Figure 1 ( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for 4 h ( as described in the Materials and Methods ) , and then incubated for 24 h ( left ) or the indicated periods of time ( right ) . Cells were fixed , stained with DAPI to visualize nuclei ( blue ) , and immunolabeled with an anti - LC3 antibody . Primary antibody was detected using an Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( green ) . Left : representative immunofluorescence images of LC3 punctae ; right : quantification of data ( LC3 - punctated cells were counted manually ) . * * * p < 0 . 001 , vs . Mtb - WT - infected condition . Scale bars , 5 µm . ( ( B ) BMDMs were infected with Mtb - Δeis in the absence or presence of 3 - methyladenine ( 3 - MA ; 10 mM ) and subjected to confocal analysis as described in Figure 1A . LC3 - punctated cells were counted manually . Each condition was assayed in triplicate , and at least 250 cells were counted in each well . * * * p < 0 . 001 , vs . SC . ( C ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . Experimental conditions were identical to those outlined in panel A . Gel images representative of three experiments are shown . ( D ) Electron micrographs of Mtb - Δeis - infected BMDMs under low ( left ) and high ( right ) magnification show the accumulation of autophagic vesicles ( black arrow , initial autophagic vacuoles ; white arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left ) , 0 . 5 µm ( right ) . ( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs were infected with Mtb - Δeis in the presence or absence of 3 - MA ( 10 mM ) or bafilomycin A1 ( Baf - A1 ; 100 nM ) . Gel images representative of three experiments are shown . The ratio of the intensities of the LC3 - II / LC3 - I and β - actin bands is indicated below each lane ( C and E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % distilled water ( B ) , 0 . 1 % DMSO ( E ) ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p<0.001, vs. Mtb-WT-infected condition. Scale bars, 5 µm. ((B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC.(C) Immunoblot analyses performed using Abs raised to LC3 or β-actin. Experimental conditions were identical to those outlined in panel A. Gel images representative of three experiments are shown.(D) Electron micrographs of Mtb-Δeis-infected BMDMs under low (left) and high (right) magnification show the accumulation of autophagic vesicles (black arrow, initial autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left), 0.5 µm (right).(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).


Figure 2 ( A and B ) BMDMs were infected with Mtb - WT , Mtb - Δeis or Mtb - c - eis at different MOIs ( 0 . 1 , 1 and 10 ) for 18 h ( A ) or for the indicated periods of time ( B ; MOI = 10 ) . Supernatants were assessed by ELISA for levels of TNF - α and IL - 6 . Data ( A and B ) are presented as the mean ± SD of five experiments . ( C and D ) BMDMs were stimulated with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 30 min . Cells were then incubated with 10 µM DHE or 5 µM DCFH - DA for 15 min , washed thoroughly , and immediately analyzed for superoxide or H2O2 production by flow cytometry ( C , Left ) . Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry ( D , top ) . Quantitative analysis of ROS generation ( C , right ; D , bottom ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis or Mtb-c-eis at different MOIs (0.1, 1 and 10) for 18 h (A) or for the indicated periods of time (B; MOI = 10). Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data (A and B) are presented as the mean±SD of five experiments.(C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


Figure 3 ( A - C ) BMDMs were infected with Mtb - Δeis ( MOI = 10 ) for 18 h in the presence or absence of DPI ( 10 µM ) , NAC ( 20 mM ) , catalase ( Cat , 1 mU / mL ) , or tiron ( 5 mM ) . ( A ) Representative immunofluorescence images ( top ) ; percentage of endogenous LC3 - punctated cells ( bottom ) ( B ) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β - actin . Gel images are representative of three experiments . The ratio of the intensities of the LC3 - II / LC3 - I and β - actin bands is indicated below each lane . ( C ) Experimental conditions were identical to those outlined in Figure 3A . Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA . Data represent the mean ± SD of five experiments . ( D - F ) BMDMs from WT and NOX2 KO mice were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 18 h . ( D ) Numbers of LC3 - punctated cells ( counted manually ) are shown ( at least 250 cells were counted in each well ) . ( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs from WT and NOX2 KO mice were infected with Mtb - Δeis for 18 h . Gel images representative of three experiments are shown . ( F ) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA . Data are presented as the mean ± SD of at least three separate experiments , each performed in triplicate . * * * p < 0 . 001 , vs . SC ( A and C ) ; WT mice ( D and F ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom)(B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane.(C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments.(D-F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well).(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown.(F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).


Figure 4 ( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for the indicated periods of time , washed to remove unbound mycobacteria , and then incubated in complete DMEM at 37 ° C in 5 % CO2 . Cell viability was assessed by PI staining and then examined by fluorescence microscopy . ( B and C ) Experimental conditions were identical to those outlined in panel A . BMDMs were infected with the three strains of mycobacteria for 36 h . B ) Apoptosis was assessed using a TUNEL / apoptosis detection kit , according to the manufacturer ' s protocol . Cells were then examined under a laser - scanning confocal microscope ( LSM 510 ; Zeiss ) . Percentages of TUNEL - positive , PI - positive , and TUNEL - / PI - double - positive cells were calculated . Data are representative of three separate experiments . ( C ) Morphological changes in BMDMs infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis at MOIs of 1 and 10 . Representative images are shown . Scale bars : 50 µm ( low magnification ) , 20 µm ( high magnification ) . ( D ) Macrophages were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis in the presence or absence of zVAD - fmk ( 20 µM ) or 3 - MA ( 10 mM ) . Staurosporine ( STS ; 500 nM ) was used as a positive control . After 36 h , cells were stained with PI and then examined by fluorescence microscopy . Data are presented as the mean ± SD of at least three separate experiments , each performed in duplicate . * p < 0 . 05 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( A and B ) ; Mtb - Δeis - infected condition without inhibitors ( D ) . UI , uninfected .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h.B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments.(C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification).(D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.


Figure 5 ( A and B ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 1 ) for the indicated periods of time , and then subjected to Western blot analysis using Abs raised to p - ERK1 / 2 , p - p38 , p - JNK , and β - actin . Data shown are representative of three independent experiments that all yielded similar results ( A ) . Expression of phospho - MAPK / β - actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically ( B ) . ( C and D ) BMDMs were pretreated with U0126 ( 5 , 10 , 20 µM ) , SB203580 ( SB ; 1 , 5 , 10 µM ) , or SP600125 ( SP ; 5 , 10 , 20 µM ) for 45 min , and then infected with Mtb - Δeis for 30 min ( C ) or 36 h ( D ) . ( C ) Cells were then incubated with DHE for 15 min , washed rapidly and thoroughly , and analyzed immediately for superoxide levels by flow cytometry . Quantitative DHE fluorescence data represent the mean ± SD of four experiments . ( D ) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy . ( ( E ) Raw264 . 7 cells were transfected with siRNA specific for JNK1 ( siJNK ) or a non - specific control siRNA ( siNS ) . At 24 h after transfection , cells were infected with Mtb - Δeis for 36 h . Cell death was then assessed by PI staining , and then examined by flow cytometry . Transfection efficiency was assessed by RT - PCR ( inset ) . Data represent the mean ± SD of five random fields and are representative of three independent experiments ( D and E ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( B ) ; SC ( C and D ) ; siNS ( E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 1) for the indicated periods of time, and then subjected to Western blot analysis using Abs raised to p-ERK1/2, p-p38, p-JNK, and β-actin. Data shown are representative of three independent experiments that all yielded similar results (A). Expression of phospho-MAPK/β-actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically (B).(C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. ((E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).


Figure 6C57BL / 6 mice were challenged , by aerosol , with 10 - 30 CFU Mtb - WT , Mtb - Δeis , or Mtb - c - eis and sacrificed 4 weeks post - infection . ( A ) High - and low - magnification electron micrographs of lung tissue sections from mice infected with Mtb - Δeis show accumulation of autophagic vesicles ( black arrow , bacteria in autophagic vacuoles ; white arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left upper ) , 0 . 5 µm ( right ) . Numbers of autophagic vacuoles per cell in each TEM section ( left lower ) ( mean ± SD ; n = 50 ) . ( B ) Quantitative RT - PCR analysis of lung tissue from Mtb - WT - , Mtb - Δeis - , and Mtb - c - eis - infected mice . Total RNA was extracted from paraffin - embedded lung tissue sections , as described in the Materials and Methods . ( 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C57BL/6 mice were challenged, by aerosol, with 10-30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50).(B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods.(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 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 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.


Figure 7 ( 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 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 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 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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 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 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 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).


Figure 1DFL 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 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 . 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 .

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Figure 1DFL 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 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.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.


Figure 2Histogram 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 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 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 DFLDFL 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 ) .

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Figure 2Histogram 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 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 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 DFLDFL 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).


Figure 31H - 15N HSQC HMGB1 ( 0 . 1 mM ) spectrum1H - 15N HSQC HMGB1 ( 0 . 1 mM ) spectrum with 0 . 2 mM CXCL12 . 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 and ( E ) 1 mM DFL . 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 .

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Figure 31H-15N HSQC HMGB1 (0.1 mM) spectrum1H-15N HSQC HMGB1 (0.1 mM) spectrum with 0.2 mM CXCL12.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 and (E) 1 mM DFL.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.


Figure 4DFL 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 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 . 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 ) .

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Figure 4DFL 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 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.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).


Figure 1 ( 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 . Quantification of heart weight ( HW ) / tibia length ( TL ) ratio ( B ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of lung weight ( LuW / TL ) ratio ( C ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of , echocardiographic fractional area change ( D ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . Quantification of left ventricular enddiastolic area ( LVEDA ; E ) N = 4 to 18 mice / group , all 6 weeks after TAC or sham surgery . 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 . Quantification of α - MHC transcript abundance N = 4 to 18 mice / group , all 6 weeks 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 images of Sirius - red - stained heart sections ( scale bar : 1mm ) ( J ) fibrosis quantification ( K ) of indicated mice 6 weeks after TAC or sham surgery . 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 images of heart sections of indicated mice 6 weeks after TAC surgery stained for Isolectin B4 ( green ) and WGA ( red , M ) 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 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 . 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 ) . 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 ) .

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Figure 1(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.Quantification of heart weight (HW)/tibia length (TL) ratio (B) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of lung weight (LuW/TL) ratio (C) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of , echocardiographic fractional area change (D) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.Quantification of left ventricular enddiastolic area (LVEDA; E) N=4 to 18 mice/group, all 6 weeks after TAC or sham surgery.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.Quantification of α-MHC transcript abundance N=4 to 18 mice/group, all 6 weeks 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 images of Sirius-red-stained heart sections (scale bar: 1mm) (J)fibrosis quantification (K) of indicated mice 6 weeks after TAC or sham surgery.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 images of heart sections of indicated mice 6 weeks after TAC surgery stained for Isolectin B4 (green) and WGA (red, M)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 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.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).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).


Figure 2 ( A ) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes ( NRCM ) after adenoviral transduction with Ad . Control ( Ad . Con ) or Ad . TIP30 . 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 . 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 . 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 . 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 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 . 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 . * 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 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 ) .

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Figure 2(A) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes (NRCM) after adenoviral transduction with Ad.Control (Ad.Con) or Ad.TIP30.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.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.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.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 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.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. *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 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).


Figure 3 ( 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 analysis of co - immunoprecipitation ( IP ) from HEK cells transfected with GST - TIP30 and eEF1A1 - Myc . ( 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 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 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 . 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 analysis of GST - tagged eEF1A1 and GST - Control ( H ) . ( 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 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 analysis of co - immunoprecipitation ( IP ) with GST - TIP30 and eEF1A2 - His .

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Figure 3(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 analysis of co-immunoprecipitation (IP) from HEK cells transfected with GST-TIP30 and eEF1A1-Myc.(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 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 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.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 analysis of GST-tagged eEF1A1 and GST-Control (H).(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 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 analysis of co-immunoprecipitation (IP) with GST-TIP30 and eEF1A2-His.


Figure 4 ( 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 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 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 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 ) . *

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Figure 4(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 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 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 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). *


Figure 5Western 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 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification ( N = 4 mice / group ) . ( 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 .

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Figure 5Western 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 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification (N=4 mice/group).(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.


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

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


Figure 7 ( 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 ) 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 ) . 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 . 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 . 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 . 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 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 .

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Figure 7(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) 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).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.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.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.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 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.


Figure 1 ( 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 ) 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 .

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Figure 1(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) 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.


Figure 1WT 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1WT 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 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 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).


Figure 2BMT 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2BMT 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).


Figure 3in 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3in 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).


Figure 4A - 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-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 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 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.


Figure 5G - 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5G-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 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.


Figure 6Bone 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 ) 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Bone 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) 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).


Figure 7OVX 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7OVX 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 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).


Figure 8A 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 images of IKBα , P65 , PKCβ treated with or without rMYDGF for RANKL - induced osteoclastogenesisM 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A 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 images of IKBα, P65, PKCβ treated with or without rMYDGF for RANKL -induced osteoclastogenesisM 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 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).


Figure 9Representative WB images of Smad2 / 3 , mTOR , STAT3 ( S ) , STAT3 ( Y ) , MAPK1 / 3 and JAK1 / 2 proteins expressionH 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 ) 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 ) .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9Representative WB images of Smad2/3, mTOR, STAT3(S), STAT3(Y), MAPK1/3 and JAK1/2 proteins expressionH 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) 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).


Figure 1Metaphysis 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 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 . 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 analysis based on two independent experiments n = 10 - 11 / groupHO - 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Metaphysis 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 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. 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 analysis based on two independent experiments n = 10-11/groupHO-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 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 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.


Figure 2RNA - seq revealed 111 DEGs in HO - 1 - / - ECs . The identified DEGs separated HO - 1 - / - and HO - 1 + / + ECs by ( B ) hierarchical clustering and ( C ) PCA . Heatmap of selected ( E ) hematopoietic factors and ( F ) adhesion molecules expression in HO - 1 - / - and HO - 1 + / + ECs . RNA - seq revealed 100 DEGs in HO - 1 - / - CARs . The identified DEGs separated HO - 1 - / - and HO - 1 + / + CARs by ( H ) hierarchical clustering and ( I ) PCA . 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 .

O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2RNA-seq revealed 111 DEGs in HO-1-/- ECs.The identified DEGs separated HO-1-/- and HO-1+/+ ECs by (B) hierarchical clustering and (C) PCA.Heatmap of selected (E) hematopoietic factors and (F) adhesion molecules expression in HO-1-/- and HO-1+/+ ECs.RNA-seq revealed 100 DEGs in HO-1-/- CARs.The identified DEGs separated HO-1-/- and HO-1+/+ CARs by (H) hierarchical clustering and (I) PCA.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.