E : Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies , HSP60 and TOM40 served as mitochondrial loading controls . F : Densitometric analysis of the bands in ( E ) normalized to loading control , bar graph of three individual experiments ( biological replicates ) , mean ± SD , One - way ANOVA with Bonferroni ' s multiple comparisons test performed against TMBIM5WT * p < 0 . 05 , * * p < 0 . 008 , Two - way ANOVA with Bonferroni ' s multiple comparisons test performed for the OPA1 statistics against TMBIM5WT , * * * p = 0 . 0009 , * * * * p < 0 . 0001 .

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E: Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. F: Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, One-way ANOVA with Bonferroni's multiple comparisons test performed against TMBIM5WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***p=0.0009, ****p<0.0001.


( A ) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods .

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(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods.


( C ) Acoustic startle reflex test to compare hearing ability between Wt ( N = 7 ) and Hpn - / - ( N = 4 ) mice .

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(C) Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn-/- (N = 4) mice.


( F ) Immunoblot analysis of phospho - Smad2 / 3 ( TGFβ pathway signaling marker ) and total Smad2 / 3 in lysates from indicated tissues isolated from Wt , Hpn + / - and Hpn - / - mice . GAPDH was used as the loading control . The histogram depicts quantification of pSmad2 / 3 compared to Wt normalized to total Smad2 / 3 .

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(F) Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt normalized to total Smad2/3.


( B ) Immunoblot analysis of Wap - Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin ( T # denotes individual tumors ) . GAPDH was used as the loading control . Lysates were derived from Wap - Myc mammary tumors from 6 mice with and 6 mice without DOX - induced hepsin

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(B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression


( D ) Immunoblot analysis of phospho - Smad2 / 3 , total Smad2 / 3 , and GAPDH ( loading control ) expression in Wt tumors transplanted into either Wt or Hpn - / - recipients ( n = 10 tumors each ) .

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(D) Immunoblot analysis of phospho-Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn-/- recipients (n = 10 tumors each).


( E ) Western blot analysis of phospho - Smad2 / 3 , total Smad2 / 3 , hepsin , and β - tubulin ( loading control ) in MCF10A - pIND20 - HPN cells treated with doxycycline ( DOX ; overexpression of hepsin ) and the TGFβR1 inhibitor Galunisertib ( TGFβR1i , 10µM ) as indicated .

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(E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated.


( G ) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A - pIND20 - HPN pL6 - TGFβ1 cells with ( DOX + ) or without ( DOX - ) hepsin overexpressing . The TGFβ ELISA assay detects active TGFβ levels ( first 2 columns ) , but heat treatment of the conditioned medium activates all TGFβ , thus effectively measuring total TGFβ ( right two columns ) .

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(G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns).


( H ) Silver - stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin . SLC was incubated with increasing concentrations of recombinant hepsin ( numbers indicate the concentration of hepsin in nM ) .

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(H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).


( A ) Coomassie - stained protein gels with cell lysates and concentrated culture supernatants of MCF10A - pIND20 - HPN cells , with ( + DOX ) or without ( - DOX ) hepsin overexpression . M indicates the media only control . R and NR above the gels indicate reducing and non - reducing conditions , respectively . Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry ; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left . The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions , highlighting the part from which fibronectin was identified . Red boxes indicate the lanes that were analyzed by mass

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(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry


( C ) Immunoblot analysis of fibronectin , hepsin , and β - tubulin ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells with ( DOX + ) or without ( DOX - ) hepsin overexpression . The graph shows the quantification of fibronectin levels .

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(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.


( 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 images of vero monolayers at 3 days post - inoculation . Panels shown are ( A ) mock , ( B ) NP , and ( 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 the viral isolate showing extracellular spherical particles with cross sections through the 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 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 .

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

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


C ) Cell monolayers two days post infection in Vero E6 ( top ) and 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 assay on Vero E6 cells two days post inoculation .

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


Protein 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 % )

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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 . 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 .

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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 . 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 :

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 .

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(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 .

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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 . ) .

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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 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 . ) .

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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 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 .

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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.


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.