γH2aXhigh than young HO - 1 + / + , 378 - 991 analyzed from 7 mice / group . Young HO - 1 - / - MPPs contain more γH2aXhigh than young HO - 1 + / + , 1693 - 2790 analyzed from 7 mice / group .

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

Young HO-1-/- LT-HSCs contain more γH2aXhigh cells than young HO-1+/+, 378-991 cells analyzed from 7 mice/group. Young HO-1-/- MPPs contain more γH2aXhigh cells than young HO-1+/+, 1693-2790 cells analyzed from 7 mice/group.


Alkaline comet revealed that HO - 1 - / - LT - HSCs possess ( L ) higher oil tail moment ( M ) more in comet tail . 1068 - 1189 analyzed from 6 mice / group .

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

Alkaline comet assay revealed that HO-1-/- LT-HSCs possess (L) higher oil tail moment and (M) more DNA in the comet tail. 1068-1189 cells analyzed from 6 mice/group.


HSCs from young HO - 1 - / - mice provide worse hematopoietic reconstitution after transplantation than HO - 1 + / + HSCs . Data are shown as mean ± , n = 8 - 9 mice / group .

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

HSCs from young HO-1-/- mice provide worse hematopoietic reconstitution after transplantation than HO-1+/+ HSCs. Data are shown as mean ± SEM, n = 8-9 mice/group.


- seq revealed 1067 DEGs between young HO - 1 - / - HO - 1 + / + LT - HSCs . - seq revealed 595 DEGs between old HO - 1 - / - HO - 1 + / + LT - HSCs .

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

RNA-seq revealed 1067 DEGs between young HO-1-/- and HO-1+/+ LT-HSCs. RNA-seq revealed 595 DEGs between old HO-1-/- and HO-1+/+ LT-HSCs.


PC1 in PCA on selected 1148 genes separated young HO - 1 + / + LT - HSCs from other groups , clustering young HO - 1 - / - LT - HSCs together with old LT - HSCs .

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

PC1 in PCA analysis on selected 1148 genes separated young HO-1+/+ LT-HSCs from other groups, clustering the young HO-1-/- LT-HSCs together with old LT-HSCs.


Hierarchical clustering on 20 % of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO - 1 + / + LT - HSCs in one old LT - HSCs with young HO - 1 - / - LT - HSCs in second .

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

Hierarchical clustering on the 20% of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO-1+/+ LT-HSCs in one and old LT-HSCs with young HO-1-/- LT-HSCs in second.


Deletion of HO - 1 in myeloid lineage did not cause expansion of LT - HSCs did not alter their cycle status .

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

Deletion of HO-1 in myeloid lineage did not cause expansion of LT-HSCs and did not alter their cell cycle status.


Long - term PB chimerism derived from HSCs transplanted to HO - 1 - / - recipients is lower than from HSCs transplanted to HO - 1 + / + recipients .

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

Long-term PB chimerism derived from HSCs transplanted to HO-1-/- recipients is lower than from HSCs transplanted to HO-1+/+ recipients.


Chimerism among BM HSPCs fractions transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± , n = 5 - 12 /

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

Chimerism among BM HSPCs fractions transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group


total number of derived from HSCs transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± , n = 5 - 12 /

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

total number of cells derived from HSCs transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group


Short term homing of HSPC to HO - 1 + / + or HO - 1 - / - BM did not differ . Data are shown as mean ± , n = 5 / group , unpaired t - .

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

Short term homing of HSPC to HO-1+/+ or HO-1-/- BM did not differ. Data are shown as mean ± SEM, n = 5/group, unpaired t-test.


Transplantation of HO - 1 - / - HSCs provides lower chimerism among HSPC fractions in recipients . Data are shown as mean ± , n = 7 - 8 mice / group .

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

Transplantation of HO-1-/- HSCs provides lower chimerism among HSPC fractions in primary recipients. Data are shown as mean ± SEM, n = 7-8 mice/group.


Transplantation of same number of donor - derived BM from recipients provides same ( C ) PB

Transplantation of the same number of donor-derived BM cells from primary recipients provides the same (C) PB chimerism


Transplantation of same number of donor - derived BM from recipients provides same BM LT - HSC chimerism .

Transplantation of the same number of donor-derived BM cells from primary recipients provides the same BM LT-HSC chimerism.


Comparison of gene log fold changes in non - transplanted HO - 1 - / - LT - HSCs HO - 1 - / - LT - HSCs transplanted twice to wild - type HO - 1 + / + showed that transplantation of HO - 1 - / - LT - HSCs twice to wild type niche reverses their transcriptional alterations .

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

Comparison of gene log fold changes in non-transplanted HO-1-/- LT-HSCs and HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+ showed that transplantation of HO-1-/- LT-HSCs twice to the wild type niche reverses their transcriptional alterations.


Frequency of colonies appearing at day 8 day 12 from HSCs co - cultured with HO - 1 + / + or HO - 1 - / - MSCs . * p < 0 . 05 , * * * p < 0 . 001 , Fisher exact .

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

Frequency of colonies appearing at day 8 and day 12 from HSCs co-cultured with HO-1+/+ or HO-1-/- MSCs. *p< 0.05, ***p<0.001, Fisher exact test.


PCA cluster of colonies on initial flow cytometry gating revealed ( C ) 4 distinct clusters of colonies , that differ by frequency of ( D ) CD11b + Gr1 - , ( E ) CD11b + Gr1 +

O O O 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 B-MEASURED_VAR O B-MEASURED_VAR O

PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (C) 4 distinct clusters of colonies, that differ by frequency of (D) CD11b+Gr1-, (E) CD11b+Gr1+


PCA cluster of colonies on initial flow cytometry gating revealed ( F ) lacking expression of analyzed lineage markers ( Lin - ) .

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

PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (F) cells lacking expression of analyzed lineage markers (Lin-).


of frequency of colonies derived from HO - 1 + / + or from HO - 1 - / - MSC co - cultures among identified clusters . Enrichment in cluster analyzed by Fisher exact , * * p < 0 . 01 , two - unpaired t - .

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

Analysis of frequency of colonies derived from HO-1+/+ or from HO-1-/- MSC co-cultures among identified clusters. Enrichment in cluster analyzed by Fisher exact test, **p<0.01, two-tailed unpaired t-test.


Sdf1α mRNA in BM of HO - 1 + / + or HO - 1 - / - mice after administration of Noggin . Data are shown as mean ± . * p < 0 . 05 , two - unpaired t - , n = 10 /

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

Sdf1α mRNA levels in BM of HO-1+/+ or HO-1-/- mice after administration of Noggin. Data are shown as mean ± SEM. *p< 0.05, two-tailed unpaired t-test, n = 10/group


LT - HSC frequency of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice . Data are shown as mean ± . * p < 0 . 05 , * * p < 0 . 01 , two - unpaired t - , n = 8 - 10 /

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

LT-HSC frequency of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice. Data are shown as mean ± SEM. *p< 0.05, **p<0.01, two-tailed unpaired t-test, n = 8-10/group


cycling status of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice .

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

cell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice.


A . Assessment of in different AIFM1 knockout clones . Lysates from different AIFM1 knockout clones wild type were analyzed by reducing SDS - PAGE immunoblotting . MIA40 / CHCHD4 are not changed in AIFM1 knockout HEK293 .

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

A. Assessment of protein levels in different AIFM1 knockout clones. Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells.


D . Assessment of in HEK293 , AIFM1 knockout AIFM1 knockout with reintroduced AIFM1 - HA ( expression of AIFM1 - HA induced for one day ) . Different amounts of lysates from indicated were analyzed by reducing SDS - PAGE immunoblotting . While of MIA40 were largely unchanged , of many MIA40 / CHCHD4 substrates were diminished in AIFM1 knockout . N = 3 biological replicates .

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

D. Assessment of protein levels in HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA (expression of AIFM1-HA induced for one day). Different amounts of lysates from the indicated cells were analyzed by reducing SDS-PAGE and immunoblotting. While levels of MIA40 were largely unchanged, levels of many MIA40/CHCHD4 substrates were diminished in AIFM1 knockout cells. N = 3 biological replicates.


F . BN - PAGE analyses of HEK293 , AIFM1 knockout AIFM1 knockout with reintroduced AIFM1 - HA or AIFM1 . I was resolved from isolated mitochondria with blue native electrophoresis ( BN - PAGE ) followed by immunoblotting or in gel determination of I specific activity . of I were decreased in AIFM1 knockout . N = 3 biological

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

F. BN-PAGE analyses of HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA or AIFM1. Complex I was resolved from isolated mitochondria with blue native electrophoresis (BN-PAGE) followed by immunoblotting or in gel determination of the complex I specific activity. Levels of complex I were decreased in AIFM1 knockout cells. N = 3 biological replicates


A . In organello import with NDUFS5 , NDUFB7 as control matrix SOD2 . In vitro translated radioactive were incubated with mitochondria isolated from HEK293 AIFM1 knockout . Non - imported were removed by treatment with Proteinase K . Imported were analyzed by reducing SDS - PAGE autoradiography . Signals were quantified using ImageQuantTL amount of imported was plotted . NDUFS5 import is strongly affected by loss of AIFM1 . Import of NDUFB7 is less affected SOD2 import remained unaffected . This is despite fact that domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar .

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

A. In organello import assay with NDUFS5, NDUFB7 and as control the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar.


B . Oxidation kinetics of endogenous NDUFB7 NDUFS5 . HEK293 , AIFM1 knockout AIFM1 knockout complemented with AIFM1 - HA were analyzed by - chase experiments coupled to redox state determination . To this end , were incubated with 35S - Met for 5 min ( ) . After removal of 35S - Met , were left in " cold " Met for different chase times . Further oxidation was stopped by addition of trichloroacetic acid ( TCA ) . Free thiols but not thiols in disulfide bonds were modified using mmPEG24 then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , analyzed by non - reducing SDS - PAGE autoradiography . Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout . NDUFS5 dropped rapidly only little oxidized NDUFS5 could be observed . N = 4 biological replicates for NDUFB7 NDUFS5 .

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

B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in "cold" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5.


C . Synthesis of NDUFS5 NDUFB7 in wild type AIFM1 knockout . were incubated with 35S - Met for 5 min ( ) , then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , analyzed by non - reducing SDS - PAGE autoradiography . NDUFS5 NDUFB7 are synthesized in equal amounts in wildtype AIFM1 knockout .

O O O O B-MEASURED_VAR I-MEASURED_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 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

C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.


D . Interaction between endogenous MIA40 / CHCHD4 its substrates in wild type AIFM1 knockout . were subjected to native immunoprecipitation ( IP ) against endogenous MIA40 / CHCHD4 . Precipitates were analyzed by SDS - PAGE immunoblotting against indicated . In AIFM1 knockout interaction between MIA40 / CHCHD4 its substrates is strongly affected with interaction between MIA40 / CHCHD4 NDUFS5 almost completely abolished . This is despite fact that NDUFS5 NDUFB7 are synthesized in equal amounts in wildtype AIFM1 knockout , indicating that lowered steady state of these are consequence of a lacking interaction with MIA40 / CHCHD4 rather than cause . using Image lab . Data from 2 experiments were combined standard deviations are presented . Asterisk indicates background band orange arrow indicates signal of NDUFS5 NDUFB7 in AIFM1 KO , respectively .

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

D. Interaction between endogenous MIA40/CHCHD4 and its substrates in wild type and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively.