( d ) Immunoblot analyses of mitochondria - containing pellet fractions from plasma - - permeabilized HeLa . Permeabilized were treated with or without tBid ( 50 nM ) for outer mitochondrial permeabilization appearance of cytochrome c was verified . Intact outer mitochondrial permeabilized samples were exposed to proteinase K for 10 min . These samples were probed using antibodies against HSP60 , OXA1 , Flag MCUR1 .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O

(d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma-membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified. Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1.


( e ) Reciprocal co - immunoprecipitation ( IP ) of MCU - GFP MCUR1 - Flag transiently expressed in COS7 . Representative of four independent experiments . WB , western blot .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(e) Reciprocal co-immunoprecipitation (IP) of MCU-GFP and MCUR1-Flag transiently expressed in COS7 cells. Representative of four independent experiments. WB, western blot.


( f ) Co - immunoprecipitation of MICU1 - Flag with MCU - GFP but not with MCUR1 - GFP transiently expressed in COS7 . Representative of four independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O

(f) Co-immunoprecipitation of MICU1-Flag with MCU-GFP but not with MCUR1-GFP transiently expressed in COS7 cells. Representative of four independent experiments.


( g ) Immunoprecipitation of LETM1 - GFP with anti - LETM1 failed to pull down MCUR1 - Flag in transiently transfected COS7 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O

(g) Immunoprecipitation of LETM1-GFP with anti-LETM1 failed to pull down MCUR1-Flag in transiently transfected COS7 cells.


( h ) Immunoprecipitation with Flag antibody pulled down LETM1 - Flag or MCU - Flag transiently expressed in COS7 ( immunoprecipitation lanes 2 3 , lower panel ) , but only co - immunoprecipitated MCUR1 - V5 in MCU - Flag - expressing ( immunoprecipitation lanes 4 versus 5 , upper panel ) , despite lower expression of MCU - Flag in MCUR1 - co - transfected ( compare MCU - Flag LETM1 - Flag expression in MCUR1 - co - transfected in lysate lanes 4 5 , bottom panel ) . Representative of three independent experiments . Uncropped of blots / gels are shown in Supplementary Fig . S6 .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Immunoprecipitation with Flag antibody pulled down LETM1-Flag or MCU-Flag transiently expressed in COS7 cells (immunoprecipitation lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1-V5 in the MCU-Flag-expressing cells (immunoprecipitation lanes 4 versus 5, upper panel), despite lower expression of MCU-Flag in MCUR1-co-transfected cells (compare MCU-Flag and LETM1-Flag expression in MCUR1-co-transfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. S6.


( a ) qrtPCR of MCU mRNA from wild - type stable MCUR1 - overexpressing HeLa that were transiently transfected with scrambled siRNA or siRNA against MCU . * * * P0 . 001 ( mean ± s . e . m . ) .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(a) qrtPCR of MCU mRNA from wild-type and stable MCUR1-overexpressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU. ***P0.001 (mean±s.e.m.).


( b ) [ Ca2 + ] m responses to histamine ( 100 μM ) in HeLa stably overexpressing MCUR1 in transiently transfected with scrambled siRNA or MCU siRNA , in stable MCUR1 - overexpressing HeLa transfected with MCU siRNA . After 48 h of siRNA transfection , were loaded with rhod - 2 [ Ca2 + ] m responses were visualized by confocal microscopy . Solid lines are mean ; shaded regions are ± s . e . m . ; n = 3 . ( c ) of peak rhod - 2 following histamine stimulation . * P0 . 05 , * * * P0 . 001 ( mean ± s . e . m . ; NS , not significant ; n = 3 ) .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O 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) [Ca2+]m responses to histamine (100 μM) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA, and in stable MCUR1-overexpressing HeLa cells transfected with MCU siRNA. After 48 h of siRNA transfection, cells were loaded with rhod-2 and [Ca2+]m responses were visualized by confocal microscopy. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (c) Quantification of peak rhod-2 fluorescence following histamine stimulation. *P0.05, ***P0.001 (mean±s.e.m.; NS, not significant; n = 3).


( d ) qrtPCR of MCU mRNA from wild - type MCUR1 - knockdown HeLa that were transiently transfected with MCU cDNA . * * P0 . 01 , * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O 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) qrtPCR of MCU mRNA from wild-type and MCUR1-knockdown HeLa cells that were transiently transfected with MCU cDNA. **P0.01, ***P0.001 (mean±s.e.m.; n = 3).


( e ) [ Ca2 + ] m responses to histamine ( 100 μM ) in wild - type MCUR1 ( shHe2 ) - knockdown HeLa overexpressing MCU . Negative shRNA ( Neg shRNA ) MCUR1 - shHe2 were used as controls . Solid lines are mean ; shaded regions are ± s . e . m . ; n = 3 . ( f ) of peak rhod - 2 following histamine stimulation . * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

(e) [Ca2+]m responses to histamine (100 μM) in wild-type and MCUR1 (shHe2)-knockdown HeLa cells overexpressing MCU. Negative shRNA (Neg shRNA) and MCUR1-shHe2 cells were used as controls. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (f) Quantification of peak rhod-2 fluorescence following histamine stimulation. ***P0.001 (mean±s.e.m.; n = 3).


( a ) AMP / ATP ratios in stable HeLa lines stably expressing negative shRNA ( Neg shRNA ) , MCUR1 shRNA ( clone shHe2 ) or shHe2 with MCUR1 re - expressed . * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O 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) AMP/ATP ratios in stable HeLa cell lines stably expressing negative shRNA (Neg shRNA), MCUR1 shRNA (clone shHe2) or shHe2 with MCUR1 re-expressed. **P0.01 (mean±s.e.m.; n = 3).


( b ) O2 consumption rates ( OCR ) in stable HeLa expressing irrelevant shRNA , clone shHe2 , clone shHe2 re - expressing MCUR1 , exposed sequentially to oligomycin ( i ) , FCCP ( ii ) rotenone plus myzothiazol ( iii ) .

O O O B-SMALL_MOLECULE O O O O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O

(b) O2 consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone shHe2, and clone shHe2 re-expressing MCUR1, exposed sequentially to oligomycin (i), FCCP (ii) and rotenone plus myzothiazol (iii).


( c ) Basal maximal O2 consumption rates in as described in b . * P0 . 05 ( mean ± s . e . m . ; n = 3 ) .

O O O O O B-SMALL_MOLECULE O O O O O 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) Basal and maximal O2 consumption rates in cells as described in b. *P0.05 (mean±s.e.m.; n = 3).


( d ) Western blot of phosphorylated total AMPK ( top ) densitometric ( bottom ) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA ( clone shHe2 ) clone shHe2 re - expressing MCUR1 . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) . ( e ) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA ( clone shHe2 ) clone shHe2 re - expressing MCUR1 ( top ) of LC3 - II / ( LC3 - I + LC3 - II ) ( bottom ) expressed as fold increase over in expressing irrelevant shRNA . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Western blot of phosphorylated and total AMPK (top) and densitometric analysis (bottom) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1. *P0.05, **P0.01 (mean±s.e.m.; n = 3). (e) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1 (top) and quantification of LC3-II/(LC3-I + LC3-II) (bottom) expressed as fold increase over levels in cells expressing irrelevant shRNA. *P0.05, **P0.01 (mean±s.e.m.; n = 3).


( f , g ) , same as in d e . Activation of AMPK ( f ) autophagy ( * * P0 . 001 ; mean ± s . e . m . ; n = 3 ) ( g ) in absence presence of InsP3R inhibitor xestospongin B ( XeB ) . * P0 . 05 , * * P0 . 001 , NS , not significant ( mean ± s . e . m . ; n = 3 ) . Uncropped of blots / gels are shown in Supplementary Fig . S6 .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f,g), The same as in d and e. Activation of AMPK (f) and autophagy (**P0.001; mean±s.e.m.; n = 3) (g) in the absence and presence of InsP3R inhibitor xestospongin B (XeB). *P0.05, **P0.001, NS, not significant (mean±s.e.m.; n = 3). Uncropped images of blots/gels are shown in Supplementary Fig. S6.


B Box plots of Em / Ad expression ratios of GATA6 ( left ) NKX2 - 1 ( right ) in FFPE lung tissue sections from controls ( Ctrl , n = 61 ) or lung cancer ( LC , n = 51 ) . Isoform - specific expression of indicated genes was analyzed by qRT - PCR after total isolation from tissue samples . Each point represents one sample .

O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

B Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in FFPE lung tissue sections from controls (Ctrl, n=61) or lung cancer (LC, n=51) patients. Isoform-specific expression of the indicated genes was analyzed by qRT-PCR after total RNA isolation from tissue samples. Each point represents one sample.


C Box plots of Em / Ad of GATA6 ( top ) or NKX2 - 1 ( bottom ) show that high Em / Ad ratios in LC samples are maintained among ethnic groups ( left ) gender ( right ) . GER , samples collected in Germany ; MEX , samples collected in Mexico .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O 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 Box plots of Em/Ad of GATA6 (top) or NKX2-1 (bottom) show that high Em/Ad ratios in LC samples are maintained among ethnic groups (left) and gender (right). GER, samples collected in Germany; MEX, samples collected in Mexico.


D Box plots of Em / Ad of GATA6 ( left ) or NKX2 - 1 ( right ) in FFPE lung tissue sections from controls or LC . Samples were staged according to TNM Classification ( UICC , 7th edition ) .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O

D Box plots of Em/Ad of GATA6 (left) or NKX2-1 (right) in FFPE lung tissue sections from controls or LC patients. Samples were staged according to the TNM Classification (UICC, 7th edition).


A Box plots of Em / Ad expression ratios of GATA6 ( left ) NKX2 - 1 ( right ) in EBCs from controls ( Ctrl , n = 65 ) or lung cancer ( LC , n = 48 ) ( training set ) . Each point represents one sample .

O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O

A Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in EBCs from controls (Ctrl, n=65) or lung cancer (LC, n=48) patients (training set). Each point represents one sample.


Expression of Bhlhe40 ( grey ) Bhlhe41 ( orange ) in indicated populations , data are from Immgen Database ( microarray data set ) . CMP - common myeloid progenitors . MΦ - macrophages .

B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O

Expression of Bhlhe40 (grey) and Bhlhe41 (orange) in the indicated cell populations, data are from the Immgen Database (microarray data set). CMP - common myeloid progenitors. MΦ - macrophages.


Flow cytometry detection of Bhlhe40 ( grey ) Bhlhe41 ( orange ) transcripts by PrimeFlow in alveolar ( right ) peritoneal ( left ) macrophages . A probe specific for Cd8a mRNA ( black ) was used as a negative control . Results representative of at least four independent experiments .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Flow cytometry detection of the Bhlhe40 (grey) and Bhlhe41 (orange) transcripts by the PrimeFlow RNA assay in alveolar (right) and peritoneal (left) macrophages. A probe specific for the Cd8a mRNA (black line) was used as a negative control. Results representative of at least four independent experiments.


surface phenotype numbers of alveolar ( C ; digested lung ) peritoneal ( D ) macrophages in WT DKO mice . Additional gating for CD11b + was applied for peritoneal macrophages as indicated . of CD11b expression on WT DKO AMs are shown , median for CD11b expression was quantified ( C , bottom left ; 3 mice per genotype ) . Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs ( C ) CD11b + F4 / 80hi peritoneal macrophages ( D ) from WT DKO mice are shown ( bottom ; 4 mice per genotype ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-TISSUE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O

Cell surface phenotype and numbers of alveolar (C; digested lung) and peritoneal (D) macrophages in WT and DKO mice. Additional gating for CD11b+ cells was applied for peritoneal macrophages as indicated. Levels of CD11b expression on WT and DKO AMs are shown, and median fluorescent intensity for CD11b expression was quantified (C, bottom left; 3 mice per genotype). Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs (C) and CD11b+F4/80hi peritoneal macrophages (D) from WT and DKO mice are shown (bottom; 4 mice per genotype).


Frequency of WT donor ( CD45 . 1 ) , DKO donor ( CD45 . 2 ) recipient ( CD45 . 1 / 2 ) in indicated BM ( LSK , CMP ) , splenic ( monocytes ) , liver ( Kupffer ) , peritoneal alveolar macrophage populations of lethally irradiated recipients ≥ 6 after transfer of a 1 : 1 mixture of lineage - depleted BM from WT DKO mice . P values < 0 . 05 ( paired Student ' s t - ) between LSK other populations are shown for frequencies of WT . Three mixed BM chimeras analyzed ; error bars represent s . d . Representative results of three independent experiments .

O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-TISSUE O B-CELL_TYPE O B-CELL_TYPE O O B-TISSUE O B-CELL_TYPE O O B-TISSUE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O

Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells in the indicated BM (LSK, CMP), splenic (monocytes), liver (Kupffer cells), peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice. P values < 0.05 (paired Student's t-test) between LSK and other populations are shown for frequencies of WT cells. Three mixed BM chimeras analyzed; error bars represent s.d. Representative results of three independent experiments.


Representative flow - cytometric of lung ( B ) macrophages as in A ( left ) . expression of CD45 . 1 CD45 . 2 on donor recipient is shown next to indicated gates ( right ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O

Representative flow-cytometric analysis of lung (B) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).


Representative flow - cytometric of peritoneal ( C ) macrophages as in A ( left ) . expression of CD45 . 1 CD45 . 2 on donor recipient is shown next to indicated gates ( right ) .

Representative flow-cytometric analysis of peritoneal (C) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).


Frequency of WT donor ( CD45 . 1 ) , DKO donor ( CD45 . 2 ) recipient ( CD45 . 1 / 2 ) among BM LSK , splenic monocytes alveolar macrophages of recipients that were irradiated with lung shielding , treated with one dose of busulfan as described in Materials Methods , transferred with a 1 : 1 mixture of lineage - depleted BM from WT DKO mice analyzed ≥ 8 after transfer . Representative flow cytometry results for alveolar macrophages ( left ) ( right ) is shown .

O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE B-CELL_TYPE O B-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-TISSUE O 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-TISSUE O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O

Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells among the BM LSK cells, splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding, treated with one dose of busulfan as described in Materials and Methods, transferred with a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer. Representative flow cytometry results for alveolar macrophages (left) and quantification (right) is shown.


Surface phenotype of from digested lungs ( right ) of donor contribution to AM compartment ( left ) of AM - deficient Csf2rb - / - Csf2rb2 - / - recipients ( CD45 . 2 ) that were subjected to intranasal transfer of a 1 : 1 mixture of sorted liver monocytes from WT ( CD45 . 1 / 2 ) DKO ( CD45 . 2 ) E18 . 5 at days 1 or 2 after birth analyzed ≥ 7 after transfer . surface phenotype of lung from a Csf2rb - / - Csf2rb2 - / - mouse that was not subjected to transfer is shown for comparison .

B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O

Surface phenotype of cells from the digested lungs (right) and quantification of donor contribution to the AM compartment (left) of AM-deficient Csf2rb-/-Csf2rb2-/-recipients (CD45.2) that were subjected to intranasal transfer of a 1:1 mixture of sorted fetal liver monocytes from WT (CD45.1/2) and DKO (CD45.2) E18.5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer. Cell surface phenotype of lung cells from a Csf2rb-/-Csf2rb2-/- mouse that was not subjected to the transfer is shown for comparison.


Frequency of donor - recipient - derived AMs in WT DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45 + sorted from neonatal lungs analyzed ≥ 7 after transfer . Results for ( lower panel ) are pooled from 3 independent experiments with a total of 8 WT 9 DKO recipients ; various combinations of CD45 . 1 CD45 . 2 were used to distinguish donor recipient .

O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O

Frequency of donor- and recipient- derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45+ cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer. Results for quantification (lower panel) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients; various combinations of CD45.1 and CD45.2 were used to distinguish donor and recipient cells.


Mixed WT : DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days . EdU incorporation was analyzed one day after last injection . Steady - state WT DKO mice were injected with EdU analyzed as in A . Representative flow cytometry plots of EdU incorporation by AMs ( C ) their ( results pooled from two independent experiments , 7 WT 6 DKO mice were analyzed in total ) ( B ) is shown .

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Mixed WT:DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days. EdU incorporation was analyzed one day after the last injection. Steady-state WT and DKO mice were injected with EdU and analyzed as in A. Representative flow cytometry plots of EdU incorporation by AMs (C) and their quantification (results pooled from two independent experiments, 7 WT and 6 DKO mice were analyzed in total) (B) is shown.


WT ( CD45 . 1 ) DKO ( CD45 . 2 ) AMs were sorted , mixed in a 1 : 1 , labeled with CellTrace Violet cultured in presence of GM - CSF . of CD45 . 1 CD45 . 2 CellTrace Violet dilution was analyzed prior to culture ( day 0 ) after 7 days of culture as described in Materials Methods .

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WT (CD45.1) and DKO (CD45.2) AMs were sorted, mixed in a 1:1 ratio, labeled with CellTrace Violet and cultured in the presence of GM-CSF. The ratio of CD45.1 and CD45.2 cells and CellTrace Violet dilution was analyzed prior to culture (day 0) and after 7 days of culture as described in Materials and Methods.


- seq of Maf Mafb expression in WT DKO AMs , which were isolated by flow cytometry from mice in steady state ( top ) or from mixed BM chimeras ( bottom ) .

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RNA-seq analysis of Maf and Mafb expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).


of bronchoalveolar lavage cytospins from WT DKO mice with lipophilic dye Oil Red O . of Oil Red O - positive in WT DKO mice for . Results pooled from two independent experiments ; 4 WT 5 DKO mice were analyzed in total .

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Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O. Quantification of Oil Red O-positive cells in WT and DKO mice for individual animals. Results pooled from two independent experiments; 4 WT and 5 DKO mice were analyzed in total.


Flow cytometric of AMs from WT DKO mice stained with lipophilic dye BODIPY 493 / 503 . Representative results of two independent experiments with three mice per group . as in C performed with WT : DKO mixed BM chimeras . Gating on CD45 . 1 CD45 . 2 was applied to identify of WT DKO donor origin . Representative results of two independent experiments with at least three chimeras .

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Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493/503. Representative results of two independent experiments with three mice per group. Analysis as in C performed with WT:DKO mixed BM chimeras. Gating on CD45.1 and CD45.2 was applied to identify cells of WT and DKO donor origin. Representative results of two independent experiments with at least three chimeras.


- seq of Acaa1b Hsd17b4 expression in WT DKO AMs , which were isolated by flow cytometry from mice in steady state ( top ) or from mixed BM chimeras ( bottom ) .

RNA-seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).


Comparison of changes in gene expression induced by Bhlhe40 / Bhlhe41 - deficiency in steady - state knockout mice ( horizontal axis ) in mixed BM chimeras ( vertical axis ) . Log2 transformed DKO / WT fold changes are plotted . Genes with expression of > 5 TPM in at least one of samples are displayed . Genes highlighted in magenta indicate known putative regulators of AMs . Closed symbols - Bhlhe40 - bound genes ( Bhlhe40 ChIP - seq peak assigned to nearest gene within a 50 kb window ) . Open symbols - genes without Bhlhe40 binding .

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Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis). Log2 transformed DKO/WT fold changes are plotted. Genes with expression of > 5 TPM in at least one of the samples are displayed. Genes highlighted in magenta indicate known and putative regulators of AMs. Closed symbols - Bhlhe40-bound genes (Bhlhe40 ChIP-seq peak assigned to the nearest gene within a 50 kb window). Open symbols - genes without Bhlhe40 binding.


Presence of transcripts ( - seq ; top ) at Gas7 Acaa1b genes in WT DKO AMs ; ChIP - seq of H3K27 acetylation in WT DKO AMs ( middle ) Bhlhe40 binding in WT AMs ( bottom ) . analyses performed on ex vivo AMs .

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Presence of RNA transcripts (RNA-seq; top) at the Gas7 and Acaa1b genes in WT and DKO AMs; the ChIP-seq analysis of H3K27 acetylation in WT and DKO AMs (middle) and Bhlhe40 binding in WT AMs (bottom). All analyses performed on ex vivo AMs.


Gene set enrichment ( GSEA ) on dataset ranked by DKO / WT fold change in steady - state AM - seq using set of genes assigned to top 1 , 000 Bhlhe40 peaks ( ranked by P value ) ( left ) or a random set of 1 , 000 genes ( right ) . Similar results were obtained with three random sets tested . NES - normalized enrichment score . FDR False Discovery Rate .

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Gene set enrichment analysis (GSEA) on dataset ranked by DKO/WT fold change in steady-state AM RNA-seq using set of genes assigned to top 1,000 Bhlhe40 peaks (ranked by P value) (left) or a random set of 1,000 genes (right). Similar results were obtained with all three random sets tested. NES - normalized enrichment score. FDR False Discovery Rate.


Pairwise comparison of H3K27 acetylation in WT DKO AMs performed on 2 - kb regions around Bhlhe40 peak summits ( right - for Bhlhe40 peaks called with P value of < 10 − 10 ; center - for top 3 , 000 Bhlhe40 peaks ( ranked by P value ) ) around WT AM ATAC - seq peaks ( Immgen ) that did not have a Bhlhe40 peak in their close proximity ( filtered as described in Materials Methods ) ( left ) . Volcano plots show adjusted P values ( vertical axes ) log2 fold change ( horizontal axes ) for H3K27ac pairwise comparison in WT vs DKO AMs ; genes assigned to some of Bhlhe40 - binding regions are labeled on left plot .

O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O

Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2-kb regions around the Bhlhe40 peak summits (right - for all Bhlhe40 peaks called with P value of < 10−10; center - for top 3,000 Bhlhe40 peaks (ranked by P value)) and around WT AM ATAC-seq peaks (Immgen) that did not have a Bhlhe40 peak in their close proximity (filtered as described in Materials and Methods) (left). Volcano plots show adjusted P values (vertical axes) and log2 fold change (horizontal axes) for H3K27ac pairwise comparison in WT vs DKO AMs; genes assigned to some of the Bhlhe40-binding regions are labeled on the left plot.


Comparison of changes in gene expression induced by Bhlhe40 / Bhlhe41 - deficiency in steady - state knockout mice ( horizontal axis ) in mixed BM chimeras ( vertical axis ) as shown in Fig . 5 A , but filtered for indicated signatures ( no TPM filter applied ) . Closed symbols - genes with associated Bhlhe40 binding ( as in Fig . 5 A ) revealed by ChIP - seq . Open symbols - genes without Bhlhe40 binding . Inserts show Bhlhe40 ChIP - seq track for selected dysregulated genes .

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Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis) as shown in Fig. 5 A, but filtered for the indicated signatures (no TPM filter applied). Closed symbols - genes with associated Bhlhe40 binding (as in Fig. 5 A) revealed by ChIP-seq. Open symbols - genes without Bhlhe40 binding. Inserts show Bhlhe40 ChIP-seq track for selected dysregulated genes.


Flow cytometric comparison of expression of indicated - surface markers ( identified in panel A ) on WT DKO AMs . A narrow forward / side scatter gate was applied to correct for possible differences in autofluorescence . Representative results of two independent experiments with three four mice per group .

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Flow cytometric comparison of expression of the indicated cell-surface markers (identified in panel A) on WT and DKO AMs. A narrow forward/side scatter gate was applied to correct for possible differences in autofluorescence. Representative results of two independent experiments with three and four mice per group.


( A ) Relative share of three PIKKs in NCS - responsive phosphorylations .

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(A) Relative share of the three PIKKs in NCS-responsive phosphorylations.


( C ) profile of a group of substrates targeted by ATM 20 min after neocarzinostatin ( NCS ) addition by ATR 1 hr after hydroxyurea ( HU ) addition . Box plots depict 23 phosphopeptides measured in two independent biological replicates . box indicates range from first to third quartiles , central band represents median . Upper lower whiskers extend to maximum minimum values which are not farther than 1 . 5 times interquartile range ( IQR ) .

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(C) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin (NCS) addition and by ATR 1 hr after hydroxyurea (HU) addition. Box plots depict 23 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).


( D ) Western blotting confirming that ATM substrate , pS824 / KAP1 is targeted by ATR in response to HU . pS345 / CHK1 - an established ATR substrate - served as a positive control for ATR activation .

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(D) Western blotting analysis confirming that the ATM substrate, pS824/KAP1 is targeted by ATR in response to HU. pS345/CHK1 - an established ATR substrate - served as a positive control for ATR activation.


( A ) A cluster of 71 neocarzinostatin ( NCS ) - induced phosphorylations that were enhanced upon continuous inhibition of - PK . Untreated are marked UT . This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition . Clusters were obtained using K - means algorithm implemented in Perseus on Z - scored intensities . Box plots depict 71 phosphopeptides measured in two independent biological replicates . box indicates range from first to third quartiles , central band represents median . Upper lower whiskers extend from box to maximum minimum values which are not farther than 1 . 5 times interquartile range ( IQR ) .

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(A) A cluster of 71 neocarzinostatin (NCS)-induced phosphorylations that were enhanced upon continuous inhibition of DNA-PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K-means algorithm implemented in Perseus on Z-scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).


( B ) Depicted are phosphopeptides that responded to NCS treatment in A - T . No significant elevation was observed in these phosphorylations in A - T following continuous inhibition of - PK . Box plots depict 71 phosphopeptides measured in two independent biological replicates . box indicates range from first to third quartiles , central band represents median . Upper lower whiskers extend from box to maximum minimum values which are not farther than 1 . 5 time interquartile range ( IQR ) .

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(B) Depicted are phosphopeptides that responded to NCS treatment in A-T cells. No significant elevation was observed in these phosphorylations in A-T cells following continuous inhibition of DNA-PK. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR).


( E ) Western blotting of selected phosphorylations in above cluster showing - PKi - induced elevation .

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(E) Western blotting analysis of selected phosphorylations in the above cluster showing DNA-PKi-induced elevation.


( F ) Temporal dynamics of - PK - dependent attenuation of pS824 / KAP - 1 .

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(F) Temporal dynamics of DNA-PK-dependent attenuation of pS824/KAP-1.


( C ) first time points at which ' compensated sites ' responded to NCS in WT A - T are presented .

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(C) The first time points at which 'compensated sites' responded to NCS in WT and A-T cells are presented.


( D ) Temporal kinase dependencies of ' compensated sites ' in A - T . Shown are numbers of ' compensated sites ' in A - T , which depended on each kinase at 20 240 min time points . early ones depend mainly on - PK , while later ones - on ATR .

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(D) Temporal kinase dependencies of the 'compensated sites' in A-T cells. Shown are the numbers of 'compensated sites' in A-T cells, which depended on each kinase at the 20 and 240 min time points. The early ones depend mainly on DNA-PK, while the later ones - on ATR.


( F ) Western blotting confirming high - throughput results for selected ATM substrates .

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(F) Western blotting analysis confirming high-throughput results for selected ATM substrates.


( A ) A group of exclusively ATM - dependent sites that were ATM - dependent one hr after NCS addition , were still phosphorylated or dephosphorylated 4 hr after treatment , but were not affected by ATMi at that time point . Box plots depict 18 phosphopeptides measured in two independent biological replicates . box indicates range from first to third quartiles , central band represents median . Upper lower whiskers extend from box to maximum minimum values which are not farther than 1 . 5 times interquartile range ( IQR ) .

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(A) A group of exclusively ATM-dependent sites that were ATM-dependent one hr after NCS addition, were still phosphorylated or dephosphorylated 4 hr after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).


( B ) This group was enriched for sites that were compensated in an ATR - dependent manner in A - T . Box plots depict 9 phosphopeptides measured in two independent biological replicates . box indicates range from first to third quartiles , central band represents median . Upper lower whiskers extend from box to maximum minimum values which are not farther than 1 . 5 times interquartile range ( IQR ) .

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(B) This group was enriched for sites that were compensated in an ATR-dependent manner in A-T cells. Box plots depict 9 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).


( C ) Western blotting of pS343 / NBS1 , which is compensated upon ATM inhibition , pS824 / KAP - 1 pS395 / NUMA , which are not compensated under these conditions .

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(C) Western blotting analysis of pS343/NBS1, which is compensated upon ATM inhibition, and pS824/ KAP-1 and pS395/NUMA, which are not compensated under these conditions.


( A - F ) were either untreated ( UT ) or treated with neocarzinostatin ( NCS ) in presence or absence of PIKK inhibitors . Intensities from 3 independent biological replicates were normalized against reference synthetic standards are depicted as mean ± .

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(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.


( C ) Time - lapse microscopy of containing a PsigV - YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress . stress ( red ) was added between 200 mins 300 min time points ( at 240 min ) . red arrow highlights a with a delayed activation of σV . Scale bar : 5 μm .

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(C) Time-lapse microscopy of cells containing a PsigV-YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress. The stress (red line) was added between the 200 mins and 300 min time points (at 240 min). The red arrow highlights a cell with a delayed activation of σV. Scale bar: 5 μm.


( B ) A priming ( 30 min ) stress of 1 µg / ml lysozyme followed by high lysozyme stress ( 20 µg / ml ) improves survival . solid blue lines are biological repeats for no priming experiment ( n = 2 ) whereas solid red lines are biological repeats for priming experiment ( n = 4 ) . ( Total number of shown for No Priming , N = 2013 Priming , N = 4937 ) .

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(B) A priming (30 min) stress of 1 µg/ml lysozyme followed by the high lysozyme stress (20 µg/ml) improves survival. The solid blue lines are the biological repeats for the no priming experiment (n=2) whereas the solid red lines are the biological repeats for the priming experiment (n=4). (Total number of cells shown for No Priming, N = 2013 and Priming, N = 4937).


( B , C ) - seq experiment on WT ( JLB130 ) ΔsigV ( JLB154 ) strains , showing of absolute expression of genes in presence absence of lysozyme stress . shaded grey box represents a ± 5 fold change . DEseq was used to identify genes which were differentially expressed with a 5 % p - value cutoff between WT ΔsigV mutant in response to lysozyme treatment ( default DEseq was used ) . ( B ) Only sigV operon is strongly activated ( > 5 fold change ) in response to lysozyme stress in WT ( JLB130 ) , as previously reported ( Guariglia - Oropeza Helmann 2011 ) . ( C ) No genes were strongly upregulated in ΔsigV ( JLB154 ) by lysozyme stress .

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(B, C) RNA-seq experiment on WT (JLB130) and ΔsigV (JLB154) strains, showing quantification of the absolute expression of individual genes in the presence and absence of lysozyme stress. The shaded grey box represents a ± 5 fold change. DEseq was used to identify genes which were differentially expressed with a 5% p-value cutoff between the WT and ΔsigV mutant in response to lysozyme treatment (default DEseq test was used). (B) Only the sigV operon is strongly activated (>5 fold change) in response to lysozyme stress in WT (JLB130), as previously reported (Guariglia-Oropeza and Helmann 2011). (C) No genes were strongly upregulated in ΔsigV (JLB154) by lysozyme stress.


( D ) Effect of overexpression of components of σV pathway ( Biological repeats : WT : n = 8 , sigV + : n = 4 , rsiV + : n = 3 oatA + : n = 4 , yrhK + : n = 3 , sipS + : n = 3 rasP + : n = 3 , pbpX + : n = 6 ) on fraction of activated . Only overexpression of sigV , rsiV or oatA changed observed dynamics compared to WT . histograms of shown data are shown in Appendix Figure S16 . ( E ) Deleting oatA did not alter σV activation dynamics . However , deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug / ml lysozyme . n > = 3 biological repeats for data shown . histograms of shown data are shown in Appendix Figure S17 . (

O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(D) Effect of the overexpression of individual components of the σV pathway (Biological repeats: WT: n=8, sigV+: n=4, rsiV+: n=3 oatA+: n=4, yrhK+: n=3, sipS+: n=3 and rasP+: n=3, pbpX+: n=6) on the fraction of activated cells. Only overexpression of sigV, rsiV or oatA changed the observed dynamics compared to WT. The histograms of the shown data are shown in Appendix Figure S16. (E) Deleting oatA did not alter the σV activation dynamics. However, deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug/ml lysozyme. n>=3 biological repeats for all data shown. The histograms of the shown data are shown in Appendix Figure S17. (


( C ) As predicted , 2xsigV or 2xsigV - rsiV stains have a homogenous σV response to 1 µg / ml lysozyme , while 2xrsiV has increased heterogeneity . Each bar plot is average of three biological repeats . bars correspond to mean ± s . d . .

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(C) As predicted, the 2xsigV or 2xsigV-rsiV stains have a homogenous σV response to 1 µg/ml lysozyme, while the 2xrsiV strain has increased heterogeneity. Each bar plot is the average of three biological repeats. The bars correspond to the mean ± s.d..


( E , F ) For very long intervals between stress ( 12h ) , heterogeneity is same as if had not experienced a prior lysozyme stress ( control ) . N > 50 single - traces .

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(E, F) For very long intervals between stress (12h), the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress (control). N>50 single-cell traces.


( A ) Yeast clones harbouring empty bait vector ( pYTH9 , mock ) or those encoding ScAtg8 , LC3B , Atg12 SUMO1 were transformed with empty prey vector ( pACT2 , mock ) or those encoding fragments of ATG4A , ATG4B , NBR1 , p62 full - Nix . Interaction was assessed by yeast growth on SD ‐ W / ‐ L / ‐ H medium with a β ‐ gal .

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(A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD‐W/‐L/‐H medium with a β‐gal assay.


( B ) GST pulldown assays using extracts of Flag ‐ Nix ‐ expressing COS7 immobilized GST or indicated GST fusions . Coprecipitated Nix was detected with Flag antibodies .

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(B) GST pulldown assays using cell extracts of Flag‐Nix‐expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies.


( C ) Purified GST ‐ tagged NixΔTM was from GST moiety by thrombin , purified used for precipitation by GST fusion . Precipitated were analysed by western blotting ( WB ) with Nix antibodies . Ponceau S was used to visualize GST ‐ fusion .

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(C) Purified GST‐tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST‐fusion proteins.


( D ) HeLa were treated as indicated endogenous were immunoprecipitated ( IP ) from extracts using Nix antibody analysed by using Nix LC3 antibodies . β ‐ gal , β ‐ galactosidase ; BafA1 , bafilomycin ‐ A1 ; CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; GST , glutathione ‐ S ‐ transferase ; IgG , immunoglobulin G ; NixΔTM , recombinant Nix lacking a TM ; SUMO1 , small ubiquitin ‐ like modifier 1 ; TCL , total lysate ; TM , transmembrane domain .

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(D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.


( C ) Lysates of COS7 transfected with Flag ‐ wt ‐ Nix or indicated mutants were incubated with immobilized GST ‐ LC3A . coprecipitated were detected by western blotting ( WB ) with Flag antibodies . BNIP3 , Bcl2 / E1B 19kDa ‐ interacting 3 ‐ like ; Dr , Danio reio ; GST , glutathione ‐ S ‐ transferase ; Hs , Homo sapiens ; LIR , LC3 ‐ interacting region ; Mm , Mus musculus ; wt , wild type ; Xl , Xenopus laevis .

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(C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.


Nix recruits EGFP ‐ GABARAP ‐ L1 to stressed mitochondria in reconstituted Nix − / − MEFs . Nix − / − MEFs cotransfected with EGFP ‐ GABARAP ‐ L1 wt ‐ Nix ( A ) , or Nix ‐ W35A ( B ) , were incubated with without mitochondrial poison CCCP for 3 h . were then fixed analysed for colocalization between EGFP ‐ GABARAP ‐ L1 Nix ( stained with Flag antibodies ) . final panel in each row shows colocalization between Flag ‐ Nix ‐ Cy3 GABARAP ‐ L1 using ' colocalization ' highlighter plug ‐ in for ImageJ software ( NIH , Bethesda , MA , USA ) .

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Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA).


( C ) was achieved by counting number of complete colocalizations , EGFP / Cy3 per for approximately 30 per condition . Results are expressed as a of colocalization ( EGFP : Cy3 ) in control CCCP ‐ treated . Error bars in ( C ) show standard deviation ( s . d . ) of three independent experiments . Statistical was performed using Mann - Whitney t ‐ . CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; DMSO , dimethyl sulfoxide ; EGFP , enhanced green ; MEF , mouse embryonic fibroblast ; wt , wild type .

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(C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.


( A ) Flow cytometry of reticulocyte ‐ enriched blood , cultured in vitro for 3 days , stained with MTR . blood was taken from mice transplanted with Nix − / − bone marrow reconstituted with wt ‐ Nix Nix ‐ W35A . Reticulocytes were induced by phenylhydrazine treatment . also express GFP .

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(A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP.


( B ) Mitochondrial clearance in GFP ‐ positive fraction of reticulocyte ‐ enriched blood , as described in panel ( A ) . Mice were reconstituted with wt ‐ Nix , LIR ‐ W35A or empty vector . Data from Nix − / − Nix + / + mice are shown for comparison .

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(B) Mitochondrial clearance in the GFP‐positive fraction of the reticulocyte‐enriched blood, as described in panel (A). Mice were reconstituted with wt‐Nix, LIR‐W35A or empty vector viruses. Data from Nix−/− and Nix+/+ mice are shown for comparison.


( C ) Anti ‐ Flag , anti ‐ GFP anti ‐ β ‐ actin western blots of liver transduced with Flag ‐ Nix Flag ‐ W35A ‐ Nix , normalized to total . GFP , green ; LIR , LC3 ‐ interacting region ; MTR , MitoTracker Red ; wt , wild type .

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(C) Anti‐Flag, anti‐GFP and anti‐β‐actin western blots of fetal liver cells transduced with Flag‐Nix and Flag‐W35A‐Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3‐interacting region; MTR, MitoTracker Red; wt, wild type.


A , B MARS ( A ) MARS2 ( B ) mRNA expression were measured by real - time PCR in blood samples with 2 3 - 4 copies . Relative expression were normalized to GAPDH , data were quantified relative to mRNA expression of 2 copy samples . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t

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A, B MARS (A) and MARS2 (B) mRNA expression levels were measured by real-time PCR in blood samples with 2 and 3-4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test


C , D Immunoblotting was performed to detect MARS in samples with 2 3 - 4 copies . - level shown in ( D ) was quantified using ImageJ divided by - level of actin . results were then graphed relative to samples with 2 copies . E , F MARS2 expression were detected ( E ) quantified ( F ) . Actin was used as a loading control . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t was

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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, D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3-4 copies. Protein-level intensity shown in (D) was quantified using ImageJ and divided by the protein-level intensity of actin. The results were then graphed relative to samples with 2 copies. E, F MARS2 protein expression levels were detected (E) and quantified (F). Actin was used as a loading control. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was used


G proliferation was assessed by Ki67 ( red ) immunofluorescent in mice neural tube sections at E10 . MARS ( green ) was co - stained . Scale bar : 150 μm .

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G Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co-stained. Scale bar: 150 μm.


H Double immunofluorescent for MARS ( green ) Ki67 ( red ) in developing heart sections at E10 . Scale bar : 150 μm .

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H Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm.


I MARS expression was detected in E80 rhesus monkey brain sections ( n = 3 ) by . Local large magnification is shown as framed . Red arrow indicates germinar layer , blue indicates immature neurons . Scale bar : 1mm .

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I MARS expression was detected in E80 rhesus monkey brain sections (n=3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinar layer, blue indicates immature neurons. Scale bar: 1mm.


MARS in embryonic chorion ( C ) their corresponding maternal deciduae ( D ) were determined by western blot . Each D / C lane pair was quantified relative to D as shown at bottom .

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MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. Each D/C lane pair was quantified relative to D as shown at the bottom.


MARS in embryonic chorion ( C ) their corresponding maternal deciduae ( D ) were determined by western blot . mean MARS in D C groups were quantified by ImageJ shown in ( K ) ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


L MARS2 mRNA expression in embryonic chorion ( C ) maternal deciduae ( D ) were detected by qPCR ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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L MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


M MARS in brains of foetal rats ( F ) their corresponding mothers ( M ) from experiment ( O ) ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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M MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


N MARS mRNA ( n = 6 ) in brains of foetal rats ( F ) their corresponding mothers ( M ) were determined by qPCR ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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N MARS mRNA levels (n=6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


O MARS in foetal rats ( F ) their corresponding mothers ( M ) were determined by western blot . Each M / F lane pair was quantified relative to M as shown at bottom .

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O MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by western blot. Each M/F lane pair was quantified relative to M as shown at the bottom.


P Representative result of MARS expression was detected by in tissues ( T ) adjacent noncancer tissues ( N ) of UCC samples . Scale bar : 50 μm .

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P Representative result of MARS expression was detected by IHC in tumor tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm.


Q Ki67 MARS expression was analysed by in HCC samples . Scale bar : 20 μm .

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Q Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.


B growth was determined in absence presence of 0 . 5 mM homocysteine or 0 . 5 mM methionine in culture media of NE4C transfected with pcDNA3 . 1 vector or pcDNA3 . 1 - Flag - Bcl - 2 plasmids ( n = 3 ) . 5μg plasmids were transfected in each 1 × 106 . index responds to changes in number adhesion . caspase 3 ( c - caspase3 ) were detected to determine apoptosis ( right ) . Data are presented as mean ± were compared using an unpaired Student ' s t .

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B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test.


C , D Apoptotic were detected by flow cytometry . apoptotic rates of untreated , MARS ( C ) - , MARS2 ( D ) - over - expressing NE4C when cultured in absence presence of 20 μM homocysteine were normalized to untreated NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


E were treated with different methionine concentrations as indicated . Apoptotic rates were detected by flow cytometry normalized to those of untreated ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


F Apoptotic rates were determined in absence presence of 20 μM homocysteine in culture media of control MARS / MARS2 knockdown NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


HTL ROS ( superoxide ) were compared ( n = 4 ) between NE4C transfected with empty vector , MARS ( A ) plasmids . Data were normalized against that of vector - transfected . HTL were determined by LC - MS , ROS were determined by DHE .

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HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.


HTL ROS ( superoxide ) were compared ( n = 4 ) between NE4C transfected with empty vector or MARS2 ( B ) plasmids . Data were normalized against that of vector - transfected . HTL were determined by LC - MS , ROS were determined by DHE .

HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.


C MARS was knocked down by small hairpin RNAs in NE4C . MARS knockdown efficiencies were confirmed by western blot . HTL superoxide were quantified ( n = 4 ) relative to vector - transfected NE4C . were cultured in 20 μM Hcy - containing media .

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C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.


D Relative superoxide were determined by DHE compared ( n = 4 ) between NE4C with without MARS knockdown by shRNA . were cultured in 20 μM Hcy - containing media . MARS2 knockdown efficiencies were confirmed by western blot .

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D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.


E NE4C with without MARS knockdown were treated with either Hcy ( 20 μM ) or Met ( 75 μM ) , superoxide were detected 6 h after start of respective treatment quantified relative to untreated NE4C ( n = 4 ) .

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E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).


F ROS ( superoxide ) were determined ( n = 4 ) by DHE of NE4C treated with various HTL concentrations for 4 h . ROS were normalized to those of untreated NE4C .

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F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.


G Superoxide in response to Hcy treatment ( 20 μM , 4 h ) in MARS - expressing control were determined relative to untreated NE4C .

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G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.


H NE4C were cultured in DMEM supplemented with different HTL as indicated . N - Hcy in HTL - treated control were detected by western blot .

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H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.


I N - Hcy in response to MARS knockdown ( by shRNA ) over - expression were detected by western blot .

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I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.


J , K Untreated MARS / MARS2 knockdown NE4C were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , N - Hcy ( J ) ( n = 4 ) in were detected 6 h after start of respective treatment quantified relative to untreated NE4C . Error bars indicate . Data were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


J , K Untreated MARS / MARS2 knockdown NE4C were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , superoxide ( K ) ( n = 4 ) in were detected 6 h after start of respective treatment quantified relative to untreated NE4C . Error bars indicate . Data were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


C SOD activities were down - regulated by in vitro N - homocysteinylation . Flag - tagged SOD1 , SOD1K23W , K122W , K128W ( 3KW ) mutant , SOD2 , SOD2K44W , K51W , K98W , K106W , K178W ( 5KW ) mutant were expressed in HEK293T , affinity purified , incubated with or without 10 μM HTL in solution ( in vitro ) . After 12 h , reaction mixture was desalted concentrated with filter tubes . N - Hcy SOD activities were determined normalized to untreated WT SOD1 SOD2 , respectively ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


D Upon switching N - Hcy substrate lysine ( K ) sites to nonmodifiable tryptophan ( W ) , N - Hcy unmodifiable mutants displayed lower specific activity than wide - type ( WT ) SOD . SOD1 ( WT , K23W , K122W , K128W , 3KW ) SOD2 ( WT , K44W , K51W , K98W , K106W , K178W , 5KW ) were expressed in HEK293T . catalytic activity of affinity - purified SOD was determined normalized to . Wild - type SOD1 SOD2 activities were set at 100 % ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_LINE O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


E , F Hcy HTL treatment in NE4C increased N - homocysteinylation of SODs decreased their activities . Flag - tagged SOD1 SOD2 were each ectopically expressed in NE4C . were transferred to serum - free DMEM with or without 20 μM Hcy ( E ) or 10 μM HTL ( F ) 6 h before harvesting . After affinity purification , specific activities of SOD1 SOD2 were determined normalized to SOD1 SOD2 activity in untreated . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


G N - Hcy SOD were down - regulated in MARS / MARS2 knockdown . Flag - tagged SOD1 SOD2 were each over - expressed in untreated MARS / MARS2 knockdown NE4C . After affinity purification , N - Hcy relative specific activities of purified SOD1 SOD2 were determined quantified relative to NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


A of β - catenin in response to Hcy treatment in NE4C were detected by western blot ( n = 3 ) . Representative western blots are shown , band intensities were quantified relative to untreated group . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Levels of β-catenin in response to Hcy treatment in NE4C cells were detected by western blot (n=3). Representative western blots are shown, and band intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


B of β - catenin in response to HTL treatment in NE4C were detected . Band intensities were normalized to that of untreated . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Levels of β-catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


C TCF activities were measured with a Topflash / Fopflash luciferase reporter system ( n = 4 ) . TCF activities responding to either 20 μM Hcy - or 10 μM HTL - treated were measured by quantifying Topflash / Fopflash relative to untreated groups . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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C TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n=4). TCF activities responding to either 20 μM Hcy- or 10 μM HTL-treated were measured by quantifying Topflash/Fopflash relative to the untreated groups. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


D of β - catenin in untreated MARS knockdown NE4C were detected ( n = 3 ) with presence or absence of 20 μM Hcy in culture media . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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D Levels of β-catenin in untreated and MARS knockdown NE4C cells were detected (n=3) with the presence or absence of 20 μM Hcy in the culture media. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


NE4C were co - transfected with NRX - Flag DVL1 - Myc plasmids treated with different concentrations of Hcy ( E ) relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of Hcy (E) The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


NE4C were co - transfected with NRX - Flag DVL1 - Myc plasmids treated with different concentrations of HTL ( F ) . relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of HTL (F). The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


G NRX - Flag DVL1 - Myc were co - transfected in untreated or MARS knockdown NE4C . relative DVL1 that co - IPed with NRX - Flag were determined quantified ( n = 3 ) for treated control cultured with or without 20 μM Hcy . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NRX-Flag and DVL1-Myc were co-transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co-IPed with NRX-Flag were determined and quantified (n=3) for treated and control cells cultured with or without 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Flag - tagged SOD1 SOD2 were co - over - expressed in NE4C cultured in DMEM supplemented with Hcy or HTL . ( H ) of β - catenin in with without SOD1 / 2 expression were determined ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β-catenin in cells with and without SOD1/2 expression were determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Flag - tagged SOD1 SOD2 were co - over - expressed in NE4C cultured in DMEM supplemented with HTL . ( I ) Relative of DVL1 that co - IPed with NRX - Flag were quantified in SOD - expressing control cultured with or without 10 μM HTL ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with HTL. (I) Relative levels of DVL1 that co-IPed with NRX-Flag were quantified in SOD-expressing and control cells cultured with or without 10 μM HTL (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


B MARS - or MARS2 - catalysed HTL production from Hcy ( 20 μM ) was carried out in vitro in presence of different AHT concentrations . amount of HTL produced in each reaction was determined by thin layer chromatography .

O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.


C MARS MARS2 activities were determined ( n = 4 ) in presence of indicated AHT concentrations in reactions . AHT IC50 values were determined for MARS MARS2 with 20 μM Hcy . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.