G MAGE - A10 and MAGE - A11 are not altered upon nutrient deprivation or DCAF12 deletion . A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times . Cell lysates were probed for the indicated proteins .

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G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.


A - B U2OS GFP - LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . Cells were then fixed , imaged , and the number of GFP - LC3 puncta were quantitated per cell . Representative images ( A ) and quantitation of > 50 cells from three independent experiments ( B ) are shown . Scale bar indicates 50 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - test .

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A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.


C - D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . Cells were then fixed , endogenous LC3 was immunostained , and the number of LC3 puncta were quantitated per cell . Representative images ( C ) and quantitation of > 50 cells from three independent experiments ( D ) are shown . Scale bar indicates 20 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - test .

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C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.


E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE - A3 / 6 depletion and monitor the autophagy adaptor / substrate p62 / SQSTM1 .

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E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.


a , Expression levels of ISX , TWIST1 , Snail1 , Fibronectin ( FN1 ) , VEGF , and E - cadherin ( CDH1 ) mRNA were examined in a ISX - GFP inducible Tet - ON transformants at 8 h after addition of DOX . Data are presented as mean ± SD in graph ( p < 0 . 001 compared to time point 0h ; Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate .

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

a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate.


b , Western blotting analysis of the protein levels of ISX , HIF1α , TWIST1 , Snail1 , Slug , ZEB1 , Bmi1 , E - cadherin ( E - Cad . ) , Fibronectin , N - cadherin ( N - Cad . ) , Vimentin , and VEGF in A549 and H1299 cells with DOX - inducible ISX expression system 8hs after DOX induction .

b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction.


ISX transcriptionally activates luciferase activity driven by TWIST1 ( c ) promoter regions in A549 cells . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate .

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ISX transcriptionally activates luciferase activity driven by TWIST1 (c) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


d , ISX transcriptionally activates luciferase activity driven by Snail1 ( d ) promoter regions in A549 cells . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate .

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d, ISX transcriptionally activates luciferase activity driven by Snail1 (d) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


e , ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells ( 10 % input of each groups were pull down and applied to qPCR ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate .

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e, ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


f , ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter . Data are presented as mean ± SD in graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate .

O O B-CONTROLLED_VAR 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

f, ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


g , ChIP analysis of ISX binding to the endogenous promoters of TWIST1 ( - 180 / + 35 ) and Snail1 ( - 160 / + 40 ) in A549 and cells ( 10 % input of each groups were pull down and applied to qPCR ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate .

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g, ChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.


h , Effect of forced expression and knockdown of ISX on cell migration ( wound healing ) measured in A549 cells . ISXi , ISX - specific shRNAi Data are presented as mean ± SD in graph ( * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm .

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h, Effect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi Data are presented as mean ± SD in graph (**, p < 0.01, ***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.


i , Effect of forced expression and knockdown of ISX on cell invasion ( transwell ) activity measured in A549 cells . Data are presented as mean ± SD in bar graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm .

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i, Effect of forced expression and knockdown of ISX on cell invasion (transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.


a , BRD4 peptide ( 291 - 314 a . a ) obtained from anti - ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography ‒ tandem mass spectrometry ( LC - tandem - MS ) . Data information : Each experiment was repeated at least three times .

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a, BRD4 peptide (291-314 a.a) obtained from anti-ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography‒tandem mass spectrometry (LC-tandem -MS). Data information: Each experiment was repeated at least three times.


b , ISX association proteins ( PCAF , BRD4 , CBP , CREB , RNA Pol II , and Histone H3 ) determined by Western blot in immunoprecipitation of A549 cells . Data information : Each experiment was repeated at least three times .

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

b, ISX association proteins (PCAF, BRD4, CBP, CREB, RNA Pol II, and Histone H3) determined by Western blot in immunoprecipitation of A549 cells. Data information: Each experiment was repeated at least three times.


c , ISX association proteins ( PCAF , BRD4 , CBP , CREB , and RNA Pol II ) determined by Western blot in anti - ISX immunoprecipitates of tumor tissues from patients with lung cancer . Data information : Each experiment was repeated at least three times .

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c, ISX association proteins (PCAF, BRD4, CBP, CREB, and RNA Pol II) determined by Western blot in anti-ISX immunoprecipitates of tumor tissues from patients with lung cancer. Data information: Each experiment was repeated at least three times.


d , Proximity ligation assay ( PLA ) of ISX and BRD4 ( PCAF ) interaction in A549 cells . Red foci indicate close proximity of the two proteins . NC : negative control . Blank arrows indicate interactions of ISX with BRD4 ( PCAF ) in the cells . Data information : Each experiment was repeated at least three times .

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d, Proximity ligation assay (PLA) of ISX and BRD4(PCAF) interaction in A549 cells. Red foci indicate close proximity of the two proteins. NC: negative control. Blank arrows indicate interactions of ISX with BRD4 (PCAF) in the cells. Data information: Each experiment was repeated at least three times.


e , Western blotting analysis of the protein levels of ISX , PCAF , and EMT markers in DOX - inducible ISX expression system A549 cells treated with TH1834 , Garcinol , C646 , MB - 3 for 8 h after DOX induction . Data information : Each experiment was repeated at least three times .

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

e, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in DOX-inducible ISX expression system A549 cells treated with TH1834, Garcinol, C646, MB-3 for 8 h after DOX induction. Data information: Each experiment was repeated at least three times.


f , Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol . Red foci indicate close proximity of the two proteins . Blank arrows indicate interactions of ISX with BRD4 in the cells . NC : negative control . Data information : Each experiment was repeated at least three times .

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

f, Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol. Red foci indicate close proximity of the two proteins. Blank arrows indicate interactions of ISX with BRD4 in the cells. NC: negative control. Data information: Each experiment was repeated at least three times.


g , Western blotting analysis of the protein levels of ISX , PCAF , and EMT markers in A549 cells with PCAF knockdown and DOX - inducible ISX expression system at 8 h time point after DOX induction . Data information : Each experiment was repeated at least three times .

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

g, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in A549 cells with PCAF knockdown and DOX-inducible ISX expression system at 8 h time point after DOX induction. Data information: Each experiment was repeated at least three times.


Cell migration ( wound healing , h ) ssay of A549 lung cancer cells treated with Garcinol . V , vehicle . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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Cell migration (wound healing, h) ssay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


i , Cell invasion ( transwell , i ) assay of A549 lung cancer cells treated with Garcinol . V , vehicle . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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i, Cell invasion (transwell, i) assay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


c , Recombinant PCAF acetylates His6 - ISX at lysine residue 69 by in vitro acetylation assay . Acetylated ISX was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times .

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c, Recombinant PCAF acetylates His6-ISX at lysine residue 69 by in vitro acetylation assay. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.


d and e , The protein levels of GFP - tagged WT or mutant ISX , PCAF and BRD4 were determined in cytosol , nuclei , and anti - GFP immunoprecipites of A549 cells by Western blotting . Acetylated ISX was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times .

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d and e, The protein levels of GFP-tagged WT or mutant ISX, PCAF and BRD4 were determined in cytosol, nuclei, and anti-GFP immunoprecipites of A549 cells by Western blotting. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.


f , The protein levels of total and acetylated histone H3 were determined in anti - histone H3 immunoprecipites of A549 cells by Western blotting . Data information : Each experiment was repeated at least three times .

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f, The protein levels of total and acetylated histone H3 were determined in anti-histone H3 immunoprecipites of A549 cells by Western blotting. Data information: Each experiment was repeated at least three times.


The cell migration ( wound healing , g ) activity were determined in A549 cells with GFP - tagged wild or ISX mutants . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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The cell migration (wound healing, g) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


h , The cell invasion ( transwell , h ) activity were determined in A549 cells with GFP - tagged wild or ISX mutants . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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h, The cell invasion (transwell, h) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


j . Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information : Each experiment was repeated at least three times .

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j. Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information: Each experiment was repeated at least three times.


k , Kaplan - Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP , ISX - GFP and ISX Ac - GFP ( AC3 ) ( n = 10 ) . p = 0 . 0003 . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) test comparing the two Kaplan - Meier curves . Data information : Each experiment was repeated at least three times .

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k, Kaplan-Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP, ISX-GFP and ISX Ac-GFP (AC3) (n = 10). p = 0.0003. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves. Data information: Each experiment was repeated at least three times.


c , Recombinant PCAF acetylates His6 - BRD4 at lysine residue 332 . Acetylated BRD4 was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times .

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c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.


d , The mCherry - tagged WT and BRD4 mutants were detected in anti - GFP immunoprecipitates by Western blotting in A549 cells . Data information : Each experiment was repeated at least three times .

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d, The mCherry-tagged WT and BRD4 mutants were detected in anti-GFP immunoprecipitates by Western blotting in A549 cells. Data information: Each experiment was repeated at least three times.


e and f , The mRNA levels of TWIST1 ( e ) and Snail1 ( f ) were verified in A549 cells co - expressing mCherry - tagged WT or mutant BRD4 and GFP - tagged ISX by RT - PCR . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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e and f, The mRNA levels of TWIST1(e) and Snail1(f) were verified in A549 cells co-expressing mCherry-tagged WT or mutant BRD4 and GFP-tagged ISX by RT-PCR. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


g and h , The DNA binding activity of TWIST1 and Snail1 were evaluated in anti - GFP - mCherry ChIP - ChIP immunoprecipitates by RT - PCR in A549 cells . Red , Hprt1 promoter ( - 190 / + 40 bp , negative control ) ( 10 % input of each groups were pulled down and used for qPCR analysis ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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g and h, The DNA binding activity of TWIST1 and Snail1 were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190/+40 bp, negative control) (10% input of each groups were pulled down and used for qPCR analysis). Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


i , The cell invasion ( transwell ) activity were determined in A549 cells co - transfected with cDNA coding for GFP - tagged ISX and mCherry - tagged BRD4 mutants . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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i, The cell invasion (transwell) activity were determined in A549 cells co-transfected with cDNA coding for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


a , Schematic depiction of ISX and deletion constructs used . b GFP - tagged truncated ISX mutant proteins or HA - tagged BRD4 were detected in anti - HA or GFP immunoprecipitates by Western blotting analysis . Data information : Each experiment was repeated at least three times .

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a, Schematic depiction of ISX and deletion constructs used. b GFP-tagged truncated ISX mutant proteins or HA-tagged BRD4 were detected in anti-HA or GFP immunoprecipitates by Western blotting analysis. Data information: Each experiment was repeated at least three times.


a mCherry - tagged truncated BRD4 mutant proteins and GFP - tagged ISX were detected in anti - GFP immunoprecipitates by Western blot . Data information : Each experiment was repeated at least three times .

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a mCherry-tagged truncated BRD4 mutant proteins and GFP-tagged ISX were detected in anti-GFP immunoprecipitates by Western blot. Data information: Each experiment was repeated at least three times.


The DNA binding activity of Snail ( b ) were evaluated in anti - GFP - mCherry ChIP - ChIP immunoprecipitates by RT - PCR in A549 cells . Red , Hprt1 promoter ( - 190 - + 40 bp , negative control ) . ( 10 % input of each groups were pull down and applied to qPCR ) Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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The DNA binding activity of Snail (b) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


c , The DNA binding activity of TWIST1 ( c ) were evaluated in anti - GFP - mCherry ChIP - ChIP immunoprecipitates by RT - PCR in A549 cells . Red , Hprt1 promoter ( - 190 - + 40 bp , negative control ) . ( 10 % input of each groups were pull down and applied to qPCR ) Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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c, The DNA binding activity of TWIST1 (c) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


The cell migration ( wound healing , d ) activity were determined in A549 cells co - transfected with cDNAs for GFP - tagged ISX and mCherry - tagged BRD4 mutants . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

The cell migration (wound healing, d) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


e , The cell invasion ( transwell , e ) activity were determined in A549 cells co - transfected with cDNAs for GFP - tagged ISX and mCherry - tagged BRD4 mutants . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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e, The cell invasion (transwell, e) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


a , IHC staining with ISX ( brown ) or PCAF ( brown ) antibody in lung tumors from patients with lung cancer . N , normal tissue ; T , tumor mass ; Nuclei , hematoxylin ( blue ) ; Blue and red arrow , cytosol and nuclear ISX ( PCAF ) . Scale bar , 500μm ( 400X ) ; Scale bar , 2mm ( 100X ) .

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a, IHC staining with ISX (brown) or PCAF (brown) antibody in lung tumors from patients with lung cancer. N, normal tissue; T, tumor mass; Nuclei, hematoxylin (blue); Blue and red arrow, cytosol and nuclear ISX (PCAF). Scale bar, 500μm (400X); Scale bar, 2mm (100X).


b , Confocal immunofluorescence detection of ISX ( green ) and BRD4 ( red ) in lung tumors from patients with NSCLC . Cell nuclei were visualized by DAPI ( blue ) ; Yellow arrows indicate co - localization ; IgG , negative control . N , normal tissue ; T , tumor mass .

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b, Confocal immunofluorescence detection of ISX (green) and BRD4 (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.


c , Confocal immunofluorescence detection of ISX ( green ) and PCAF ( red ) in lung tumors from patients with NSCLC . Cell nuclei were visualized by DAPI ( blue ) ; Yellow arrows indicate co - localization ; IgG , negative control . N , normal tissue ; T , tumor mass .

c, Confocal immunofluorescence detection of ISX (green) and PCAF (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.


Correlation analysis of mRNA expression for , ( d ) ISX and BRD4 , in 157 lung cancer samples .

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Correlation analysis of mRNA expression for, (d) ISX and BRD4, in 157 lung cancer samples.


e , Correlation analysis of mRNA expression for , ( e ) ISX and PCAF in 157 lung cancer samples .

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e, Correlation analysis of mRNA expression for, (e) ISX and PCAF in 157 lung cancer samples.


f , Protein expression of ISX , ISX ( 69Kac ) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting . N , normal tissue ; T , tumor mass ; Arrow , target protein ; * , unspecific band .

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f, Protein expression of ISX, ISX(69Kac) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting. N, normal tissue; T, tumor mass; Arrow, target protein; *, unspecific band.


The Kaplan - Meier survival curve was used to analyze survival correlation between patients ( n = 157 ) with NSCLC and ISX ( g ) levels . On the basis of the cut - off values of fold differences , the study population was dichotomized into the " high " and " low " expression group . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) test comparing the two Kaplan - Meier curves .

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The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and ISX (g) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.


g and h , The Kaplan - Meier survival curve was used to analyze survival correlation between patients ( n = 157 ) with NSCLC and PCAF ( h ) levels . On the basis of the cut - off values of fold differences , the study population was dichotomized into the " high " and " low " expression group . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) test comparing the two Kaplan - Meier curves .

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g and h, The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and PCAF (h) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.


A . Immunoblot analysis of ACE2 expression in the lysate of human lung A549 cells overexpressing ACE2 compared to non - transduced ( - ) A549 cells . Calnexin served as loading control .

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A. Immunoblot analysis of ACE2 expression in the lysate of human lung A549 cells overexpressing ACE2 compared to non-transduced (-) A549 cells. Calnexin served as loading control.


B , C . Illustration of the Incucyte readout . Infection of A549 or ACE2 - expressing A549 cells with SARS - CoV - 2 encoding a GFP expression cassette . Infection of cells was monitored microscopically as green fluorescence through live - cell imaging at 24 hours post infection ( hpi ) ( B ) or during the time - course of 48 h and shown as the mean of the GFP - positive area relative to the whole area covered by cells in the same well ( C ) . Error bars represent means ± SD derived from biological triplicates ( N = 3 ) . Scale bar , 500 µm .

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B, C. Illustration of the Incucyte readout. Infection of A549 or ACE2-expressing A549 cells with SARS-CoV-2 encoding a GFP expression cassette. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 24 hours post infection (hpi) (B) or during the time-course of 48 h and shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well (C). Error bars represent means ± SD derived from biological triplicates (N = 3). Scale bar, 500 µm.


D . A549 - ACE2 cells were treated for 6 hours with different concentrations of indicated inhibitors and then infected with SARS - CoV - 2 - GFP . GFP - fluorescence relative to DMSO - treated control cells was determined 48 hpi as described in ( C ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed for every indicated concentration compared to DMSO ( 0 µM ) . Asterisks indicate significance compared to DMSO ( N = 3 - 6 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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D. A549-ACE2 cells were treated for 6 hours with different concentrations of indicated inhibitors and then infected with SARS-CoV-2-GFP. GFP-fluorescence relative to DMSO-treated control cells was determined 48 hpi as described in (C). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


E . A549 - ACE2 cells were treated for 30 min with PMA at indicated concentrations and then infected with SARS - CoV - 2 - GFP . Infection of cells was monitored as in C . Two - way ANOVA with Tukey ' s post - hoc multiple comparison test . Asterisks indicate significance compared to the DMSO control of each time point ( N = 9 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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E. A549-ACE2 cells were treated for 30 min with PMA at indicated concentrations and then infected with SARS-CoV-2-GFP. Infection of cells was monitored as in C. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Asterisks indicate significance compared to the DMSO control of each time point (N = 9). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


F , A549 - ACE2 cells were treated with apratastat , DPC - 333 at indicated concentrations . Experiment was conducted and analyzed as in ( D ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed for every indicated concentration compared to DMSO ( 0 µM ) . Asterisks indicate significance compared to DMSO ( N = 3 - 6 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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F, A549-ACE2 cells were treated with apratastat, DPC-333 at indicated concentrations. Experiment was conducted and analyzed as in (D). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


G . A549 - ACE2 cells were treated with apratastat , DPC - 333 or BB94 at indicated concentrations . Experiment was conducted and analyzed as in ( D ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed for every indicated concentration compared to DMSO ( 0 µM ) . Asterisks indicate significance compared to DMSO ( N = 3 - 6 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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G. A549-ACE2 cells were treated with apratastat, DPC-333 or BB94 at indicated concentrations. Experiment was conducted and analyzed as in (D). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


H , I . A549 - ACE2 cells were treated for 1 h with indicated inhibitors ( 10 µM ) or DMSO before inoculation with GFP - encoding spike pseudoparticles ( VSV pseudotyped with SARS - CoV - 2 spike protein ( VSV - S ) ) ( H ) or as a control LCMV glycoprotein ( VSV - GP ) ( I ) . At 16 hpi , infection was analyzed by counting GFP - positive cells . Data are normalized to the number of infected cells in virus - only control wells without DMSO ( N = 3 - 5 ) . One - way ANOVA with Tukey ' s post - hoc multiple comparison test . Asterisks indicate significance compared to DMSO . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H, I. A549-ACE2 cells were treated for 1 h with indicated inhibitors (10 µM) or DMSO before inoculation with GFP-encoding spike pseudoparticles (VSV pseudotyped with SARS-CoV-2 spike protein (VSV-S)) (H) or as a control LCMV glycoprotein (VSV-GP) (I). At 16 hpi, infection was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without DMSO (N = 3-5). One-way ANOVA with Tukey's post-hoc multiple comparison test. Asterisks indicate significance compared to DMSO. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


A , RT - qPCR quantification of viral RNA ( SARS - CoV - 2 N gene relative to RPLP0 ) upon infection of A549 - ACE2 cells with indicated SARS - CoV - 2 strains at 24 hpi . Cells were pretreated with BB94 ( 10 µM ) or DMSO for 6 h . Data are normalized to DMSO ( N = 3 - 7 ) . A two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed . Data information : Data are represented as means ± 95 % CI from at least three ( A , biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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A, RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three (A, biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


B . RT - qPCR quantification of viral RNA ( SARS - CoV - 2 N gene relative to RPLP0 ) upon infection of A549 - ACE2 cells with indicated SARS - CoV - 2 strains at 24 hpi . Cells were pretreated with BB94 ( 10 µM ) , DPC - 333 ( 10 µM ) or DMSO for 6 h . Data are normalized to DMSO ( N = 3 - 7 ) . A two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed . Data information : Data are represented as means ± 95 % CI from at least three , B ) biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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B. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94 (10 µM), DPC-333 (10 µM) or DMSO for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three , B) biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


C . Normal Human Bronchial Epithelial cells ( NHBE ) from ten donors were infected with SARS - CoV - 2 . NHBE cells were pre - treated with camostat mesylate ( 10µM ) , BB94 ( 1 µM or 10 µM ) or DMSO for 6 hours . RNA was isolated at 24 hpi and levels of SARS - CoV - 2 N relative to RPLP0 from total cellular RNA was measured by RT - qPCR . Data are normalized to the DMSO control ( N = 5 - 10 ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least five / ten biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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C. Normal Human Bronchial Epithelial cells (NHBE) from ten donors were infected with SARS-CoV-2. NHBE cells were pre-treated with camostat mesylate (10µM), BB94 (1 µM or 10 µM) or DMSO for 6 hours. RNA was isolated at 24 hpi and levels of SARS-CoV-2 N relative to RPLP0 from total cellular RNA was measured by RT-qPCR. Data are normalized to the DMSO control (N = 5-10). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


D , NHBE cells from five donors were infected with GFP - expressing SARS - CoV - 2 . Infection of cells was monitored microscopically as green fluorescence through live - cell imaging at 72 hours post - infection ( hpi ) and is shown as the mean of the GFP - positive area relative to the whole area covered by cells in the same well . In ( D ) NHBE cells were pre - treated with the indicated drugs for 6 hours before infection Data are normalized to the DMSO control ( N = 5 ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least five / ten biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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D, NHBE cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 72 hours post-infection (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. In (D) NHBE cells were pre-treated with the indicated drugs for 6 hours before infection Data are normalized to the DMSO control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


E . NHBE cells from five donors were infected with GFP - expressing SARS - CoV - 2 . Infection of cells was monitored microscopically as green fluorescence through live - cell imaging at 72 hours post - infection ( hpi ) and is shown as the mean of the GFP - positive area relative to the whole area covered by cells in the same well . NHBE cells were were treated in ( E ) with the indicated drugs 4 hours after infection . Data are normalized to the DMSO control ( N = 5 ) . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least five / ten biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E. NHBE cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically as green fluorescence through live-cell imaging at 72 hours post-infection (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. NHBE cells were were treated in (E) with the indicated drugs 4 hours after infection. Data are normalized to the DMSO control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.


A . Western Blot ( WB ) analysis of A549 - ACE2 knock - out ( KO ) lines . Two different KO lines were generated using distinct CRISPR guide RNAs for both ADAM10 and ADAM17 . A non - targeting control ( NTC ) guide RNA served as control . Shown are two replicates for each cell line . Mature ( m ) and immature ( im ) forms of ADAM10 and ADAM17 are indicated . ACE2 levels remained unaffected . α - tubulin served as loading control . * : remaining , low amount of immature ADAM10 and ADAM17 . The double KO ( dKO ) was generated from guide 1 of each ADAM10 and ADAM17 . B . Quantification of the abundance of mature ADAM10 and ADAM17 protein forms from ( A ) . Data are normalized to loading control tubulin and to NTC ( N = 3 - 4 ) . The band of mADAM17 runs very close to three unspecific bands ( marked with arrowheads ) - one closely above and two closely below mADAM17 ( A ) . Thus , quantification of remaining mADAM17 in the ADAM10 KO includes also these three unspecific bands . Two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A. Western Blot (WB) analysis of A549-ACE2 knock-out (KO) lines. Two different KO lines were generated using distinct CRISPR guide RNAs for both ADAM10 and ADAM17. A non-targeting control (NTC) guide RNA served as control. Shown are two replicates for each cell line. Mature (m) and immature (im) forms of ADAM10 and ADAM17 are indicated. ACE2 levels remained unaffected. α-tubulin served as loading control. *: remaining, low amount of immature ADAM10 and ADAM17. The double KO (dKO) was generated from guide 1 of each ADAM10 and ADAM17. B. Quantification of the abundance of mature ADAM10 and ADAM17 protein forms from (A). Data are normalized to loading control tubulin and to NTC (N = 3-4). The band of mADAM17 runs very close to three unspecific bands (marked with arrowheads) - one closely above and two closely below mADAM17 (A). Thus, quantification of remaining mADAM17 in the ADAM10 KO includes also these three unspecific bands. Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


C . RT - qPCR quantification of viral RNA ( SARS - CoV - 2 N gene relative to RPLP0 ) upon infection of A549 - ACE2 NTC or KO cells with SARS - CoV - 2 . Cells were treated with BB94 ( 10 µM ) or DMSO for 6 h prior to infection and RNA was isolated at 24 hpi . Two - way ANOVA with Tukey ' s post - hoc multiple comparison test . Data are normalized to NTC DMSO ( N = 3 ) . Data information : Data are represented as means ± 95 % CI from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 NTC or KO cells with SARS-CoV-2. Cells were treated with BB94 (10 µM) or DMSO for 6 h prior to infection and RNA was isolated at 24 hpi. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Data are normalized to NTC DMSO (N = 3). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


D - G . UMAP of 63 , 103 cells from BAL fluid derived from infected individuals ( D ) Individual cell types are indicated in color . ( E ) Normalized counts of SARS - CoV - 2 reads , ADAM10 , ADAM17 , and TMPRSS2 gene expression levels . ( F ) Dot plot of ADAM10 , ADAM17 , and TMPRSS2 expression , grouped by disease severity . Expression levels are color - coded , the percentage of cells expressing the aforementioned genes is size coded . ( G ) Cells that are infected and additionally express ADAM17 ( ADAM17 + , red ) , ADAM10 ( ADAM10 + , blue ) , both ADAMs ( ADAM10 + ADAM17 + , purple ) , or TMPRSS2 ( TMPRSS2 + , orange ) are indicated .

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

D-G. UMAP of 63,103 cells from BAL fluid derived from infected individuals (D) Individual cell types are indicated in color. (E) Normalized counts of SARS-CoV-2 reads, ADAM10, ADAM17, and TMPRSS2 gene expression levels. (F) Dot plot of ADAM10, ADAM17, and TMPRSS2 expression, grouped by disease severity. Expression levels are color-coded, the percentage of cells expressing the aforementioned genes is size coded. (G) Cells that are infected and additionally express ADAM17 (ADAM17+, red), ADAM10 (ADAM10+, blue), both ADAMs (ADAM10+ ADAM17+, purple), or TMPRSS2 (TMPRSS2+, orange) are indicated.


B . Representative images of syncytia formation assay . Large syncytia were observed for NTC and ADAM17 KO cells , but were strongly reduced when ADAM10 was knocked - out , either alone or together with ADAM17 or upon inhibition with BB94 ( 10 µM ) . For both ADAMs , the cell lines obtained with gRNA sequence 1 were used . Images show GFP fluorescence and Hoechst staining . Scale bars , 50 µm . C . Quantification of images from fluorescence microscopy ( B ) . Left plot : the area of the fused cells in green ( GFP ) was quantified and normalized to the Hoechst signal ( blue ) of the entire image to account for differences in cell density . Right plot : The nuclei within syncytia were determined by calculating the ratio between the Hoechst signal within syncytia and the total Hoechst signal in the entire image . The data are normalized to NTC DMSO ( N > = 8 ) . Two - way ANOVA with Tukey ' s correction for multiple comparisons . All data are represented as means ± 95 % CI of at least 3 independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 .

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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of syncytia formation assay. Large syncytia were observed for NTC and ADAM17 KO cells, but were strongly reduced when ADAM10 was knocked-out, either alone or together with ADAM17 or upon inhibition with BB94 (10 µM). For both ADAMs, the cell lines obtained with gRNA sequence 1 were used. Images show GFP fluorescence and Hoechst staining. Scale bars, 50 µm. C. Quantification of images from fluorescence microscopy (B). Left plot: the area of the fused cells in green (GFP) was quantified and normalized to the Hoechst signal (blue) of the entire image to account for differences in cell density. Right plot: The nuclei within syncytia were determined by calculating the ratio between the Hoechst signal within syncytia and the total Hoechst signal in the entire image. The data are normalized to NTC DMSO (N >= 8). Two-way ANOVA with Tukey's correction for multiple comparisons. All data are represented as means ± 95% CI of at least 3 independent experiments. **p < 0.01, ***p < 0.001.


In vitro cleavage assay of SARS - CoV - 2 spike protein . Recombinant spike protein ( 1 µg ) was digested with ADAM10 , TMPRSS2 ( A ) for 24 hours at a 1 : 1 ratio and analyzed by Western Blot . Camostat mesylate or BB94 ( 10 µM ) were pre - incubated with TMPRSS2 or ADAM proteases , respectively , for 15 min before addition of spike protein . The spike protein and fragments were detected using an antibody against its C - terminal 6xHIS tag .

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

In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM10, TMPRSS2 (A) for 24 hours at a 1:1 ratio and analyzed by Western Blot. Camostat mesylate or BB94 (10 µM) were pre-incubated with TMPRSS2 or ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.


B . In vitro cleavage assay of SARS - CoV - 2 spike protein . Recombinant spike protein ( 1 µg ) was digested with ADAM17 ( B ) for 24 hours at a 1 : 1 ratio and analyzed by Western Blot . BB94 ( 10 µM ) were pre - incubated with ADAM proteases , respectively , for 15 min before addition of spike protein . The spike protein and fragments were detected using an antibody against its C - terminal 6xHIS tag .

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

B. In vitro cleavage assay of SARS-CoV-2 spike protein. Recombinant spike protein (1 µg) was digested with ADAM17 (B) for 24 hours at a 1:1 ratio and analyzed by Western Blot. BB94 (10 µM) were pre-incubated with ADAM proteases, respectively, for 15 min before addition of spike protein. The spike protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.


C . Representative images and quantification of a proximity ligation assay ( PLA ) between the SARS - CoV - 2 Spike and ADAM17 in A549 - ACE2 cells . Cells were infected with SARS - CoV - 2 and placed on ice ( 4 ° C ) . After 2 h , cells were shifted to 37 ° C for 15 min or left on ice . Images are presented as overlay between the Nuclei ( Hoechst , blue ) and the PLA ( pseudo - color : red ) channel . Scale bar , 20 µm . The number of PLA foci per cell was determined for the acquired images ( N = 28 - 30 ) . A two - sided independent Student ' s t - test with Benjamini - Hochberg FDR correction was performed . Data information : Data are represented as means ± 95 % CI of at least 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C. Representative images and quantification of a proximity ligation assay (PLA) between the SARS-CoV-2 Spike and ADAM17 in A549-ACE2 cells. Cells were infected with SARS-CoV-2 and placed on ice (4°C). After 2 h, cells were shifted to 37°C for 15 min or left on ice. Images are presented as overlay between the Nuclei (Hoechst, blue) and the PLA (pseudo-color: red) channel. Scale bar, 20 µm. The number of PLA foci per cell was determined for the acquired images (N = 28-30). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


D . A549 - ACE2 cells were treated for 48 h with BB94 ( 10 µM ) , DPC - 333 ( 5 µM ) or DMSO . PMA ( 25 ng / ml ) was added for 3 h before harvest to stimulate ACE2 shedding . Concentration of the soluble ACE2 fragment was determined in the conditioned medium by ELISA and is shown relative to the concentration in the DMSO control ( N = 6 ) . One - way ANOVA with Tukey ' s post - hoc multiple comparison test . Data information : Data are represented as means ± 95 % CI of at least 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

D. A549-ACE2 cells were treated for 48 h with BB94 (10 µM), DPC-333 (5 µM) or DMSO. PMA (25 ng/ml) was added for 3 h before harvest to stimulate ACE2 shedding. Concentration of the soluble ACE2 fragment was determined in the conditioned medium by ELISA and is shown relative to the concentration in the DMSO control (N = 6). One-way ANOVA with Tukey's post-hoc multiple comparison test. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


E . GFP - encoding spike pseudoparticles ( VSV pseudotyped with SARS - CoV - 2 spike protein ( VSV - S ) were incubated with indicated amounts of recombinant , soluble ACE2 ( sACE2 ) or bovine serum albumin ( BSA ) as a control prior to infection of A549 - ACE2 cells . At 16 hpi , infection was analyzed by counting GFP - positive cells . Data are normalized to the number of infected cells in virus - only control wells without sACE2 or BSA ( N = 3 ) . Two - sided independent Student ' s t test with Benjamini - Hochberg FDR correction . Data information : Data are represented as means ± 95 % CI of at least 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E. GFP-encoding spike pseudoparticles (VSV pseudotyped with SARS-CoV-2 spike protein (VSV-S) were incubated with indicated amounts of recombinant, soluble ACE2 (sACE2) or bovine serum albumin (BSA) as a control prior to infection of A549-ACE2 cells. At 16 hpi, infection was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without sACE2 or BSA (N = 3). Two-sided independent Student's t test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.


( a ) Expression of Ulk1 and Atg5 - 12 in the indicated erythroid cells .

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

(a) Expression of Ulk1 and Atg5-12 in the indicated erythroid cells.


( b - e ) Induction of conventional macroautophagy in WT and Ulk1gt / gt , but not in Atg5 − / − , embryonic erythroid cells . Ter119 + erythroid cells from WT , Atg5 − / − , and Ulk1gt / gt embryonic mice ( E18 . 5 ) were treated with 1 μM rapamycin ( b , c ) and 1 μM STS ( d , e ) , and then harvested at the indicated times . ( b , d ) Representative protein expression of LC3 and p62 measured by western blot . Actin was a loading control . Uncropped images are shown in Supplementary Fig . 10 . ( c , e ) Semi - quantitative analysis of LC3 - II / LC3 - I and p62 protein expression ( n = 3 , mean ± s . d . ) . Asterisks indicate a significant difference at P0 . 05 ( analysis of variance ( ANOVA ) ) . # P0 . 05 versus value of WT 24 h ( ANOVA ) . ' NS ' indicates not significant ( ANOVA ) .

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

(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).


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

(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).


( a ) Induction of alternative macroautophagy in WT and Atg5 − / − , but not Ulk1gt / gt and DKO , erythroid cells . Erythroblasts and reticulocytes from the liver of embryonic mice ( E18 . 5 ) were incubated with or without STS ( 1 μM ) for 24 h , followed by staining with anti - Lamp2 ( red ) , anti - Ter119 ( green ) and DAPI ( blue ) . DAPI - positive erythroblasts and DAPI - negative reticulocytes are shown . Lamp2 image and merged image ( DAPI , Ter119 and Lamp2 ) are shown . Scale bar , 1 μm . Large dots for Lamp2 are observed in STS - treated WT and Atg5 − / − cells , but not STS - treated Ulk1gt / gt or DKO cells .

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

(a) Induction of alternative macroautophagy in WT and Atg5−/−, but not Ulk1gt/gt and DKO, erythroid cells. Erythroblasts and reticulocytes from the liver of embryonic mice (E18.5) were incubated with or without STS (1 μM) for 24 h, followed by staining with anti-Lamp2 (red), anti-Ter119 (green) and DAPI (blue). DAPI-positive erythroblasts and DAPI-negative reticulocytes are shown. Lamp2 image and merged image (DAPI, Ter119 and Lamp2) are shown. Scale bar, 1 μm. Large dots for Lamp2 are observed in STS-treated WT and Atg5−/− cells, but not STS-treated Ulk1gt/gt or DKO cells.


( b ) Representative electron micrographs of EC incubated with or without STS . Erythroid cells were harvested from the liver of embryonic mice ( E18 . 5 ) , incubated with or without STS ( 1 μM ) for 24 h , and analysed by electron microscopy ( EM ) . Scale bar , 1 μm . Insets of WT and Atg5 − / − cells show mitophagy ( Scale bar , 0 . 5 μm ) . Inset of a Ulk1gt / gt cell showing mitochondria that have not been engulfed ( Scale bar , 0 . 5 μm ) . Arrows point to non - engulfed mitochondria and the arrowheads indicate engulfed mitochondria . ( c - e ) Quantitative analysis of mitophagy after STS treatment , calculated from EM photos . Population of reticulocytes with mitophagy ( c ) , number of mitochondria per reticulocytes ( d ) and population of reticulocytes showing macroautophagy ( e ) were calculated ( n > 35 cells per mouse ) . The data are shown as mean ± s . d . ( n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

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

(b) Representative electron micrographs of EC incubated with or without STS. Erythroid cells were harvested from the liver of embryonic mice (E18.5), incubated with or without STS (1 μM) for 24 h, and analysed by electron microscopy (EM). Scale bar, 1 μm. Insets of WT and Atg5−/− cells show mitophagy (Scale bar, 0.5 μm). Inset of a Ulk1gt/gt cell showing mitochondria that have not been engulfed (Scale bar, 0.5 μm). Arrows point to non-engulfed mitochondria and the arrowheads indicate engulfed mitochondria. (c-e) Quantitative analysis of mitophagy after STS treatment, calculated from EM photos. Population of reticulocytes with mitophagy (c), number of mitochondria per reticulocytes (d) and population of reticulocytes showing macroautophagy (e) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


( a ) Three - colour flow cytometry of erythroid cells from WT embryonic liver ( E14 . 5 ) stained with anti - Ter119 , anti - CD71 and Mitotracker Deep Red . ( Left upper panel ) Ter119 versus CD71 fluorescence . ( Other panels ) FSC versus Mitotracker fluorescence of the R1 ( right upper panel ) , R2 ( left lower panel ) and R3 ( right lower panel ) fractions . ( b , c ) Percentage of cells with a low number of mitochondria ( mitochondrialow cells ) among CD71 − Ter119 + EC . Erythroid cells from the liver ( E14 . 5 and E18 . 5 ) and peripheral blood ( E18 . 5 ) of indicated mice were stained with anti - Ter119 , anti - CD71 and Mitotracker Deep 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O

(a) Three-colour flow cytometry of erythroid cells from WT embryonic liver (E14.5) stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red. (Left upper panel) Ter119 versus CD71 fluorescence. (Other panels) FSC versus Mitotracker fluorescence of the R1 (right upper panel), R2 (left lower panel) and R3 (right lower panel) fractions. (b,c) Percentage of cells with a low number of mitochondria (mitochondrialow cells) among CD71−Ter119+ EC. Erythroid cells from the liver (E14.5 and E18.5) and peripheral blood (E18.5) of indicated mice were stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red.


( b ) Representative histograms of mitochondrial content in CD71 − Ter119 + cells . Numbers indicate the population of mitochondrialow cells .

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

(b) Representative histograms of mitochondrial content in CD71−Ter119+ cells. Numbers indicate the population of mitochondrialow cells.


( c ) The percentage of CD71 − Ter119 + cells without mitochondria was determined by gating the mitochondrialow fraction , as shown in ( b ) ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

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(c) The percentage of CD71−Ter119+ cells without mitochondria was determined by gating the mitochondrialow fraction, as shown in (b) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


( d ) Percent mitochondrialow cells in the CD71 − Ter119 + EC of Atg7F / FCre embryo . Erythroid cells from the liver and peripheral blood of WT and Atg7F / FCre embryo ( E18 . 5 ) were stained with anti - Ter119 , anti - CD71 , and Mitotracker Deep Red ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) .

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

(d) Percent mitochondrialow cells in the CD71−Ter119+ EC of Atg7F/FCre embryo. Erythroid cells from the liver and peripheral blood of WT and Atg7F/FCre embryo (E18.5) were stained with anti-Ter119, anti-CD71, and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test).


( e ) Percentage of mitochondrialow cells in Syto16low cells . Liver erythroid cells ( E18 . 5 ) were stained with Syto16 ( DNA ) and Mitotracker Deep Red . Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig . 5 , the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( ANOVA ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

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

(e) Percentage of mitochondrialow cells in Syto16low cells. Liver erythroid cells (E18.5) were stained with Syto16 (DNA) and Mitotracker Deep Red. Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig. 5, the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction (mean±s.d., n=6). *P0.05 versus value of WT (ANOVA); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


( f ) Immunofluorescent analysis of mitochondrial and lysosomal proteins . Liver Ter119 + cells ( E18 . 5 ) of the indicated mice were stained with anti - Tom20 ( mitochondrial marker ) and anti - Lamp2 ( lysosomal marker ) antibodies , and observed by fluorescent microscopy . Scale bar , 2 μm . Green , red , white , and blue indicate Tom20 , Lamp2 , Ter119 and DAPI ( DNA ) , respectively . Mitochondrial and lysosomal markers have separate distributions in Ulk1gt / gt cells and DKO cells .

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

(f) Immunofluorescent analysis of mitochondrial and lysosomal proteins. Liver Ter119+ cells (E18.5) of the indicated mice were stained with anti-Tom20 (mitochondrial marker) and anti-Lamp2 (lysosomal marker) antibodies, and observed by fluorescent microscopy. Scale bar, 2 μm. Green, red, white, and blue indicate Tom20, Lamp2, Ter119 and DAPI (DNA), respectively. Mitochondrial and lysosomal markers have separate distributions in Ulk1gt/gt cells and DKO cells.


( a - f ) Representative EM of WT ( a ) , ATG5 − / − ( b ) , Ulk1gt / gt ( c , d ) and DKO ( e , f ) reticulocytes from embryonic liver ( E14 . 5 ) . ( a , b ) Mitophagy was detected in WT and ATG5 − / − cells ( scale bar , 1 μm ) . Insets indicate mitophagy ( scale bar , 0 . 2 μm ) . ( c , e ) In Ulk1gt / gt and DKO cells , numerous mitochondria were in contact with membrane structures ( scale bar , 1 μm ) . Enlarged images are shown on the right side ( scale bar , 0 . 2 μm ) . ( d , f ) Some Ulk1gt / gt and DKO cells showed plasma membrane blebs containing mitochondria ( scale bar , 1 μm ) . ( g , h ) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18 . 5 . Population of reticulocytes with mitophagy ( g ) , and number of mitochondria per reticulocytes ( h ) were calculated ( n > 35 cells per mouse ) . The data are shown as mean ± s . d . ( n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

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

(a-f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.5). (a,b) Mitophagy was detected in WT and ATG5−/− cells (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (c,e) In Ulk1gt/gt and DKO cells, numerous mitochondria were in contact with membrane structures (scale bar, 1 μm). Enlarged images are shown on the right side (scale bar, 0.2 μm). (d,f) Some Ulk1gt/gt and DKO cells showed plasma membrane blebs containing mitochondria (scale bar, 1 μm). (g,h) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18.5. Population of reticulocytes with mitophagy (g), and number of mitochondria per reticulocytes (h) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


( a ) Erythroblasts ( EB ; mitochondriahigh Syto16high ) , reticulocytes ( RC ; mitochondriahigh Syto16low ) and EC ( EC ; Ter119 + PBC ) were collected from the indicated embryos ( E18 . 5 ) , and the expression of each protein was examined by western blot . The asterisk indicates a nonspecific band . Uncropped images are shown in Supplementary Figs 11 and 12 .

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

(a) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated embryos (E18.5), and the expression of each protein was examined by western blot. The asterisk indicates a nonspecific band. Uncropped images are shown in Supplementary Figs 11 and 12.


( b ) Semi - quantitative analysis of the expression level of each protein and the LC3 - II / LC3 - I ratio ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT EC ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − EC ( ANOVA ) ; ' NS ' indicates not significant versus value of WT EC ( ANOVA ) .

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

(b) Semi-quantitative analysis of the expression level of each protein and the LC3-II/LC3-I ratio (mean±s.d., n=3). *P0.05 versus value of WT EC (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− EC (ANOVA); 'NS' indicates not significant versus value of WT EC (ANOVA).


( c ) Modification of Ulk1 and Atg13 during erythrocyte maturation . Cell lysates were treated with λ - phosphatase ( 400 U ) for 45 min at 30 ° C , and band shift was examined by anti - Ulk1 and anti - Atg13 antibodies . Asterisk indicates non - specific band . Uncropped images are shown in Supplementary Fig . 12 .

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(c) Modification of Ulk1 and Atg13 during erythrocyte maturation. Cell lysates were treated with λ-phosphatase (400 U) for 45 min at 30 °C, and band shift was examined by anti-Ulk1 and anti-Atg13 antibodies. Asterisk indicates non-specific band. Uncropped images are shown in Supplementary Fig. 12.


( a ) Morphology of cultured erythroid cells on day 0 , 1 and 3 of differentiation . Erythroblasts were purified from embryonic liver ( E14 . 5 ) and incubated for the indicated times in erythrocyte differentiation medium . The cells were stained with a Diff - quick kit . ( b ) Percentage of cells without nuclei ( Syto16low cells ) on the indicated days of differentiation ( mean ± s . d . , n = 6 ) . Loss of nuclei occurred normally in all mice .

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

(a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation. Erythroblasts were purified from embryonic liver (E14.5) and incubated for the indicated times in erythrocyte differentiation medium. The cells were stained with a Diff-quick kit. (b) Percentage of cells without nuclei (Syto16low cells) on the indicated days of differentiation (mean±s.d., n=6). Loss of nuclei occurred normally in all mice.


( c , d ) Percentage of Syto16low cells without mitochondria . Differentiated cells were stained with Syto16 and Mitotracker Deep Red . Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in ( c ) . Numbers indicate the population of mitochondrialow cells . Representative dot plots are also available in Supplementary Fig . 13 . ( d ) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in ( c ) ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

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

(c,d) Percentage of Syto16low cells without mitochondria. Differentiated cells were stained with Syto16 and Mitotracker Deep Red. Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in (c). Numbers indicate the population of mitochondrialow cells. Representative dot plots are also available in Supplementary Fig. 13. (d) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in (c) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


( e - h ) Impact of various drugs on mitochondrial clearance during in vitro differentiation . Differentiated cells from WT embryos were incubated with or without wortmannin ( e ) , 3 - methyladenine ( 3 - MA ) ( f ) , rapamycin ( g ) or compound C ( h ) at the indicated concentrations and time periods . Percentage of mitochondrialow cells / Syto16low cells was measured by flow cytometry ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of no drug ( ANOVA ) .

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(e-h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).


( i ) Impact of various drugs on mitochondrial clearance during in vitro differentiation . with cells from Atg5 − / − embryos exposed to wortmannin or 3 - MA ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of no drug ( ANOVA ) .

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(i)Impact of various drugs on mitochondrial clearance during in vitro differentiation.with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).


( a ) Number of EC and haemoglobin content in the blood of the indicated mice ( 10 - 20 weeks old ; mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) .

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(a) Number of EC and haemoglobin content in the blood of the indicated mice (10-20 weeks old; mean±s.d., n=6). *P0.05 versus value of WT (Student's t-test).


( b ) Analysis of early erythrocyte development . Bone marrow cells were harvested from the indicated mice ( 10 - 20 weeks old ) and stained with anti - cKit and anti - Sca1 antibodies . The population of each cell type was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) .

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

(b) Analysis of early erythrocyte development. Bone marrow cells were harvested from the indicated mice (10-20 weeks old) and stained with anti-cKit and anti-Sca1 antibodies. The population of each cell type was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)).


( c ) Percentage of mitochondria - containing Ter119 + blood cells . Cells in peripheral blood from the indicated mice ( 10 - 20 weeks old ) were stained with anti - Ter119 antibody together with Mitotracker Deep Red . The population of mitochondriahigh cells among Ter119 - positive cells was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( ANOVA ) .

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(c) Percentage of mitochondria-containing Ter119+ blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with anti-Ter119 antibody together with Mitotracker Deep Red. The population of mitochondriahigh cells among Ter119-positive cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).


( d ) Percentage of mitochondria - containing Syto16low blood cells . Cells in peripheral blood from the indicated mice ( 10 - 20 weeks old ) were stained with Syto16 and Mitotracker Deep Red . The population of mitochondriahigh cells among Syto16 - negative cells was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( ANOVA ) .

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

(d) Percentage of mitochondria-containing Syto16low blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with Syto16 and Mitotracker Deep Red. The population of mitochondriahigh cells among Syto16-negative cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).


( e ) Representative electron micrographs of bone marrow reticulocytes in 10 week - old WT and Ulk1gt / gt mice ( scale bar , 1 μm ) . Insets indicate mitophagy ( scale bar , 0 . 2 μm ) .

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(e) Representative electron micrographs of bone marrow reticulocytes in 10 week-old WT and Ulk1gt/gt mice (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm).


( f , g ) Erythroblasts ( EB ; mitochondriahigh Syto16high ) , reticulocytes ( RC ; mitochondriahigh Syto16low ) and EC ( EC ; Ter119 + PBC ) were collected from the indicated mice ( 10 - 20 weeks old ) , and the expression of each protein was examined by western blot . Uncropped images are shown in Supplementary Fig . 14 . ( g ) Semi - quantitative analysis of the expression level of each protein ( mean ± s . d . , n = 3 ) . ' NS ' indicates not significant versus value of WT EC ( ANOVA ) .

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(f,g) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated mice (10-20 weeks old), and the expression of each protein was examined by western blot. Uncropped images are shown in Supplementary Fig. 14. (g) Semi-quantitative analysis of the expression level of each protein (mean±s.d., n=3). 'NS' indicates not significant versus value of WT EC (ANOVA).


( h , i ) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis . ( h ) Representative images of erythrocyte in PHZ - treated WT and Ulk1gt / gt mice ( 14 weeks old ) . Blood cells were stained with anti - Ter119 ( erythroid cell marker ) and anti - Tom20 ( mitochondrial marker ) antibodies and observed by fluorescence microscopy . Green and red indicate Ter119 and Tom20 , respectively . Scale bar , 10 μm . ( i ) Percentage of mitochondrialow cells among Syto16low cells . Erythroid cells were stained with Syto16 ( DNA ) and Mitotracker Deep Red ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . Mitochondrial retention was observed in Ulk1gt / gt mice .

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(h,i) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis. (h) Representative images of erythrocyte in PHZ-treated WT and Ulk1gt/gt mice (14 weeks old). Blood cells were stained with anti-Ter119 (erythroid cell marker) and anti-Tom20 (mitochondrial marker) antibodies and observed by fluorescence microscopy. Green and red indicate Ter119 and Tom20, respectively. Scale bar, 10 μm. (i) Percentage of mitochondrialow cells among Syto16low cells. Erythroid cells were stained with Syto16 (DNA) and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test). Mitochondrial retention was observed in Ulk1gt/gt mice.