( D ) Silver - stained gel with samples from an in vitro protease activity with recombinant hepsin either full purified plasma fibronectin ( upper panel ) or 70 kDa most N - terminal fragment of fibronectin ( lower panel ) . Full - fibronectin ( 1μg ) or 70 kDa fibronectin fragment ( 1μg ) were incubated with increasing concentrations of recombinant hepsin . Arrowheads indicate full - fibronectin , 70kDa fragment , 50 kDa cleavage fragment generated by hepsin , hepsin . schematic figure shows full - fibronectin 70KDa N - terminal fragment . red arrow indicates location of putative hepsin cleavage site . RGD indicated RGD - binding domain in fibronectin .

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(D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.


( A ) Immunoblot of fibronectin in indicated tissues isolated from Wt , Hpn + / - Hpn - / - mouse ( representative of at least three repeats ) . GAPDH blot is used here as loading control .

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(A) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mouse (representative of at least three repeats). GAPDH blot is used here as the loading control.


( C ) Immunoblot of fibronectin , hepsin , β - tubulin ( loading control ) in prostate lysates of control mice or mice with prostate - specific hepsin overexpression N = 2 mice each , two prostate lobes were run separately ( a b ) .

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(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in prostate lysates of control mice or mice with prostate-specific hepsin overexpression N=2 mice each, the two prostate lobes were run separately (a and b).


( A ) Immunoblot of fibronectin , pSmad2 / 3 , total Smad2 / 3 , GAPDH ( loading control ) in lysates from MCF10A - pIND20 - HPN without hepsin overexpression . graph depicts of pSmad2 / 3 normalized to total Smad2 / 3 , compared to siCtrl ( N = 3 biological repeats ) . ( B ) Immunoblot of lysates ( pSmad2 / 3 , total Smad2 / 3 vinculin as loading control ) concentrated culture supernatant ( anti - V5 for TGFβ ) from MCF10A - pIND20 - HPN pL6 - TGFβ1 - V5 without hepsin overexpression . Ponceau is shown as a loading control for western blot with concentrated culture supernatant . graph depicts of pSmad2 / 3 normalized to total Smad2 / 3 , compared to siCtrl ( N = 3 biological repeats ) .

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(A) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (B) Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3 and vinculin as loading control) and concentrated cell culture supernatant (anti-V5 for TGFβ) from MCF10A-pIND20-HPN pL6-TGFβ1-V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats).


( E ) Immunoblot of fibronectin , pSmad2 / 3 , total Smad2 / 3 , hepsin , GAPDH ( loading control ) in lysates from MCF10A - pIND20 - HPN with ( DOX + ) without ( DOX - ) hepsin overexpression , with or without fibronectin silencing ( siFN1 ) . graph depicts of pSmad2 / 3 normalized to total Smad2 / 3 ( N = 3 biological repeats ) .

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(E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats).


( A - C ) 10X phase contrast of vero monolayers at 3 days post - inoculation . Panels shown are ( A ) mock , ( B ) NP , ( C ) OP .

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(A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) NP, and (C) OP.


( D ) Electron microscopic image of viral isolate showing extracellular spherical particles with cross sections through nucleocapsids , seen as black dots .

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(D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.


A ) Two SARS - CoV - 2 passage 4 stocks ( black gray ) were quantified utilizing plaque at day two ( closed circles ) day three ( open circles ) post infection of Vero E6 Vero CCL81 .

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A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81.


B ) Plaque on Vero E6 Vero CCL81 at day 2 day 3 post inoculation .

B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation.


C ) monolayers two days post infection in Vero E6 ( top ) Vero CCL81 ( bottom row ) at multiple dilutions .

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C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.


Viral titer for SARS - CoV - 2 was quantitated by plaque on Vero E6 two days post inoculation .

Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation.


lysates were probed by western blot with B ) anti - SARS nucleocapsid ( N ) antibody or C ) anti - SARS - CoV spike ( S ) antibody .

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Protein lysates were probed by western blot with B) anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody.


A . NIH3T3 transfected with KV10 . 1 * EGFP did not show cilia . were transiently transfected , after 24 h serum was removed for additional 24 hours to induce ciliogenesis , finally were stained with anti - acetylated α - tubulin . While most were ciliated , those showing green were devoid of cilia . Scale bar : 10µm .

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A. NIH3T3 cells transfected with KV10.1*EGFP did not show primary cilia. Cells were transiently transfected, after 24 h serum was removed for additional 24 hours to induce ciliogenesis, and finally cells were stained with anti-acetylated α-tubulin. While most cells were ciliated, those showing green fluorescence were devoid of cilia. Scale bar: 10µm.


B . NIH3T3 transfected with KV10 . 1 ( red bars ) showed markedly less cilia than control ( empty vector , bars ) . Subconfluent cultures grown in presence of FCS were serum starved for 24 hours to induce ciliogenesis . Cilia were stained with anti acetylated α - tubulin antibody . To determine ciliary disassembly , were starved for 24 hours then incubated for 4h in FCS to induce cycle re - entry ciliary resorption .

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B. NIH3T3 cells transfected with KV10.1 (red bars) showed markedly less cilia than control cells (empty vector, white bars). Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis. Cilia were stained with anti acetylated α-tubulin antibody. To determine ciliary disassembly, cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re-entry and ciliary resorption.


C . Similarly , hTERT - RPE1 transfected with KV10 . 1 also showed less cilia . Ciliogenesis ciliary disassembly were induced as in B cilia were stained using anti acetylated α - tubulin quantified as in A . Inset shows equivalent experiment using structurally related potassium channel KV10 . 2 , which did not alter frequency of expression of cilia . D . Examples of fields of view of hTERT - RPE1 transfected with KV10 . 1 , serum starved for 24 hours cilia were revealed with anti Arl13B antibody ( arrows ) . A majority of control - transfected showed cilia , while KV10 . 1 transfected did not . Scale bar : 10µm .

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C. Similarly, hTERT-RPE1 cells transfected with KV10.1 also showed less cilia. Ciliogenesis and ciliary disassembly were induced as in B and cilia were stained using anti acetylated α-tubulin and quantified as in A. The Inset shows the equivalent experiment using the structurally related potassium channel KV10.2, which did not alter the frequency of expression of cilia.D. Examples of fields of view of hTERT-RPE1 cells transfected with KV10.1, serum starved for 24 hours and cilia were revealed with anti Arl13B antibody (arrows). A majority of control-transfected cells showed cilia, while KV10.1 transfected did not. Scale bar: 10µm.


A . Representative showing hTERT - RPE1 grown in presence of serum stained with anti acetylated α - tubulin antibody . There were more ciliated after treatment with siRNA against KV10 . 1 ( KV10 . 1 KD ) , as compared to scrambled siRNA - transfected , which were devoid of cilia ( control ) . Scale bar : 10 µm . B . of effect depicted in A for number of indicated at base of columns .

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A. Representative images showing hTERT-RPE1 grown in the presence of serum and stained with anti acetylated α-tubulin antibody. There were more ciliated cells after treatment with siRNA against KV10.1 (KV10.1 KD), as compared to scrambled siRNA-transfected cells, which were devoid of cilia (control). Scale bar: 10 µm.B. Quantification of the effect depicted in A for the number of images indicated at the base of the columns.


C . Time course of ciliary disassembly . hTERT - RPE1 were serum starved for 24 hours , fixed at times indicated after serum reintroduction , stained with anti acetylated α - tubulin to reveal cilia . While in presence of scrambled siRNA abundance of cilia decreased rapidly ( black ) , it remained virtually unchanged in where KV10 . 1 was knocked down ( red ) . N = 8 - 13 . D . Time course of ciliary shortening after serum reintroduction . In same experiments as in C , of remaining cilia was measured automatically using ImageJ ( FIJI ) . While ciliary decreased with a similar time course in siRNA - treated as in controls , cilia were longer in KV10 . 1 - knockdown . A re - elongation of cilia was evident in both populations after 6 hours . Values are mean ± for a number of cilia between 62 ( control , 4 . 5 h ) 308 ( both groups , 1 . 5 h ) .

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C. Time course of ciliary disassembly. hTERT-RPE1 cells were serum starved for 24 hours, fixed at the times indicated after serum reintroduction, and stained with anti acetylated α-tubulin to reveal cilia. While in the presence of scrambled siRNA the abundance of cilia decreased rapidly (black line), it remained virtually unchanged in cells where KV10.1 was knocked down (red). N=8-13.D. Time course of ciliary shortening after serum reintroduction. In the same experiments as in C, the length of the remaining cilia was measured automatically using ImageJ (FIJI). While ciliary length decreased with a similar time course in the siRNA-treated cells as in controls, cilia were longer in KV10.1-knockdown cells. A re-elongation of cilia was evident in both populations after 6 hours. Values are mean ± SEM for a number of cilia between 62 (control, 4.5 h) and 308 (both groups, 1.5 h).


E . Blockade of permeation through K + channel altered speed of deciliation . After serum starvation , medium containing serum Astemizole ( 10 µM ) was added , were fixed stained at he indicated time points ( N = 12 ) .

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E. Blockade of permeation through the K+ channel altered the speed of deciliation. After serum starvation, medium containing serum and Astemizole (10 µM) was added, and cells were fixed and stained at he indicated time points (N=12).


A . of amount of ciliated in embryonic fibroblasts from KV10 . 1 knockout mice . Actively growing cultures , cultures after 24 h serum starvation , four hours after subsequent serum reintroduction were stained for presence of cilia using anti - acetylated α - tubulin . An increase in frequency of cilia in knockout was evident under conditions , but especially marked in ciliary resorption ( 4h after serum reintroduction ) , indicating an influence of KV10 . 1 expression on deciliation .

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A. Quantification of the amount of ciliated cells in primary embryonic fibroblasts from KV10.1 knockout mice. Actively growing cultures, cultures after 24 h serum starvation, and four hours after subsequent serum reintroduction were stained for the presence of cilia using anti-acetylated α-tubulin. An increase in frequency of cilia in the knockout cells was evident under all conditions, but especially marked in the ciliary resorption test (4h after serum reintroduction), indicating an influence of KV10.1 expression on deciliation.


B . Exogenous expression of human KV10 . 1 reverted phenotype in MEFs . obtained from three were transfected with KV10 . 1 or empty vector ( mock ) , serum starved for 48 h cilia were revealed by immunocytochemistry using anti - acetylated α - tubulin in indicated number of . KV10 . 1 expression decreased frequency of ciliated .

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B. Exogenous expression of human KV10.1 reverted the phenotype in MEFs. Cells obtained from three animals were transfected with KV10.1 or empty vector (mock), serum starved for 48 h and cilia were revealed by immunocytochemistry using anti-acetylated α-tubulin in the indicated number of images. KV10.1 expression decreased the frequency of ciliated cells.


C . MEFs from knockout mice did not show cilia when transfected with KV10 . 1 . were co - transfected with human KV10 . 1 mVenus , serum starved for 48 hours then fixed stained for cilia ( AcTub ) . Venus - positive did not show cilia . Scale bar : 25 µm .

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C. MEFs from knockout mice did not show primary cilia when transfected with KV10.1. Cells were co-transfected with human KV10.1 and mVenus, serum starved for 48 hours and then fixed and stained for cilia (AcTub). Venus-positive cells did not show cilia. Scale bar: 25 µm.


D . In wild type MEFs , transfection with KV10 . 1 reduced presence of cilia . When a gain - of - function mutant ( L352V ) that originates developmental defects in human was transfected , frequency of with cilia was further reduced .

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D. In wild type MEFs, transfection with KV10.1 reduced the presence of primary cilia. When a gain-of-function mutant (L352V) that originates developmental defects in human patients was transfected, the frequency of cells with cilia was further reduced.


E . Sensitivity to SHH signaling was significantly increased in fibroblasts from KV10 . 1 knockout mice , in agreement with higher abundance of cilia . were serum starved SHH was stimulated after 24 hours by addition of culture supernatant of SHH - expressing HEK293 . Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT - PCR .

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E. Sensitivity to SHH signaling was significantly increased in primary fibroblasts from KV10.1 knockout mice, in agreement with the higher abundance of primary cilia. Cells were serum starved and SHH was stimulated after 24 hours by addition of culture supernatant of SHH-expressing HEK293 cells. Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT-PCR.


A . Actively proliferating hTERT - RPE1 were transfected with scrambled or KV10 . 1 siRNA , cycle distribution was determined using acridine orange . Knockdown of KV10 . 1 induces an increase in with / content compatible with G0 ( rectangle ) . However , quantitative increase ( to approximately 10 % ) was insufficient to explain abundance of ciliated ( over 40 % )

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A. Actively proliferating hTERT-RPE1 cells were transfected with scrambled or KV10.1 siRNA, and cell cycle distribution was determined using acridine orange staining. Knockdown of KV10.1 induces an increase in cells with DNA/RNA content compatible with G0 (rectangle). However, the quantitative increase (to approximately 10%) was insufficient to explain the abundance of ciliated cells (over 40%)


B . proliferation marker Ki67 frequently coexists with cilia in KV10 . 1 knockdown . Exponentially growing were stained for acetylated α - tubulin ( red , AvTub ) Ki67 ( green ) . Nuclei were counterstained with Draq5 ( blue ) . Scale bar : 10 µm . C . of effects observed in B . Although positivity for Ki67 ( proliferating ) presence of cilia were not always excluding each other even in transfected with control siRNA , KV10 . 1 knockdown increased frequency of ciliated , while it reduced amount of Ki67 positive , actively proliferating . Of those presenting a cilium , fraction of simultaneously Ki - 67 - positive was much larger upon KV10 . 1 knockdown . Scale bar , 10 µm .

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B. The proliferation marker Ki67 frequently coexists with primary cilia in KV10.1 knockdown cells. Exponentially growing cells were stained for acetylated α-tubulin (red, AvTub) and Ki67 (green). Nuclei were counterstained with Draq5 (blue). Scale bar: 10 µm.C. Quantification of the effects observed in B. Although the positivity for Ki67 (proliferating cells) and the presence of cilia were not always excluding each other even in cells transfected with control siRNA, KV10.1 knockdown increased the frequency of ciliated cells, while it reduced the amount of Ki67 positive, actively proliferating cells. Of those cells presenting a cilium, the fraction of simultaneously Ki-67-positive cells was much larger upon KV10.1 knockdown. Scale bar, 10 µm.


D . cilia were present in actively synthesizing ( measured by EdU incorporation ) . were transfected with siRNA against KV10 . 1 , 24 hours later were incubated in presence of EdU for 4h before fixation . KV10 . 1 knockdown resulted in presence of abundant EdU - positive simultaneously ciliated , suggesting a decoupling between deciliation cycle progression . Scale bar :

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D. Primary cilia were present in cells actively synthesizing DNA (measured by EdU incorporation). Cells were transfected with siRNA against KV10.1, and 24 hours later were incubated in the presence of EdU for 4h before fixation. KV10.1 knockdown resulted in the presence of abundant EdU-positive and simultaneously ciliated cells, suggesting a decoupling between deciliation and cell cycle progression. Scale bar: 10µm


A . An anti - KV10 . 1 antibody directed against an extracellular epitope labels a structure related to centrosome ( pericentrin - positive ) positive for Arl13b with an appearance suggestive of cilium in HFF ( human foreskin fibroblast ) . were serum starved for 24 or 48h , fixed , blocked ( 10 % BSA ) then stained without permeabilization with anti - KV10 . 1 antibody recognizing an extracellular epitope . Immunostaining for pericentrin was performed after subsequent permeabilization 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 . After serum starvation for 48h , serum was reintroduced for 4h to induce KV10 . 1 expression ciliary resorption . were then fixed stained with a mixture of anti - KV10 . 1 mouse monoclonals 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 monoclonal anti KV10 . 1 ( mAb 62 ) as revealed by MEFs from wild type ( left ) or knockout ( 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 . from cilia of MEFs from KV10 . 1 knockout mice obtained using mAb62 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 , 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 . expressing a KV10 . 1 * mVenus fusion under control of endogenous promoter to render a mild overexpression were fixed immunostained for pericentrin . Venus 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 extracts followed by immunoblot revealed presence of KV10 . 1 in same fractions as Aurora A , phospho - aurora A 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 . fractions obtained as in A were ultracentrifuged to sediment membranes . In those preparations , immunoprecipitation revealed presence of full - KV10 . 1 in both centrosomal 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 CLS does not preclude functional expression at plasma . Two electrode - voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10 . 1 channel lacking CLS at C - terminus revealed voltage - gated currents after stimulation using potential depicted in inset . C . Conductance / voltage relationship of deleted channel was similar to that of wild - type ( shown for comparison as a solid ) .

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

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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 . CLS is required for KV10 . 1 - induced tumorigenesis . CHO were transfected with indicated constructs implanted into flank of nude mice . KV10 . 1ΔCLS transfected did not induce larger tumors than 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 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 domain of KV10 . 1 responsible for interaction with cortactin abolished effects of KV10 . 1 overexpression on 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 lack of KV10 . 1 .

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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 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 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 - lipid - coated microspheres with profilin - actin , CP Arp2 / 3 in solution . ( D ) Widefield epifluorescence 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 exception of additional presence of 200nM CP 50nM Arp2 / 3 ( E ) Scheme of dendritic network growth from square WAVE1 - micropatterns on a 2 - dimensional coverslip with profilin - actin , CP 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 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 ) outside ( red arrows ) of WAVE1 - micropatterns ( magenta ) in presence of 1uM profilin - actin visualized by a filament - binding probe ( 5nM Alexa488 - UTRNN ) .

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(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 corresponding kymographs of barbed end growth for indicated WAVE1 , N - WASP or 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 ) 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 absence ( upper panel ) or presence of near - saturating amounts of WAVE1 WCA .

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(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 WAVE1 - actin ratios . red 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 ( 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 ) using indicated WAVE1 variants . Conditions are 5μM profilin - actin , 200nM CP 50nM Arp2 / 3 . For conditions , actin network assembly was quenched by adding 5M excess Latrunculin B phalloidin 10 minutes after initiating 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 .

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(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 ) 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 .

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


( A ) weighted mass average of WAVE1 PRD ( 15 μM ) determined by sedimentation equilibrium ultracentrifugation with LatB - stabilized monomers ( 15 μM ) various profilin concentrations as indicated . lower dashed indicates expected mass of WAVE1 PRD ; 5 - 7 kDa upshift is due to non - specific actin binding ( WAVE1 PRD [ null ] , black symbols ) . upper dashed mark equilibrium mass observed for 15 μM WAVE1 PRD [ B ] in 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 . 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 ) 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 . of LatB - stabilized , Atto540Q - actin monomers was constant ( 175nM ) at 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 type annotations of three samples . B ) composition of three healthy kidney samples .

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 type classification .

Dotplot exhibition of canonical markers used for cell type classification.


gene expression patterns of ACE2 of detected types in kidney .

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


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

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


of canonical markers applied for type assignment .

Heatmap of canonical markers applied for cell type assignment.


Violin plot displaying expression signals of kidney types .

Violin plot displaying the expression signals of kidney cell types.


two - dimensional visualization of bladder atlas by UMAP .

two-dimensional visualization of bladder cell atlas by UMAP.


Expression pattern of canonical markers used for identifying bladder types .

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


ACE2 expression across 12 subgroups in bladder dataset .

ACE2 expression across 12 cell subgroups in the bladder dataset.


( a , b ) Changes in HEK293T ( a ) mitochondrial ( b ) [ Ca2 + ] in response to ionomycin ( 2 . 5 μM ) were simultaneously measured by fluo - 4 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 MICU1 mRNA from mouse tissues ( n = 3 ; mean ± s . e . m . ) .

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


( d ) qrtPCR of MCUR1 mRNA from HEK293T 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 clones of rescued MCUR1 mRNA in shHe2 clone ( n = 3 ; mean ± s . e . m . ) . same lentiviral shRNAs were used to generate shHK4 shHe1 shHK5 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 expression ( top ) densitometric ( bottom left ) ( n = 3 ; ± s . e . m . ) . Bottom right , Flag - tagged MCUR1 expression in clone shHe2 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 from movies of HEK293T negative shRNA or shHK5 showing ( green ) mitochondrial ( red ) [ Ca2 + ] before , during 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 ) ( green ) mitochondrial matrix ( red ) [ Ca2 + ] responses in HEK293T ( i - l ) challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( i ) Wild - type HEK293T . ( j ) expressing negative shRNA . ( k ) Clone shHK5 ( n = 4 ) . ( l ) of peak rhod - 2 . * * 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 ) ( green ) mitochondrial matrix ( red ) [ Ca2 + ] responses in HeLa ( m - p ) challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( m ) HeLa expressing negative shRNA . ( n ) Clone shHe2 . ( o ) Clone shHe2 re - expressing MCUR1 ( n = 3 ) . ( p ) of peak rhod - 2 . * 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 [ Ca2 + ] m signals evoked by ATP ( 100 μM ) thapsigargin ( Tg , 2 μM ) were monitored simultaneously using fura - 2 / AM mitopericam ( ipcam490 ) , respectively in control ( left ) MCUR1 - knockdown ( right ) HeLa . [ Ca2 + ] c is calibrated in nanomolar concentrations ( black ) , whereas mitopericam is inversely normalized to baseline ( F0 / F ; red ) .

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

(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 [ Ca2 + ] m peaks during ATP stimulation ( negative shRNA n = 29 ; MCUR1 knockdown n = 36 ) . * 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 ) [ Ca2 + ] m ( Ripcam ) signals in response to CaCl2 ( 1 μM ) InsP3 ( IP3 , 7 . 5 μM ) addition in permeabilized . Uncropped 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 bathed in intracellular - like solution containing thapsigargin ( Tg ) were loaded with ΔΨm indicator JC - 1 Ca2 + indicator Fura2FF , to which pulses of 10 μM Ca2 + were added before addition of mitochondrial uncoupler CCCP ( carbonyl cyanide m - chloro phenyl hydrazone ) . ( a - e ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] ΔΨm in expressing negative shRNA ( Neg shRNA ; a ) , clone shHe1 ( b ) , clone shHe2 ( c ) , clone shHe2 re - expressing MCUR1 ( d ) HeLa stably overexpressing MCUR1 ( e ) . Under similar conditions , 1 μM Ru360 was added before 10 μM Ca2 + pulses until addition of CCCP .

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

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 + ] ΔΨm in negative shRNA ( f ) , MCUR1 - knockdown clone shHe1 ( g ) shHe2 ( h ) , in control 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 ) MCUR1 - overexpressing ( k ) HEK293T were permeabilized with digitonin in intracellular - like medium containing thapsigargin bath [ Ca2 + ] indicator Fura2FF , 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 was maintained by a balance of MCU - mediated Ca2 + uptake CGP37157 ( 10 μM ) - sensitive Na + - Ca2 + exchanger - mediated extrusion . CCCP was added as indicated . solid is 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 k during 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 of HeLa transiently co - transfected for 48 h with GFP - tagged MCUR1 DsRed - Mito plasmids ( a ) or mRFP - tagged MCUR1 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 of Flag - tagged MCUR1 in HeLa crude mitochondrial fraction , outer mitochondrial ( OMM ) mitoplasts , using antibodies against VDAC1 ( outer mitochondrial ) , COII ( integral marker ) 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 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 .

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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 MCUR1 - Flag transiently expressed in COS7 . 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 . 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 .

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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 ( 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 .

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

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

(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 ) .

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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 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 ) .

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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 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 expressing irrelevant shRNA , clone shHe2 , clone shHe2 re - expressing MCUR1 , exposed sequentially to oligomycin ( i ) , FCCP ( ii ) 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 maximal O2 consumption rates in 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 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 ) .

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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 ) , 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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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 ) 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 . Samples were staged according to 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 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 .

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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 ) Bhlhe41 ( orange ) in indicated populations , data are from 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 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 .

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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.


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 ) .

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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 ) 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 .

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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 of lung ( B ) macrophages as in A ( left ) . expression of CD45 . 1 CD45 . 2 on donor recipient is shown next to 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 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 .

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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 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 .

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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 - 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 .

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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 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 .

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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 .

(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).