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

G-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 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

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 B-CONTROLLED_VAR 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 ) .

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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 ) .

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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 .

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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 .

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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 /

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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 ± .

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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 - .

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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 .

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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 .

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


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

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 /

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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 /

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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 - .

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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 .

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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 .

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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 .

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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 +

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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 - .

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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 .

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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 .

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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 .

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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

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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 .

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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 .

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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 .

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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 .

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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.


A . Assessment of MIA40 / CHCHD4 variant - AIFM1 interaction . indicated MIA40 / CHCHD4 - Strep variants were affinity precipitated ( AP ) under native conditions after stopping thiol - disulfide exchange reactions by NEM incubation . Precipitates were tested for AIFM1 , ALR MIA40 / CHCHD4 by reducing SDS - PAGE immunoblotting . 1 % of total lysate was loaded as input control . M , mock control not expressing MIA40 - Strep . Both , wildtype MIA40 / CHCHD4 MTS - MIA40 / CHCHD4 but not MIA40 / CHCHD4 ∆ 1 - 40 variants coprecipitated AIFM1 . mitochondria - localized variants precipitate ALR indicating redox functionality . Arrowhead indicates endogenous MIA40 / CHCHD4 .

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A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.


Emetine - chase experiments to assess stability of AIFM1 / CHCHD4 . HEK293 stably expressing MIA40 / CHCHD4 - Strep were treated for indicated times with ribosome inhibitor emetine . Then , were lysed under native conditions ( triton X - 100 ) , MIA40 / CHCHD4 - Strep ( B ) were precipitated . Precipitates were analysed by reducing SDS - PAGE immunoblotting . MIA40 / CHCHD4 interact over a period of at least 6 - 8 hours while two MIA40 / CHCHD4 substrates , NDUFB7 AK2 , are quickly released from MIA40 / CHCHD4 ( B , gray box ) . AP , affinity precipitation ; asterisk , background band .

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Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4-Strep were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and MIA40/CHCHD4-Strep (B) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 interact over a period of at least 6-8 hours while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band.


C . Emetine - chase experiments to assess stability of AIFM1 / CHCHD4 . AIFM1 knockout stably expressing AIFM1 - HA ( C ) were treated for indicated times with ribosome inhibitor emetine . Then , were lysed under native conditions ( triton X - 100 ) , AIFM1 - HA ( C ) were precipitated . Precipitates were analysed by reducing SDS - PAGE immunoblotting . MIA40 / CHCHD4 AIFM1 interact over a period of at least 6 - 8 hours AP , affinity precipitation ; asterisk , background band .

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C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours AP, affinity precipitation; asterisk, background band.


Eluted fractions were subjected to SDS - PAGE TCE ( E ) . AIFM1 full - MIA40 / CHCHD4 migrate together indicating formation of a . MIA40 / CHCHD4 was present in excess over AIFM1 resulting in presence of free MIA40 / CHCHD4 . AIFM1 ∆ 1 - 40 MIA40 / CHCHD4 do not migrate together emphasizing importance of N - terminal in MIA40 for interaction wth AIFM1 .

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Eluted fractions were subjected to SDS-PAGE and TCE staining (E). AIFM1 and full-length MIA40/CHCHD4 migrate together indicating formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and ∆1-40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N-terminal amino acids in MIA40 for interaction wth AIFM1.


A . Gel filtration to assess AIFM1 - MIA40 in HEK293 . HEK293 , lacking AIFM1 ( AIFM1 knockout , AIFM1 KO ) or AIFM1 knockout complemented with AIFM1 - HA were lysed under native conditions ( triton X - 100 ) , cleared lysates subjected to gel filtration . Eluted fractions were subjected to TCA precipitation , resuspension in loading buffer containing SDS DTT , subsequent immunoblotting against AIFM1 MIA40 . Endogenous AIFM1 endogenous MIA40 migrate in a with a larger than 150 kDa ( as judged by comparison to markers : apoferritin 443 kDa ; β - amylase 200 kDa ; alcohol dehydrogenase 150 kDa ; bovine serum albumin 66 kDa ; carbonic anhydrase 29 kDa ) . Absence of AIFM1 results in migration of MIA40 at height of monomeric MIA40 . This behavior is partially rescued by complementation with AIFM1 - HA . B . Gel filtration of a MIA40 variant lacking first 40 ( ∆ 1 - 40 MIA40 / CHCHD4 - Strep ) , which constitute AIFM1 interaction motif . Experiment was performed as described in ( A ) . AIFM1 endogenous MIA40 / CHCHD4 migrate in a with a larger than 150 kDa , while N - terminally truncated MIA40 / CHCHD4 migrates as monomer . For number of biological replicates quantifications , see 5C . C . of 5A , B Appendix Figure S9 . of areas representing region of monomeric MIA40 / CHCHD4 AIFM1 - MIA40 / CHCHD4 , respectively was performed . Quantifications consistently show that AIFM1 knockout , siRNA - mediated depletion of AIFM1 overexpression of MIA40 / CHCHD4 resulted in an increase of monomeric MIA40 / CHCHD4 indicating that amounts of available AIFM1 with respect to amounts of MIA40 / CHCHD4 are tightly regulated . numbers below bars indicate numbers of biological replicates performed . Averages standard deviations are presented .

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A. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C. C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented.


D . of AIFM1 MIA40 / CHCHD4 in mitochondria isolated from indicated tissues . of AIFM MIA40 / CHCHD4 was formed normalized to in HEK293 ( = 1 ) . AIFM1 is in liver kidney more abundant than MIA40 / CHCHD4 .

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D. Levels of AIFM1 and MIA40/CHCHD4 in mitochondria isolated from the indicated tissues. Ratio of AIFM and MIA40/CHCHD4 was formed and normalized to ratio in HEK293 cells (= 1). AIFM1 is in liver and kidney more abundant than MIA40/CHCHD4.


E . Gel filtration of lysates prepared from mitochondria isolated from indicated tissues from HepG2 HEK293 . Experiment was performed as described in ( A ) . AIFM1 MIA40 / CHCHD4 co - migrate in samples . Excess AIFM1 migrates at lower molecular mass . Number of biological replicates in plot . Where appropriate , standard deviations are presented .

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E. Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells. Experiment was performed as described in (A). AIFM1 and MIA40/CHCHD4 co-migrate in all samples. Excess AIFM1 migrates at lower molecular mass. Number of biological replicates in quantification plot. Where appropriate, standard deviations are presented.


F . Assessing solubility of MIA40 / CHCHD4 in IMS . Mitochondria were isolated form indicated lines outer destroyed by hypoxic swelling . MIA40 / CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype or AIFM1 knockout that had been complemented with AIFM1 - HA . It was released from lacking AIFM1 . This indicates that MIA40 / CHCHD4 in wildtype is - associated via AIFM1 .

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F. Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling. MIA40/CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1-HA. It was released from cells lacking AIFM1. This indicates that MIA40/CHCHD4 in wildtype cells is membrane-associated via AIFM1.


A . Oxidation kinetics of NDUFB7 NDUFS5 in presence of proteasome inhibitor MG132 . HEK293 , AIFM1 knockout AIFM1 knockout complemented with AIFM1 - HA were analysed by - chase experiments coupled to redox state determination . occurrence of oxidized NDUFB7 was not changed in presence of MG132 . Conversely , NDUFS5 were strongly increased . In AIFM1 knockout , large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation - dependent import . fraction of oxidized accumulating is slightly larger than in absence of MG132 . N = 1 biological replicate for NDUFB7 2 biological replicates for NDUFS5 .

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A. Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analysed by pulse-chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation-dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.


( a - d ) Formation of inclusions in SY5Y expressing FL ( a ) or indicated truncated forms of Sph1 ( b - d ) was induced by 12 h lactacystin treatment . After extensive washes , were maintained in serum - supplemented ( serum + ) or serum - free ( serum − ) media , in absence ( Control ) or presence of 10 mM 3 - MA , an inhibitor of macroautophagy . Graphs show relative fold difference in number of containing either Sph1 Agm ( left ) or Agg ( right ) observed under different recovery conditions . Values are mean + s . e . ( n = 4 ) . * Significant versus serum + condition . See also Supplementary Figs S1 - S5 .

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 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 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

(a-d) Formation of protein inclusions in SY5Y cells expressing FL (a) or the indicated truncated forms of Sph1 (b-d) was induced by 12 h lactacystin treatment. After extensive washes, cells were maintained in serum-supplemented (serum+) or serum-free (serum−) media, and in the absence (Control) or presence of 10 mM 3-MA, an inhibitor of macroautophagy. Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (left) or Agg (right) observed under different recovery conditions. Values are mean+s.e. (n=4). *Significant versus serum+ condition. See also Supplementary Figs S1-S5.


( a - c ) Top : of Agm Agg observed in MEFs from WT Atg5 KO mice expressing FL ( a ) , or indicated truncated Sph1 ( b - c ) treated with lactacystin ( 12 h ) recovered in basal conditions ( serum + ) . Graphs show relative fold difference in number of containing either Sph1 Agm ( middle ) or Agg ( bottom ) . Values are mean + s . e . ( n = 3 ) . * Significant versus control .

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

(a-c) Top: fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL (a), or the indicated truncated Sph1 proteins (b-c) treated with lactacystin (12 h) and recovered in basal conditions (serum+). Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (middle) or Agg (bottom). Values are mean+s.e. (n=3). *Significant versus control.


( d - f ) Relative fold difference in number of FL ( d ) or truncated ( e - f ) Sph1 Agm ( left ) Agg ( right ) in WT Atg5 KO mice MEFs incubated in serum + or serum − conditions . Values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition .

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(d-f) Relative fold difference in the number of FL (d) or truncated (e-f) Sph1 Agm (left) and Agg (right) in WT and Atg5 KO mice MEFs incubated in serum+ or serum− conditions. Values are mean+s.e. (n=3). *Significant versus serum+ condition.


( a - d ) Left : of SY5Y co - expressing HA - p38 ( red ) GFP ( green ) forms of FL ( a ) or indicated truncated Sph1 ( b - d ) . were treated with lactacystin ( 12 h ) recovered in presence or absence of serum . Graphs : of p38 existing alone in Agm or co - aggregating with Sph1 in forms of Agm or Agg ( left ) , susceptibility of p38 existing on its own in Agm or co - aggregating with Sph1 in Agm toward serum - stimulated autophagic clearance ( right ) . Values are expressed as folds Agm in presence of serum are mean + s . e . ( n = 3 ) . * Significant versus serum + condition calculated using 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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 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

(a-d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b-d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student's t-test.


( e ) Autophagic susceptibility of Agm formed by p38 ANK1 - p38 fusion . Values are mean + s . e . ( n = 3 ) . * Significant versus control group calculated using Bonferroni . See also Supplementary Fig . S6 .

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(e) Autophagic susceptibility of Agm formed by p38 and ANK1-p38 fusion proteins. Values are mean+s.e. (n=3). *Significant versus control group calculated using Bonferroni test. See also Supplementary Fig. S6.


( a ) Top : colocalization of LAMP1 ( red ) with Agm Agg of ACA CACT Sph1 highlighted by anti - GFP ( green ) in SY5Y . Arrows indicate colocalization . Bottom : LAMP1 colocalization with Agm Agg in maintained in serum + or serum − media , treated or not with lysosomal inhibitors ( N / L ) .

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(a) Top: colocalization of LAMP1 (red) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti-GFP (green) in SY5Y cells. Arrows indicate colocalization. Bottom: percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum+ or serum− media, treated or not with lysosomal inhibitors (N/L).


( b ) LAMP1 colocalization with Agm in co - expressing HA - p38 indicated Sph1 maintained in absence of serum .

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(b) Percentage LAMP1 colocalization with Agm in cells co-expressing HA-p38 and the indicated Sph1 proteins maintained in the absence of serum.


( c ) Left : colocalization of LAMP1 ( red ) with GFP - p38 - ANK1 - p38 Agm in SY5Y maintained in absence of serum . Arrows indicate colocalization . Right : LAMP1 colocalization with p38 ANK1 - p38 Agm in maintained in presence or absence of serum .

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(c) Left: colocalization of LAMP1 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Right: percentage LAMP1 colocalization with p38 and ANK1-p38 Agm in cells maintained in the presence or absence of serum.


( d , e ) Correlative electron microscopy of SY5Y expressing indicated GFP - tagged Sph1 truncations ( d ) , or p38 ANK1 - p38 ( e ) . Top : phase - contrast microscopy ; middle : electron of boxed region ; bottom : higher magnification examples of autophagic vacuoles ( yellow arrows ) . # Location of Agm or Agg structures in both corresponding .

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(d,e) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP-tagged Sph1 truncations (d), or p38 and ANK1-p38 (e). Top: phase-contrast fluorescence microscopy; middle: electron micrographs of the boxed region; bottom: higher magnification examples of autophagic vacuoles (yellow arrows). #Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs.


( f ) Top : colocalization of LC3 ( red ) with GFP - p38 - ANK1 - p38 Agm in SY5Y maintained in absence of serum . Arrows indicate colocalization . Bottom : colocalization of LC3 with . Values in graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , c , f ) or p38 Agm ( b ) calculated using Bonferroni . See also Supplementary Fig . S7 .

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(f) Top: colocalization of LC3 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Bottom: percentage colocalization of LC3 with the fluorescent proteins. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,c,f) or p38 Agm (b) calculated using Bonferroni test. See also Supplementary Fig. S7.


( a ) showing colocalization patterns of indicated GFP - p38 variants Sph1 ( green ) with endogenous p62 ( red ) in SY5Y . Arrows indicate colocalization .

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(a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells. Arrows indicate colocalization.


( b ) Quantitative FRAP of mCherry - p62 expressed alone ( control ) or co - expressed with indicated GFP - Sph1 - p38 variants . Top : time course of changes in relative ( RFI ) ; bottom : of p62 in diffusing fraction diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten analysed over three different experiments . Representative are shown in Supplementary Fig . S8 .

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(b) Quantitative FRAP analysis of mCherry-p62 expressed alone (control) or co-expressed with the indicated GFP-Sph1 and -p38 variants. Top: time course of changes in relative fluorescence intensity (RFI); bottom: percentage of p62 in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Representative images are shown in Supplementary Fig. S8.


( c - d ) Top : IBs for p62 actin in control p62 knockdown ( p62 KD # 325 or # 788 ) SY5Y ( c ) ; in control p62 KO MEFs ( d ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in same in response to serum removal .

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(c-d) Top: IBs for p62 and actin in control and p62 knockdown (p62 KD #325 or #788) SY5Y cells (c); and in control and p62 KO MEFs (d). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells in response to serum removal.


( e ) of GFP - p38 , - ANK1 - p38 - NA Sph1 Agm in SY5Y immunostained for endogenous NBR1 . Arrows indicate colocalization .

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(e) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1. Arrows indicate colocalization.


( f , g ) Top : IBs for NBR1 , p62 actin in control NBR1 knockdown ( NBR1 KD # 161 ) SY5Y ( f ) , in control p62 / NBR1 double knockdown ( Double KD # A or # B ) SY5Y ( g ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in same . For densitometric analyses , values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition . See also Supplementary Fig . S8 - S11 .

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(f,g) Top: IBs for NBR1, p62 and actin in control and NBR1 knockdown (NBR1 KD #161) SY5Y cells (f), and in control and p62/NBR1 double knockdown (Double KD #A or #B) SY5Y cells (g). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells. For all densitometric analyses, values are mean+s.e. (n=3). *Significant versus serum+ condition. See also Supplementary Fig. S8-S11.


( a - d ) of GFP - p38 , - ANK1 - p38 - NA Sph1 Agm in SY5Y immunostained for endogenous beclin - 1 ( a ) , Vps34 ( b ) , Atg14L ( c ) DFCP - 1 ( d ) . Arrows indicate colocalization .

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(a-d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d). Arrows indicate colocalization.


( a ) Quantitative FRAP of indicated GFP - p38 variants . Top : representative . Middle : time course of changes in relative ( RFI ) . Bottom : of in diffusing fraction diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten analysed over three different experiments . Scale bars , 10

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(a) Quantitative FRAP analysis of the indicated GFP-p38 variants. Top: representative images. Middle: time course of changes in relative fluorescence intensity (RFI). Bottom: percentage of proteins in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Scale bars, 10 μm


( b ) Top : schematic showing lysine ( K ) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site ( s ) . Bottom : relative fold difference in number of Agm formed by WT different ' K to R ' mutants of ANK1 - p38 fusion in SY5Y maintained in presence or absence of serum .

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(b) Top: schematic showing all lysine (K) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site(s). Bottom: relative fold difference in the number of Agm formed by WT and the different 'K to R' mutants of ANK1-p38 fusion protein in SY5Y cells maintained in the presence or absence of serum.


( c ) IBs for HA ( top ) GFP ( bottom ) of total lysates ( Input ) GFP immunoprecipitates ( IP ) from lysates of SY5Y co - transfected with indicated HA - ubiquitin constructs GFP - p38 or - ANK1 - p38 WT or mutant K385R in presence of lactacystin . ( d ) of of each p38 ubiquitinated via K63 compared with K48 ubiquitination . Values were corrected by of GFP immunoprecipitated .

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(c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated.


( e ) Top : IF for GFP K63 - linked ubiquitin ( red ) of SY5Y expressing GFP - p38 or - ANK1 - p38 WT mutant K385R ( green ) . Arrows indicate colocalization . Bottom : of Agm positive for K63 - linked ubiquitin .

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(e) Top: IF for GFP and K63-linked ubiquitin (red) of SY5Y cells expressing GFP-p38 or -ANK1-p38 WT and mutant K385R (green). Arrows indicate colocalization. Bottom: percentage of Agm positive for K63-linked ubiquitin.


( f ) Quantitative FRAP of GFP - ANK1 - p38 K385R GFP - ANK1 - p38 as in ( a ) . Values are mean + s . e . of ten analysed over three different experiments .

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(f) Quantitative FRAP analysis of GFP-ANK1-p38 K385R and GFP-ANK1-p38 as in (a). Values are mean+s.e. of ten cells analysed over three different experiments.


( g ) Top : representative of GFP - ANK1 - p38 K385R Agm in SY5Y immunostained for Atg14L , Vps34 DFCP - 1 . Bottom : of p38 variant NA Sph1 Agm that colocalized with Atg14L , Vps34 DFCP - 1 under serum − conditions . Values in graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition or p38 Agm . # Significant versus ANK1 - p38 Agm . Significance calculated using Bonferroni . See also Supplementary Fig . S8 .

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(g) Top: representative images of GFP-ANK1-p38 K385R Agm in SY5Y cells immunostained for Atg14L, Vps34 and DFCP-1. Bottom: percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L, Vps34 and DFCP-1 under serum− conditions. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition or p38 Agm. #Significant versus ANK1-p38 Agm. Significance calculated using Bonferroni test. See also Supplementary Fig. S8.


( a , b ) Autophagic clearance of Agm ( a ) or Agg ( b ) formed by FL WT mutant K385R or K394R GFP - Sph1 in SY5Y , in response to serum removal ( a ) or upon blockage of basal autophagy with 3 - MA ( b ) .

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(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b).


( c , d ) Left : Agm ( c ) or Agg ( d ) of FL GFP - Sph1 ( green ) immunostained for endogenous K63 - linked ubiquitin ( red ) in SY5Y . Arrows in ( c ) indicate colocalization . Right : of Agm ( c ) or Agg ( d ) positive for K63 - linked ubiquitin .

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(c,d) Left: Agm (c) or Agg (d) of FL GFP-Sph1 (green) immunostained for endogenous K63-linked ubiquitin (red) in SY5Y cells. Arrows in (c) indicate colocalization. Right: percentage of Agm (c) or Agg (d) positive for K63-linked ubiquitin.


( e ) Formation of WT mutant K385R FL Sph1 ( left ) ANK1 - p38 ( right ) Agm in response to proteasome inhibition . Values in graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , b ) or WT FL Sph1 or ANK1 - p38 ( c - e ) .

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(e) Formation of WT and mutant K385R FL Sph1 (left) and ANK1-p38 (right) Agm in response to proteasome inhibition. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,b) or WT FL Sph1 or ANK1-p38 (c-e).


Interaction for MIA40 AK2 . Stable inducible lines that express either different variants of MIA40 - Strep ( wildtype a redox inactive mutant ( MIA40C53S , C55S ) , ( Fischer et al . , 2013 ) or AK2 - HA were treated with N - ethyl maleimide ( NEM ) to stop thiol - disulfide exchange processes lysed under denaturing conditions . Lysates were analysed by subsequent affinity purification against Strep ( MIA40 ) immunoblotting . Like classical MIA40 substrates , AK2 interacted with active site of MIA40 ( lane 6 ) . 0 . 5 % input was loaded as control .

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Interaction analysis for MIA40 and AK2. Stable inducible cell lines that express either different variants of MIA40-Strep (wildtype and a redox inactive mutant (MIA40C53S,C55S), (Fischer et al., 2013) or AK2-HA were treated with N-ethyl maleimide (NEM) to stop thiol-disulfide exchange processes and lysed under denaturing conditions. Lysates were analysed by subsequent affinity purification against Strep (MIA40) and immunoblotting. Like classical MIA40 substrates, AK2 interacted with the active site of MIA40 (lane 6). 0.5% input was loaded as control.


Redox state of endogenous AK2 by inverse redox shift . HEK293 were lysed by TCA precipitation after NEM treatment , lysates were reduced using TCEP then thiols modified using alkylating agent mmPEG12 . With this " inverse " method , previously oxidized cysteine thiols are modified with mmPEG12 , which results in a slower migration on SDS - PAGE . Treatment of intact with diamide consequently modification of cysteines served as control . Likewise , absence of NEM treatment served as ' max ' control . Treatment lysates with NEM instead of mmPEG12 served as minimal shift ( min ) control . A , B , isoforms of AK2 . Both isoforms of AK2 are semi - oxidized in intact .

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

Redox state analysis of endogenous AK2 by inverse redox shift assay. HEK293 cells were lysed by TCA precipitation after NEM treatment, lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12. With this "inverse" method, previously oxidized cysteine thiols are modified with mmPEG12, which results in a slower migration on SDS-PAGE. Treatment of intact cells with diamide and consequently modification of all cysteines served as control. Likewise, absence of NEM treatment served as 'max' control. Treatment lysates with NEM instead of mmPEG12 served as minimal shift (min) control. A, B, isoforms of AK2. Both isoforms of AK2 are semi-oxidized in intact cells.


Oxidation kinetics experiment to investigate maturation of endogenous AK2 . HEK293 were labeled for 10 min with [ 35S ] - methionine chased with cold methionine for indicated times . chase was stopped by TCA precipitation , then lysate was treated with mmPEG12 to determine AK2 redox states , followed by IP against AK2 . Eluates were analyzed by Tris - Tricine - PAGE autoradiography . With this " direct " redox state determination method reduced AK2 was modified with either three ( isoform B ) or four ( isoform A ) mmPEG12 . Semi - oxidized AK2 was modified with only one ( " B " ) or two ( " A " ) mmPEG12 , respectively . Unmodified fully reduced lysates served as controls . Endogenous AK2 becomes oxidized with a half time of less than 20 min .

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

Oxidation kinetics experiment to investigate maturation of endogenous AK2. HEK293 cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by TCA precipitation, and then the lysate was treated with mmPEG12 to determine the AK2 redox states, followed by IP against AK2. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. With this "direct" redox state determination method reduced AK2 was modified with either three (isoform B) or four (isoform A) mmPEG12. Semi-oxidized AK2 was modified with only one ("B") or two ("A") mmPEG12, respectively. Unmodified and fully reduced cell lysates served as controls. Endogenous AK2 becomes oxidized with a half time of less than 20 min.