A . An anti - KV10 . 1 antibody directed against an extracellular epitope labels a structure related to the centrosome ( pericentrin - positive ) and positive for Arl13b with an appearance suggestive of the primary cilium in HFF ( human foreskin fibroblast ) cells . Cells were serum starved for 24 or 48h , fixed , blocked ( 10 % BSA ) and then stained without permeabilization with anti - KV10 . 1 antibody recognizing an extracellular epitope . Immunostaining for pericentrin was performed after subsequent permeabilization and additional block . Scale bars : 10 µm ( pericentrin ) , 20 µm ( Arl13b ) .

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A. An anti-KV10.1 antibody directed against an extracellular epitope labels a structure related to the centrosome (pericentrin-positive) and positive for Arl13b with an appearance suggestive of the primary cilium in HFF (human foreskin fibroblast) cells. Cells were serum starved for 24 or 48h, fixed, blocked (10% BSA) and then stained without permeabilization with anti-KV10.1 antibody recognizing an extracellular epitope. Immunostaining for pericentrin was performed after subsequent permeabilization and additional block. Scale bars: 10 µm (pericentrin), 20 µm (Arl13b).


B . Anti - KV10 . 1 antibodies recognize ciliary structures in hTERT - RPE1 cells . After serum starvation for 48h , serum was reintroduced for 4h to induce KV10 . 1 expression and ciliary resorption . Cells were then fixed and stained with a mixture of anti - KV10 . 1 mouse monoclonals and a rabbit monoclonal anti acetylated α - tubulin . Scale bar : 2 µm .

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B. Anti-KV10.1 antibodies recognize ciliary structures in hTERT-RPE1 cells. After serum starvation for 48h, serum was reintroduced for 4h to induce KV10.1 expression and ciliary resorption. Cells were then fixed and stained with a mixture of anti-KV10.1 mouse monoclonals and a rabbit monoclonal anti acetylated α-tubulin. Scale bar: 2 µm.


C . Specificity of the monoclonal anti KV10 . 1 ( mAb 62 ) as revealed by staining MEFs from wild type ( left ) or knockout animals ( right ) Scale bar : 50 µm :

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C. Specificity of the monoclonal anti KV10.1 (mAb 62) as revealed by staining MEFs from wild type (left) or knockout animals (right) Scale bar: 50 µm:


D . Images from primary cilia of MEFs from KV10 . 1 knockout mice obtained using mAb62 and a rabbit polyclonal anti acetylated a - tubulin antibody . No recognizable structures were stained by mAb62 . Scale bar : 1 µm . E . In contrast , mAb62 did stain ciliary - related structures ( acetylated - tubulin - positive ) in wild type mice . Scale bar : 1 µm .

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D. Images from primary cilia of MEFs from KV10.1 knockout mice obtained using mAb62 and a rabbit polyclonal anti acetylated a-tubulin antibody. No recognizable structures were stained by mAb62. Scale bar: 1 µm.E. In contrast, mAb62 did stain ciliary-related structures (acetylated-tubulin-positive) in wild type mice. Scale bar: 1 µm.


A . KV10 . 1 localizes to pericentrin - positive structures during ciliary resorption . After short ( 4h ) reintroduction of FCS in serum starved hTERT RPE1 cells , the signal of anti - KV10 . 1 antibody co - localized with pericentrin . Scale bar : 25µm .

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A. KV10.1 localizes to pericentrin-positive structures during ciliary resorption. After short (4h) reintroduction of FCS in serum starved hTERT RPE1 cells, the signal of anti-KV10.1 antibody co-localized with pericentrin. Scale bar: 25µm.


B . Cells expressing a KV10 . 1 * mVenus fusion under the control of the endogenous promoter to render a mild overexpression were fixed and immunostained for pericentrin . Venus fluorescence colocalized with pericentrin - positive structures . Scale bar : 5µm .

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B. Cells expressing a KV10.1*mVenus fusion under the control of the endogenous promoter to render a mild overexpression were fixed and immunostained for pericentrin. Venus fluorescence colocalized with pericentrin-positive structures. Scale bar: 5µm.


A . Density centrifugation of hTERT - RPE1 cell extracts followed by immunoblot revealed the presence of KV10 . 1 in the same fractions as Aurora A , phospho - aurora A and a - tubulin ( centrosomal fractions ) .

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A. Density centrifugation of hTERT-RPE1 cell extracts followed by immunoblot revealed the presence of KV10.1 in the same fractions as Aurora A, phospho-aurora A and a-tubulin (centrosomal fractions).


B . KV10 . 1 is also present in microsomal fractions . Cytoplasmic fractions obtained as in A were ultracentrifuged to sediment membranes . In those preparations , immunoprecipitation revealed the presence of full - length KV10 . 1 in both the centrosomal and the microsomal fractions ( obtained by ultracentrifugation ) .

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B. KV10.1 is also present in microsomal fractions. Cytoplasmic fractions obtained as in A were ultracentrifuged to sediment membranes. In those preparations, immunoprecipitation revealed the presence of full-length KV10.1 in both the centrosomal and the microsomal fractions (obtained by ultracentrifugation).


B . Deletion of the CLS does not preclude functional expression at the plasma membrane . Two electrode - voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10 . 1 channel lacking the CLS at the C - terminus revealed voltage - gated currents after stimulation using the potential depicted in the inset . C . The Conductance / voltage relationship of the deleted channel was similar to that of wild - type ( shown for comparison as a solid line ) .

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B. Deletion of the CLS does not preclude functional expression at the plasma membrane. Two electrode-voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10.1 channel lacking the CLS at the C-terminus revealed voltage-gated currents after stimulation using the potential depicted in the inset.C. The Conductance/voltage relationship of the deleted channel was similar to that of wild-type (shown for comparison as a solid line).


D . In the absence of serum , the C - terminus of KV10 . 1 reduced the number of cilia ( maintained cells deciliated ) also after deletion of the CLS ( data for controls is reproduced from Figure 5A ) The CLS is required to induce deciliation . When hTERT - RPE1 and 3T3 cells transfected with KV10 . 1 or KV10 . 1ΔCLS were serum starved , and serum was reintroduced after 24 hours for 4 h , KV10 . 1ΔCLS failed to reduce the fraction of ciliated cells .

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D. In the absence of serum, the C-terminus of KV10.1 reduced the number of cilia (maintained cells deciliated) also after deletion of the CLS (data for controls is reproduced from Figure 5A)The CLS is required to induce deciliation. When hTERT-RPE1 and 3T3 cells transfected with KV10.1 or KV10.1ΔCLS were serum starved, and serum was reintroduced after 24 hours for 4 h, KV10.1ΔCLS failed to reduce the fraction of ciliated cells.


E . The CLS is required for KV10 . 1 - induced tumorigenesis . CHO cells were transfected with the indicated constructs and implanted into the flank of nude mice . KV10 . 1ΔCLS transfected cells did not induce larger tumors than the control .

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E. The CLS is required for KV10.1-induced tumorigenesis. CHO cells were transfected with the indicated constructs and implanted into the flank of nude mice. KV10.1ΔCLS transfected cells did not induce larger tumors than the control.


A . Overexpression of cortactin compensated for the knockdown of KV10 . 1 in ciliary disassembly ( 4 h serum reintroduction after starvation ) .

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A. Overexpression of cortactin compensated for the knockdown of KV10.1 in ciliary disassembly (4 h serum reintroduction after starvation).


B . Deletion of the domain of KV10 . 1 responsible for interaction with cortactin abolished the effects of KV10 . 1 overexpression on the abundance of cilia .

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B. Deletion of the domain of KV10.1 responsible for interaction with cortactin abolished the effects of KV10.1 overexpression on the abundance of cilia.


C . In mouse embryonic fibroblasts , active ( phosphorylated ) cortactin is increased as compared to wild type , possibly compensating the lack of KV10 . 1 .

C. In mouse embryonic fibroblasts, active (phosphorylated) cortactin is increased as compared to wild type, possibly compensating the lack of KV10.1.


( A ) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin - actin in solution . ( B ) Time - lapse TIRF microscopy of single filament growth from Alexa488phalloidin - stabilized seeds ( green ) in the presence of 5μM profilin - actin .

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(A) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin-actin in solution. (B) Time-lapse TIRF microscopy of single filament growth from Alexa488phalloidin-stabilized seeds (green) in the presence of 5μM profilin-actin.


( C ) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF - and lipid - coated microspheres with profilin - actin , CP and Arp2 / 3 in solution . ( D ) Widefield epifluorescence images of indicated network components of bead - attached dendritic networks after kinetic arrest at indicated times .

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(C) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF- and lipid-coated microspheres with profilin-actin, CP and Arp2/3 in solution. (D) Widefield epifluorescence images of indicated network components of bead-attached dendritic networks after kinetic arrest at indicated times.


Biochemical conditions are matching ( B ) with the exception of the additional presence of 200nM CP and 50nM Arp2 / 3 ( E ) Scheme of dendritic network growth from square WAVE1 - micropatterns on a 2 - dimensional coverslip with profilin - actin , CP and Arp2 / 3 in solution . ( F ) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth . Conditions are as in ( D )

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Biochemical conditions are matching (B) with the exception of the additional presence of 200nM CP and 50nM Arp2/3 (E) Scheme of dendritic network growth from square WAVE1-micropatterns on a 2-dimensional coverslip with profilin-actin, CP and Arp2/3 in solution. (F) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth. Conditions are as in (D)


( B ) TIRF microscopy of actin monomers ( blue , 1uM total , 30 % Cy5 labeled ) in the presence of equimolar profilin recruited to a WAVE1 pattern ( magenta ) .

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(B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta).


( C ) TIRF microscopy of actin polymerization in - ( blue arrows ) and outside ( red arrows ) of WAVE1 - micropatterns ( magenta ) in the presence of 1uM profilin - actin visualized by a filament - binding probe ( 5nM Alexa488 - UTRNN ) .

(C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN).


( D ) Kymograph of a growing filament barbed end entering a WAVE1 micropattern . Conditions as in ( C )

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(D) Kymograph of a growing filament barbed end entering a WAVE1 micropattern. Conditions as in (C)


( G ) Surface mCherry - NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1 , N - WASP or dark mCherry control constructs in 1uM profilin - actin .

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(G) Surface mCherry-NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin.


( B ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) and representative kymographs ( lower panel ) from multi - color TIRF imaging of actin polymerization on WAVE1 - coated coverslips . Conditions are 1uM actin , 5nM Alexa488 - UTRNN .

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(B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN.


( E ) TIRF microscopy of actin polymerization from 2uM actin in the absence ( upper panel ) or presence of near - saturating amounts of WAVE1 WCA .

(E) TIRF microscopy of actin polymerization from 2uM actin in the absence (upper panel) or presence of near-saturating amounts of WAVE1 WCA.


( F ) Barbed end polymerization rates as a function of actin and WAVE1 - actin ratios . The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on - rates as indicated ( see Methods ) .

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(F) Barbed end polymerization rates as a function of actin and WAVE1-actin ratios. The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on-rates as indicated (see Methods).


( B ) Widefield epifluorescence images ( Alexa488 - actin ) of bead - attached dendritic networks following kinetic arrest after 10 min of network growth for spike - in experiments ( i . e . 60 % his10 - darkCherry - PWCA , 40 % his10 - Cherry - W ; molar percentage ) using indicated WAVE1 variants . Conditions are 5μM profilin - actin , 200nM CP and 50nM Arp2 / 3 . For all conditions , actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction .

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(B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction.


( C ) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control ( N = 57 - 73 beads per condition ) . Errors bars represent SD .

(C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD.


( D ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) and representative kymographs ( lower panel ) from multi - color TIRF imaging of polymerization from profilin - actin on WAVE1 - coated coverslips . Conditions are 2µM actin , 3µM profilin , 5nM Alexa488 - UTRNN .

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(D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN.


( E ) Average barbed end polymerization rates for indicated WAVE1 variants . Errors bars represent SD .

(E) Average barbed end polymerization rates for indicated WAVE1 variants. Errors bars represent SD.


( A ) The weighted mass average of WAVE1 PRD ( 15 μM ) determined by sedimentation equilibrium ultracentrifugation with LatB - stabilized monomers ( 15 μM ) and various profilin concentrations as indicated . The lower dashed line indicates the expected mass of WAVE1 PRD ; the 5 - 7 kDa upshift is due to non - specific actin binding ( WAVE1 PRD [ null ] , black symbols ) . The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD [ B ] in the presence of 90 μM profilin alone .

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(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone.


( C ) FRET efficiencies for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P _ CA = grey ) as a function of LatB - stabilized , quencher - ( Atto540Q - ) labeled actin monomers concentration . Lines are fits to a single site binding curve .

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(C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve.


( D ) WH2 occupancies ( left axis , Methods ) and FRET efficiencies ( right axis ) for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P * WCA ( null ) = cyan , P _ CA = grey ) as a function of profilin concentration . Concentration of LatB - stabilized , Atto540Q - actin monomers was constant ( 175nM ) at all conditions . Lines are fits to a competitive inhibition model ( see Methods ) .

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(D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).


UMAP plot revealing the cell type annotations of three samples . B ) The cellular composition of three healthy kidney samples .

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UMAP plot revealing the cell type annotations of three samples. B) The cellular composition of three healthy kidney samples.


Dotplot exhibition of canonical markers used for cell type classification .

Dotplot exhibition of canonical markers used for cell type classification.


The gene expression patterns of ACE2 of all detected cell types in kidney .

The gene expression patterns of ACE2 of all detected cell types in kidney.


cell type annotations visualized by UMAP . B ) The sample specific UMAP visualization of two samples .

cell type annotations visualized by UMAP. B) The sample specific UMAP visualization of two samples.


Heatmap of canonical markers applied for cell type assignment .

Heatmap of canonical markers applied for cell type assignment.


Violin plot displaying the expression signals of kidney cell types .

Violin plot displaying the expression signals of kidney cell types.


two - dimensional visualization of bladder cell atlas by UMAP .

two-dimensional visualization of bladder cell atlas by UMAP.


Expression pattern of canonical markers used for identifying bladder cell types .

Expression pattern of canonical markers used for identifying bladder cell types.


ACE2 expression across 12 cell subgroups in the bladder dataset .

ACE2 expression across 12 cell subgroups in the bladder dataset.


( a , b ) Changes in HEK293T cell cytoplasmic ( a ) and mitochondrial ( b ) [ Ca2 + ] in response to ionomycin ( 2 . 5 μM ) were simultaneously measured by fluo - 4 and rhod - 2 imaging , respectively . Each bar represents one target gene silenced with pooled siRNA .

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(a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA.


( c ) qrtPCR of MCU , MCUR1 and MICU1 mRNA from mouse tissues ( n = 3 ; mean ± s . e . m . ) .

(c) qrtPCR of MCU, MCUR1 and MICU1 mRNA from mouse tissues (n = 3; mean ± s.e.m.).


( d ) qrtPCR of MCUR1 mRNA from HEK293T cell clones ( n = 3 ; mean ± s . e . m . ) . KD , knockdown . Neg shRNA , negative shRNA .

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(d) qrtPCR of MCUR1 mRNA from HEK293T cell clones (n = 3; mean ± s.e.m.). KD, knockdown. Neg shRNA, negative shRNA.


( e ) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells ( n = 3 ; mean ± s . e . m . ) . The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2 , respectively .

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(e) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells (n = 3; mean ± s.e.m.). The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2, respectively.


( f ) MCUR1 protein expression levels ( top ) and densitometric analysis ( bottom left ) ( n = 3 ; ± s . e . m . ) . Bottom right , Flag - tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA - resistant MCUR1 cDNA plasmid . WB , western blot .

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(f) MCUR1 protein expression levels (top) and densitometric analysis (bottom left) (n = 3; ± s.e.m.). Bottom right, Flag-tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA-resistant MCUR1 cDNA plasmid. WB, western blot.


( g , h ) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic ( green ) and mitochondrial ( red ) [ Ca2 + ] before , during and after ionomycin exposure . Scale bar , 20 μm .

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(g,h) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca2+] before, during and after ionomycin exposure. Scale bar, 20 μm.


( i - p ) Cytoplasmic ( green ) and mitochondrial matrix ( red ) [ Ca2 + ] responses in HEK293T ( i - l ) cells challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( i ) Wild - type HEK293T cells . ( j ) Cells expressing negative shRNA . ( k ) Clone shHK5 ( n = 4 ) . ( l ) Quantification of peak rhod - 2 fluorescence . * * P0 . 01 ( mean ± s . e . m . ) .

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(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HEK293T (i-l) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (i) Wild-type HEK293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n = 4). (l) Quantification of peak rhod-2 fluorescence. **P0.01 (mean±s.e.m.).


( i - p ) Cytoplasmic ( green ) and mitochondrial matrix ( red ) [ Ca2 + ] responses in HeLa ( m - p ) cells challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( m ) HeLa cells expressing negative shRNA . ( n ) Clone shHe2 . ( o ) Clone shHe2 re - expressing MCUR1 ( n = 3 ) . ( p ) Quantification of peak rhod - 2 fluorescence . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ) .

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(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HeLa (m-p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.).


( q ) [ Ca2 + ] c and [ Ca2 + ] m signals evoked by ATP ( 100 μM ) and thapsigargin ( Tg , 2 μM ) were monitored simultaneously using fura - 2 / AM and mitopericam ( ipcam490 ) , respectively in control ( left ) and MCUR1 - knockdown ( right ) HeLa cells . [ Ca2 + ] c is calibrated in nanomolar concentrations ( black ) , whereas mitopericam fluorescence is inversely normalized to baseline ( F0 / F ; red ) .

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(q) [Ca2+]c and [Ca2+]m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura-2/AM and mitopericam (ipcam490), respectively in control (left) and MCUR1-knockdown (right) HeLa cells. [Ca2+]c is calibrated in nanomolar concentrations (black), whereas mitopericam fluorescence is inversely normalized to baseline (F0/F; red).


( r ) Summary mean [ Ca2 + ] c and [ Ca2 + ] m peaks during ATP stimulation ( negative shRNA n = 29 ; MCUR1 knockdown n = 36 cells ) . * P0 . 05 ( mean ± s . e . m . ) .

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(r) Summary mean [Ca2+]c and [Ca2+]m peaks during ATP stimulation (negative shRNA n = 29; MCUR1 knockdown n = 36 cells). *P0.05 (mean±s.e.m.).


( s ) Increase in bath [ Ca2 + ] ( Rfura2 ) and [ Ca2 + ] m ( Ripcam ) signals in response to CaCl2 ( 1 μM ) and InsP3 ( IP3 , 7 . 5 μM ) addition in permeabilized cells . Uncropped images of blots / gels are shown in Supplementary Fig . S6 .

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(s) Increase in bath [Ca2+](Rfura2) and [Ca2+]m (Ripcam) signals in response to CaCl2 (1 μM) and InsP3 (IP3, 7.5 μM) addition in permeabilized cells. Uncropped images of blots/gels are shown in Supplementary Fig. S6.


Digitonin - permeabilized HeLa cells bathed in intracellular - like solution containing thapsigargin ( Tg ) were loaded with the ΔΨm indicator JC - 1 and the Ca2 + indicator Fura2FF , to which pulses of 10 μM Ca2 + were added before the addition of the mitochondrial uncoupler CCCP ( carbonyl cyanide m - chloro phenyl hydrazone ) . ( a - e ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] and ΔΨm in cells expressing negative shRNA ( Neg shRNA ; a ) , clone shHe1 ( b ) , clone shHe2 ( c ) , clone shHe2 re - expressing MCUR1 ( d ) and HeLa cells stably overexpressing MCUR1 ( e ) . Under similar conditions , 1 μM Ru360 was added before 10 μM Ca2 + pulses until the addition of CCCP .

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Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a-e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP.


( f - i ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] and ΔΨm in negative shRNA cells ( f ) , MCUR1 - knockdown clone shHe1 ( g ) and shHe2 ( h ) , and in control cells overexpressing MCUR1 ( i ) .

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(f-i) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in negative shRNA cells (f), MCUR1-knockdown clone shHe1 (g) and shHe2 (h), and in control cells overexpressing MCUR1 (i).


( j , k ) Negative shRNA ( j ) and MCUR1 - overexpressing ( k ) HEK293T cells were permeabilized with digitonin in intracellular - like medium containing thapsigargin and bath [ Ca2 + ] indicator Fura2FF , and then pulsed with 10 μM Ca2 + . After mitochondrial clearance of bath Ca2 + , Ru360 caused an elevation of bath [ Ca2 + ] , indicating that steady - state bath [ Ca2 + ] after the pulse was maintained by a balance of MCU - mediated Ca2 + uptake and CGP37157 ( 10 μM ) - sensitive Na + - Ca2 + exchanger - mediated extrusion . CCCP was added as indicated . The solid line is the mean ; shaded areas are ± s . e . m . ( n = 3 ) .

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(j,k) Negative shRNA (j) and MCUR1-overexpressing (k) HEK293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin and bath [Ca2+] indicator Fura2FF, and then pulsed with 10 μM Ca2+. After mitochondrial clearance of bath Ca2+, Ru360 caused an elevation of bath [Ca2+], indicating that steady-state bath [Ca2+] after the pulse was maintained by a balance of MCU-mediated Ca2+ uptake and CGP37157 (10 μM)-sensitive Na+-Ca2+ exchanger-mediated extrusion. CCCP was added as indicated. The solid line is the mean; shaded areas are ±s.e.m. (n = 3).


( l ) [ Ca2 + ] m efflux rate derived from j and k during the initial 60 s following Ru360 addition ( NS ; not significant ; n = 3 ) .

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(l) [Ca2+]m efflux rate derived from j and k during the initial 60 s following Ru360 addition (NS; not significant; n = 3).


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( a , b ) Confocal images of HeLa cells transiently co - transfected for 48 h with GFP - tagged MCUR1 and DsRed - Mito plasmids ( a ) or mRFP - tagged MCUR1 and EYFP - Mito ( b ) . Scale bar , 20 μm .

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(a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP-Mito (b). Scale bar, 20 μm.


( c ) Immunoblot analysis of Flag - tagged MCUR1 in the HeLa cell crude mitochondrial fraction , outer mitochondrial membrane ( OMM ) and mitoplasts , using antibodies against VDAC1 ( outer mitochondrial membrane protein ) , COII ( integral membrane marker ) and Flag .

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(c) Immunoblot analysis of Flag-tagged MCUR1 in the HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (outer mitochondrial membrane protein), COII (integral membrane marker) and Flag.


( 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 .

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(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 and MCUR1 - Flag transiently expressed in COS7 cells . Representative of four independent experiments . WB , western blot .

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(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 cells . Representative of four independent experiments .

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(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 cells .

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(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 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 .

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

(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 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 ) .

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(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 and MCUR1 - knockdown HeLa cells that were transiently transfected with MCU cDNA . * * P0 . 01 , * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) .

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(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 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 ) .

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(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 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 ) .

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(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 cells expressing irrelevant shRNA , clone shHe2 , and clone shHe2 re - expressing MCUR1 , exposed sequentially to oligomycin ( i ) , FCCP ( ii ) and rotenone plus myzothiazol ( iii ) .

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

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(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 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 ) .

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(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 ) , 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 .

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(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 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 .

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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 ) and gender ( right ) . GER , samples collected in Germany ; MEX , samples collected in Mexico .

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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 patients . Samples were staged according to the TNM Classification ( UICC , 7th edition ) .

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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 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 .

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

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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 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 .

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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.


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 ) .

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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 ) 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 .

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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 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 ) .

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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 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 ) .

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 ) 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 .

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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 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 .

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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 - 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 .

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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 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 .

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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 ) 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 .

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.


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 ) .

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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).


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 .

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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 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 .

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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.


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 ) .

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 ) 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 .

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 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 .

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 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 .

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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 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 .

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

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 ) 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 .

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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 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 .

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 the three PIKKs in NCS - responsive phosphorylations .

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


( 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 ) .

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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 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 .

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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 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 ) .

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

(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 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 ) .

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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 analysis of selected phosphorylations in the above cluster showing DNA - 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 DNA - PK - dependent attenuation of pS824 / KAP - 1 .

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


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

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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 analysis confirming high - throughput results for selected ATM substrates .

(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 . 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 ) .

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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 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 ) .

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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 analysis of pS343 / NBS1 , which is compensated upon ATM inhibition , and pS824 / KAP - 1 and 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 ) 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 .

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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 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 .

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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 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 ) .

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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 ) 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 .

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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 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 . (

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(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 , 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 . .

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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 ) , the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress ( control ) . N > 50 single - cell 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 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 .

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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 cell extracts of Flag ‐ Nix ‐ expressing COS7 cells and immobilized GST or the 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

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 ) 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 .

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

(C) Quantification 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 . 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 .

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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 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 .

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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 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 .

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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 ) 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

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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 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

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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 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 .

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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 staining for MARS ( green ) and 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 IHC . Local large magnification is shown as framed . Red arrow indicates germinar layer , blue indicates immature neurons . Scale bar : 1mm .

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 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 .

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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 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

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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 ) 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

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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 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

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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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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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 IHC in tumor tissues ( T ) and 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.