( B ) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression . mRNA expression levels are represented relatively to control cells ( mean ± SD , n = 3 , Student ' s t - test ) .

(B) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).


( C ) Soft agar colony formation assay following ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - test ) .

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(C) Soft agar colony formation assay following ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( D ) FACS analyses of p75 cell surface expression upon ZEB1 overexpression . Bar chart representing the mean percentage of p75 - high , int , low and negative cells from 2 independent experiments ( Fisher ' s exact test ) .

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(D) FACS analyses of p75 cell surface expression upon ZEB1 overexpression. Bar chart representing the mean percentage of p75-high, int, low and negative cells from 2 independent experiments (Fisher's exact test).


( E ) Clonogenic assay + / - PLX4032 ( 100 nM ) treatment for 10 days . The graph represents the mean number of colonies in 3 independent experiments ( Student ' s t - test ) .

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(E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student's t-test).


( A ) A375 cells were infected with retroviruses expressing a control or ZEB1 - shRNA . Western blot analyses of ZEB1 and p75 upon ZEB1 knock - down . GAPDH was used as a loading control . High and low exposures ( exp ) for p75 are shown .

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(A) A375 cells were infected with retroviruses expressing a control or ZEB1-shRNA. Western blot analyses of ZEB1 and p75 upon ZEB1 knock-down. GAPDH was used as a loading control. High and low exposures (exp) for p75 are shown.


( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 and JARID1B upon ZEB1 knock - down . mRNA expression levels are represented relatively to shRNA control cells ( mean ± SD , n = 3 , Student ' s t - test ) .

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(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 knock-down. mRNA expression levels are represented relatively to shRNA control cells (mean ± SD, n=3, Student's t-test).


( C ) FACS analyses of p75 expression upon ZEB1 knock - down . Bar chart representing the mean percentage of p75 - high , int and low cells from 2 independent experiments ( Fisher ' s exact test ) .

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(C) FACS analyses of p75 expression upon ZEB1 knock-down. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's exact test).


( D ) Soft agar colony formation assay upon ZEB1 knock - down . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - test ) .

(D) Soft agar colony formation assay upon ZEB1 knock-down. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( E ) 2 x 106 Control or ZEB1 - shRNA A375 cells were injected subcutaneously into nude mice . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) .

(E) 2 x 106 Control or ZEB1-shRNA A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( F ) A375 cells were infected with an IPTG - inducible ZEB1 - shRNA . Left panel : Western blot analyses of ZEB1 expression ± IPTG ( 100µM ) treatment for 6 days . Actin was used as a loading control . Right panel : 2 x 106 IPTG - inducible shRNA - Control or shRNA - ZEB1A375 cells were injected subcutaneously into nude mice . When the tumor reached 5 mm in diameter , ZEB1 expression was silenced by adding IPTG ( 10 mM ) into the drinking water for 20 days . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) .

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(F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( G ) SKMEL5 cells expressing an IPTG - inducible ZEB1 - shRNA were treated with IPTG ( 100µM ) for 10 days ( + IPTG ) , then IPTG was removed ( - IPTG ) and ZEB1 , MITF and p75 expression levels were analyzed by Western blot and / or quantitative PCR analyses . GAPDH was used as a protein loading control , mRNA expression levels are represented relatively to untreated cells , in which the levels were fixed at 1 ( mean ± SD , n = 2 ) .

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(G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).


( A ) Western blot analyses of ZEB1 and P - ERK in shRNA - control or shRNA - ZEB1 - expressing A375 cells + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control .

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(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.


( B ) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 ( 150 nM ) . Scale bar = 100 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - test ) . ( C ) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA - ZEB1 - expressing cells ( n = 3 , Student ' s t - test ) .

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(B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student's t-test).


( D ) RPMI7951 , A375 - R and SKMEL5 - R were infected with a retrovirus encoding a constitutive shRNA - ZEB1 . Short - term cultures of GOKA and ESP cells , derived from vemurafenib - resistant patients , were infected with a lentivirus encoding an IPTG - inducible shRNA - ZEB1 . Western blot analyses showing efficient ZEB1 knock - down in the different models , ± IPTG ( 200µM ) , ± PLX4032 ( 3µM ) as indicated . Induction of cell death was assessed by PARP cleavage . GAPDH or actin were used as loading control .

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

(D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control.


( E ) Clonogenic assays in the presence of 3 µM PLX4032 , and with or without IPTG ( 200 µM ) as indicated . The graphs represent the mean number of colonies in 3 independent experiments ( Student ' s t - test ) .

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(E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student's t-test).


( F ) Western blot analyses of ZEB1 , p75 and quantitative PCR analyses of MITF in shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant patient - derived short - term culture cells . Actin was used as a loading control . mRNA expression levels are represented relatively to control cells ( mean ± SD , n = 3 , Student ' s t - test ) .

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(F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).


( G ) 2 , 5 x 106 shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant cells were injected subcutaneously in nude mice . When the tumor reached 5 mm in diameter , ZEB1 expression was silenced by providing mice with IPTG ( 10 mM ) in their drinking water and orally administering vemurafenib ( 50 mg / kg ) daily for 4 weeks . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) .

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(G) 2,5 x 106 shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( H ) Upper part : Western blot analyses of ZEB1 in shRNA - Control ( 1 to 4 ) or shRNA - ZEB1 ( 5 to 8 ) ESPxenografttumors , showing efficient ZEB1 knock - down directly in the tumors , after IPTG ± PLX4032 treatment in vivo . Lower part : Representative pictures of ZEB1 immunostaining in shRNA - Control or shRNA - ZEB1tumors , after IPTG treatment in vivo . Scale bar = 40µm .

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(H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESPxenografttumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1tumors, after IPTG treatment in vivo. Scale bar = 40µm.


( A - C ) Telophase GSCs marked by H3S10p ( gray ) expressing old ( green ) and new ( red ) histone H3 show non - overlapping patterns ( A ) . Telophase GSCs expressing old ( green ) and new ( red ) histone H4 show moderate levels of overlap ( B ) . GSCs expressing old ( green ) and new ( red ) histone H2A show high levels of overlap ( C ) . ( D ) A control where the old and new fluorescent tags have been switched shows the non - overlapping patterns of old ( red ) versus new ( green ) H3Rev histone in mitotic telophase GSCs marked by H3S10p ( gray ) . ( E ) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division , where a value of 0 is equal inheritance at exactly a 1 : 1 ratio . Data gathered over 9 separate experiments for H3Rev GSCs , H2A GSCs , and H4 GSCs . Data gathered over 16 separate experiments for H3 GSCs . Values are mean + 95 % CI . ( F - H ) Telophase CBs marked by H3S10p ( gray ) expressing old ( green ) and new ( red ) histone H3 also show non - overlapping patterns . ( F ) Telophase CBs expressing old ( green ) and new ( red ) histone H4 ( G ) and H2A ( H ) , which depict more overlap like GSCs . ( I ) A control with switched old and new fluorescent tags shows the non - overlapping old old ( red ) versus new ( green ) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p ( gray ) .

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(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C). (D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray). (E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI. (F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs. (I) A control with switched old and new fluorescent tags shows the non-overlapping old old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray).


( B - C ) Non - overlapping patterns of old ( green ) versus new ( red ) histone H3 ( B ) and overlapping patterns for H2A ( C ) in mitotic prometaphase bam mutant cells marked by anti - H3S10p ( gray ) , similar to WT GSCs . Scale bars : 2μm .

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(B-C) Non-overlapping patterns of old (green) versus new (red) histone H3 (B) and overlapping patterns for H2A (C) in mitotic prometaphase bam mutant cells marked by anti-H3S10p (gray), similar to WT GSCs. Scale bars: 2μm.


( F - H ) Examples showing old ( green ) versus new ( red ) H3 distribution patterns and percentage of signal strength at a single genomic locus labeled with fluorescent probes ( gray ) for dad gene ( F ) , bgcn gene ( G ) , and ss gene ( H ) in bam mutant GSC - like cells at prometaphase marked by anti - H3S10p ( blue ) . Scale bars : 1μm . Zoomed - in images of each probe are displayed to the right of each figure panel . Scale bars : 0 . 1μm .

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(F-H) Examples showing old (green) versus new (red) H3 distribution patterns and percentage of signal strength at a single genomic locus labeled with fluorescent probes (gray) for dad gene (F), bgcn gene (G), and ss gene (H) in bam mutant GSC-like cells at prometaphase marked by anti-H3S10p (blue). Scale bars: 1μm. Zoomed-in images of each probe are displayed to the right of each figure panel. Scale bars: 0.1μm.


( A - B ) Old ( red ) versus new ( green ) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes ( cyan ) for dad ( A ) and bam ( B ) in WT telophase female GSCs , marked by anti - H3S10p ( gray ) . ( C ) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs . Data gathered over 8 separate experiments . D

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Old (red) versus new (green) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes (cyan) for dad (A) and bam (B) in WT telophase female GSCs, marked by anti-H3S10p (gray). (C) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs. Data gathered over 8 separate experiments. D


A . Labeling of non - permeabilized ( top ) and permeabilized ( bottom ) cells demonstrate that surface labeling does not label internal proteins . GluA2 labeling ( green ) is visible both on the surface and internally , while DARPP32 labeling ( red ) is only visible internally .

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A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally.


B . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no treatment or 20 min 2APB and dantrolene . Bottom . Quantified expression of GluA2 under both conditions , n = 9 replicates of 10 cells each , Student ' s t test .

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B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.


C . Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co - cultures with either no treatment , 30 min DHPG , or 2APB and dantrolene pretreatment followed by DHPG . D . Quantified expression of GluA1 under each condition , n = 9 replicates of 10 cells , p < 0 . 001 , ANOVA . E . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures under the same conditions as in C . F . Quantified expression of GluA2 under each condition , n = 12 replicates of 10 cells each , p < 0 . 001 , ANOVA . G . Representative images of surface myc - GluA2 , internal myc - GluA2 , and DARPP32 expression in striatal neurons from co - cultures under the same conditions as in C . H . Quantified expression of surface myc : total myc under each condition , n = 4 replicates of 10 cells each , p < 0 . 05 , ANOVA . All error bars represent s . e . m . Abbr . A1 = GluA1 , A2 = GluA2 , D = DARPP32 , s - myc = surface myc , i - myc = internal myc , m = merge .

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

C. Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.


A . Left . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) or for 30 min with DHPG and pretreated with Xestospungin C and S107 ( XeC / S107 + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 or 20 cells each , p < 0 . 0001 , Student ' s t test .

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

A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test.


B . Left . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) , or for 30 min with DHPG and pretreated with S107 ( S107 + DHPG ) , or for 30 min with DHPG and pretreated with Xestospungin C ( XeC + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 cells each , ANOVA .

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B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA.


C . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures under control or 10 min Thapsigargin ( TG ) treatment . Bottom . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 cells each , Student ' s t test . D . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with treatment with either no DHPG or with DHPG for 30 min , after 15 min of Thapsigargin . Bottom . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 cells each , Student ' s t test . E . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min , after 5 min of BAPTA . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 cells each , p < 0 . 001 , Student ' s t test . F . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA - AM . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 cells each , Student ' s t test . All error bars represent s . e . m .

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

C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


A . Top . Representative image of a radiograph of mature and immature bands of GluA2 after no treatment , DHPG , or 2APB / dan followed by DHPG and incubated with Endo H . Bottom . Quantified graph of the ratio of the mature , endo H insensitive GluA2 band to the total GluA2 for each condition . For DHPG , n = 16 ; for DHPG + 2APB / dan , n = 11 , p < 0 . 05 , ANOVA .

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

A. Top. Representative image of a radiograph of mature and immature bands of GluA2 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA2 band to the total GluA2 for each condition. For DHPG, n = 16; for DHPG + 2APB/dan, n = 11, p < 0.05, ANOVA.


B . Top . Representative image of a radiograph of mature and immature bands of GluA1 after no treatment , DHPG , or 2APB / dan followed by DHPG and incubated with Endo H . Bottom . Quantified graph of the ratio of the mature , endo H insensitive GluA1 band to the total GluA1 for each condition , For DHPG , n = 4 ; for DHPG + 2APB / dan , n = 4 , ANOVA .

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

B. Top. Representative image of a radiograph of mature and immature bands of GluA1 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA1 band to the total GluA1 for each condition, For DHPG, n = 4; for DHPG + 2APB/dan, n = 4, ANOVA.


C . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment . D . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 cells each , Student ' s t test . All error bars represent s . e . m .

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-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

C. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment. D. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


A Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment , 10 min DHPG , and 2APB / dantrolene followed by 10 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions . For 10 min DHPG , n = 8 ; for 10 min DHPG + 2APB / dan , n = 6 , p < 0 . 05 , ANOVA .

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

A Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and 2APB/dantrolene followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 10 min DHPG, n = 8; for 10 min DHPG + 2APB/dan, n = 6, p < 0.05, ANOVA.


B . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment , 10 min DHPG , and and cycloheximide followed by 10 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions , For 10 min DHPG , n = 8 ; for 10 min DHPG + CHX , n = 5 , p < 0 . 05 , ANOVA .

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-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 B-CONTROLLED_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

B. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and and cycloheximide followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions, For 10 min DHPG, n = 8; for 10 min DHPG + CHX, n = 5, p < 0.05, ANOVA.


C . Top . Representative immunoblots from GluA2 co - IPs probed for Sec23 and GluA2 after no treatment , 30 min DHPG , and 2APB / dantrolene followed by 30 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 17 ; For 30 min DHPG + 2APB / dan , n = 14 , p < 0 . 05 , ANOVA .

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

C. Top. Representative immunoblots from GluA2 co-IPs probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and 2APB/dantrolene followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 17; For 30 min DHPG + 2APB/dan, n = 14, p < 0.05, ANOVA.


D . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment , 30 min DHPG , and cycloheximide followed by 30 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 12 ; For 30 min DHPG + CHX , n = 11 p < 0 . 01 , ANOVA .

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 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 B-CONTROLLED_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

D. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and cycloheximide followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 12; For 30 min DHPG + CHX, n = 11 p < 0.01, ANOVA.


E . Top . Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG . Bottom . Quantification of Sec23 after subtracting the non - specific signal , n = 3 , p < 0 . 01 , Student ' s t test .

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

E. Top. Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG. Bottom. Quantification of Sec23 after subtracting the non-specific signal, n = 3, p < 0.01, Student's t test.


F . Representative immunoblot of a control coIP that demonstrates the specificity of the GluA2 coIP . All error bars represent s . e . m .

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F. Representative immunoblot of a control coIP that demonstrates the specificity of the GluA2 coIP. All error bars represent s.e.m.


A . Representative immunoblots from GluA2 coIP probed for Sec23 , Sec13 , and GluA2 after no DHPG , 10 , 30 , and 60 minutes of treatment with DHPG . B . Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A . For Sec23 , 10 min DHPG , n = 16 ; 30 min DHPG , n = 15 ; 60 min DHPG , n = 7 , p < 0 . 0001 , ANOVA . For Sec13 , 10 min DHPG , n = 7 ; 30 min DHPG , n = 14 ; 60 min DHPG , n = 6 , p < 0 . 01 , ANOVA .

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 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-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 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 B-CONTROLLED_VAR 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

A. Representative immunoblots from GluA2 coIP probed for Sec23, Sec13, and GluA2 after no DHPG, 10, 30, and 60 minutes of treatment with DHPG. B. Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A. For Sec23, 10 min DHPG, n = 16; 30 min DHPG, n = 15; 60 min DHPG, n = 7, p < 0.0001, ANOVA. For Sec13, 10 min DHPG, n = 7; 30 min DHPG, n = 14; 60 min DHPG, n = 6, p < 0.01, ANOVA.


C . Representative images of total expression levels of Sec23 ( top ) and Sec13 ( bottom ) . Expression levels do not change in the presence of DHPG . All error bars represent s . e . m .

O O O O 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

C. Representative images of total expression levels of Sec23 (top) and Sec13 (bottom). Expression levels do not change in the presence of DHPG. All error bars represent s.e.m.


A . Individual traces of mEPSCs of both control and naspm treated neurons after TTX , TTX + DHPG , and TTX + DHPG + 2APB / dan treatment .

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

A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.


B . Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A . Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey .

O O 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 B-CONTROLLED_VAR O O O O O O

B. Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A. Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey.


C . Average amplitude of mEPSCs ( pA ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 17 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 005 , ANOVA . D . Average frequency of mEPSC ( Hz ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 12 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 0001 , ANOVA .

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

C. Average amplitude of mEPSCs (pA) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 17; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.005, ANOVA. D. Average frequency of mEPSC (Hz) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 12; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.0001, ANOVA.


E . Average amplitude of mEPSCs ( pA ) for only naspm treated neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . F . Average frequency of mEPSC ( Hz ) for only naspm treated neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene .

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

E. Average amplitude of mEPSCs (pA) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. F. Average frequency of mEPSC (Hz) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene.


G . Average rise time of mEPSC ( ms ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . H . Average decay time of mEPSC ( ms ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . All error bars represent s . e . m .

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

G. Average rise time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. H. Average decay time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. All error bars represent s.e.m.


( a , b ) Dual positive cells are more prevalent in epithelial organs and cells . ( a ) Proportion of ACE2 + TMPRSS2 + cells ( y axis ) per dataset ( dots ) from 21 tissues and organs ( rows ) .

O O O O O O O O O O O O O O O O O O O O O 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,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows).


( e - i ) In situ validation of dual positive cells in the lung , airways , and submucosal gland . ( e - g ) PLISH and immunostaining in human adult lung alveoli for ( e ) ACE2 ( white ) , TMPRSS2 ( green ) and CTSL ( red ) , ( f ) ACE2 ( white ) , TMPRSS2 ( green ) and HTII - 280 ( red ) , ( g ) ACE2 ( red ) and Pro - SFTPC ( green ) . ( h ) PLISH and immunostaining in human large airway submucosal glands . ACE2 ( red ) , ACTA2 ( green ) and DAPI ( blue ) . ( i ) PLISH in human adult large airway . ACE2 ( green ) and DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).


( a , c ) Single - cell ATAC - seq of lung samples from primary carina ( 1C ) and subpleural parenchyme ( RPL ) ( n = 1 patient , k = 3 samples , 3 , 366 cells from 1C , 8 , 340 cells from RPL ) . Uniform Manifold Approximation and Projection ( UMAP ) embedding of scATAC - seq profiles ( dots ) colored by ( a , left ) cell types , ( a , right ) cells with at least 1 fragment ( indicating accessibility , open chromatin ) mapping to the ACE2 gene locus ( defined as − 2kb upstream the Transcription Start Site to Transcription End Site ) , or by sample

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location


( g ) Low expression in pediatric samples . Mean expression level ( log CPM , y axis ) of ACE2 ( blue ) , TMPRSS2 ( orange ) , and CTSL ( green ) across age bins ( x axis ) in AT2 ( left ) and ciliated ( right ) cells . Pediatric samples : 0 - 10 years . Samples from past or current smokers were removed from this plot to avoid smoking confounders . Error bars are omitted due to y - axis limitations . They are typically 10 - fold the mean value ( Supplementary Table 5 ) . Multiciliated and AT2 cells are shown as these cell types are present in fetal data , and show significant age associations with ACE2 expression .

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

(g) Low expression in pediatric samples. Mean expression level (log CPM, y axis) of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) across age bins (x axis) in AT2 (left) and ciliated (right) cells. Pediatric samples: 0-10 years. Samples from past or current smokers were removed from this plot to avoid smoking confounders. Error bars are omitted due to y-axis limitations. They are typically 10-fold the mean value (Supplementary Table 5). Multiciliated and AT2 cells are shown as these cell types are present in fetal data, and show significant age associations with ACE2 expression.


( a ) Gradual increase in Ace2 expression by airway epithelial cell type with age . Mean expression ( y axis ) of Ace2 in different airway epithelial cells ( x axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , and error bars ( standard error of the mean ( SEM ) ) .

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(a) Gradual increase in Ace2 expression by airway epithelial cell type with age. Mean expression (y axis) of Ace2 in different airway epithelial cells (x axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (standard error of the mean (SEM)).


( b ) Increase in proportion of Ace2 + Ctsl + goblet and club cells with age . Percent of Ace2 + Ctsl + cells ( x axis ) in different airway epithelial cell types ( y axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , and error bars ( SEM ) .

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(b) Increase in proportion of Ace2+Ctsl+ goblet and club cells with age. Percent of Ace2+Ctsl+ cells (x axis) in different airway epithelial cell types (y axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (SEM).


( c - j ) Increase in Ace2 expression in secretory cells with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( c , d ) UMAP of scRNA - seq profiles ( dots ) colored by experimental group ( c ) or by Ace2 + cells and indicated double positive cells ( d ) . Alveolar epithelial cells ( AT1 and AT2 ) and airway epithelial secretory and ciliated cells are marked .

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(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (c,d) UMAP of scRNA-seq profiles (dots) colored by experimental group (c) or by Ace2+ cells and indicated double positive cells (d). Alveolar epithelial cells (AT1 and AT2) and airway epithelial secretory and ciliated cells are marked.


( c - j ) Increase in Ace2 expression in secretory cells with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( e ) The relative frequency of Ace2 + cells is increased by smoking in airway secretory cells but not AT2 cells . Relative proportion ( y axis ) of Ace2 + ( red ) and Ace2 - ( grey ) cells in smoking and control mice of different cell types ( x axis ) .

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(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (e) The relative frequency of Ace2+ cells is increased by smoking in airway secretory cells but not AT2 cells. Relative proportion (y axis) of Ace2+ (red) and Ace2- (grey) cells in smoking and control mice of different cell types (x axis).


( f , g ) Expression of Ace2 is increased in airway secretory cells , but not in AT2 cells . Distribution of Ace2 expression ( y axis ) in secretory ( f ) and AT2 ( g ) cells from control and smoking mice ( x axis ) .

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(f, g) Expression of Ace2 is increased in airway secretory cells, but not in AT2 cells. Distribution of Ace2 expression (y axis) in secretory (f) and AT2 (g) cells from control and smoking mice (x axis).


( h - j ) Re - analysis of published bulk mRNA - Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of ( h ) Ace2 , ( i ) Tmprss2 , and ( j ) Ctsl after five months of chronic exposure .

(h-j) Re-analysis of published bulk mRNA-Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of (h) Ace2, (i) Tmprss2, and (j) Ctsl after five months of chronic exposure.


( k ) Expression in placenta . UMAP plots showing expression of Ace2 , Tmprss2 and Ctsl in single and double positive cells from embryonic days 9 . 5 to 18 ( left ) and embryonic day 14 . 5 ( right ) of mouse placenta

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(k) Expression in placenta. UMAP plots showing expression of Ace2, Tmprss2 and Ctsl in single and double positive cells from embryonic days 9.5 to 18 (left) and embryonic day 14.5 (right) of mouse placenta development


( a ) Multiple proteases are co - expressed with ACE2 in human lung scRNA - seq . Scatter plot of significance ( y axis , - log10 ( adjusted P value ) ) and effect size ( x axis ) of co - expression of each protease gene ( dot ) with ACE within each indicated epithelial cell type ( color ) . Dashed line : significance threshold . TMPRSS2 and PCSKs that significantly co - expressed with ACE2 are marked .

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(a) Multiple proteases are co-expressed with ACE2 in human lung scRNA-seq. Scatter plot of significance (y axis, -log10(adjusted P value)) and effect size (x axis) of co-expression of each protease gene (dot) with ACE within each indicated epithelial cell type (color). Dashed line: significance threshold. TMPRSS2 and PCSKs that significantly co-expressed with ACE2 are marked.


A Range of example RAD51 nuclear expression score ( RAD51NES ) values with respective unmixed monochrome fluorescent IHC staining images . EpCAM is used as a tumour marker and an internal sample quality control . Scale bar is 50μm .

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A Range of example RAD51 nuclear expression score (RAD51NES) values with respective unmixed monochrome fluorescent IHC staining images. EpCAM is used as a tumour marker and an internal sample quality control. Scale bar is 50μm.


C Distribution of RAD51 nuclear expression score ( RAD51NES ) in the BCC cohort . The cohort is divided into RAD51 - Low expressing cases ( first quartile , Q1 - blue ) , intermediate cases ( interquartile range , IQR - grey ) and RAD51 - High expressing cases ( fourth quartile , Q4 - brown ) . Dashed line denotes the median RAD51NES in this cohort .

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C Distribution of RAD51 nuclear expression score (RAD51NES) in the BCC cohort. The cohort is divided into RAD51-Low expressing cases (first quartile, Q1 - blue), intermediate cases (interquartile range, IQR - grey) and RAD51-High expressing cases (fourth quartile, Q4 - brown). Dashed line denotes the median RAD51NES in this cohort.


D Survival analysis of the BCC cohort . Kaplan - Meier plots for progression - free survival ( PFS ) ( left ) and overall survival ( OS ) ( right ) stratified according to fourth quartile ( Q4 ) and first quartile ( Q1 ) of RAD51NES . Log - rank test , shading denotes 95 % confidence intervals .

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D Survival analysis of the BCC cohort. Kaplan-Meier plots for progression-free survival (PFS) (left) and overall survival (OS) (right) stratified according to fourth quartile (Q4) and first quartile (Q1) of RAD51NES. Log-rank test, shading denotes 95% confidence intervals.


E Number of cases with progression at 12 and 24 months . Chi - square test .

E Number of cases with progression at 12 and 24 months. Chi-square test.


A Distribution of RAD51NES in the SCOTROC4 cohort . Dashed line denotes the median RAD51NES in this cohort .

A Distribution of RAD51NES in the SCOTROC4 cohort. Dashed line denotes the median RAD51NES in this cohort.


B Survival analysis of the SCOTROC4 cohort . Kaplan - Meier plots for PFS ( left ) and OS ( right ) stratified according to quartiles of RAD51NES . Log - rank test , shading denotes 95 % confidence intervals .

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B Survival analysis of the SCOTROC4 cohort. Kaplan-Meier plots for PFS (left) and OS (right) stratified according to quartiles of RAD51NES. Log-rank test, shading denotes 95% confidence intervals.


C Number of cases with progression at 12 and 24 months . Chi - square test .

C Number of cases with progression at 12 and 24 months. Chi-square test.


D Survival analysis of HRD positive patients according to quartile of RAD51NES . Log - rank test , shading denotes 95 % confidence intervals . E , Survival analysis of HRD negative patients . Log - rank test , shading denotes 95 % confidence intervals .

D Survival analysis of HRD positive patients according to quartile of RAD51NES. Log-rank test, shading denotes 95% confidence intervals. E, Survival analysis of HRD negative patients. Log-rank test, shading denotes 95% confidence intervals.


A In - vitro cell survival assay of HGSOC cell lines upon stable RAD51 overexpression . Mean with standard deviation is shown of at least three biological replicates per point . Extra - sum - of - squares F test .

A In-vitro cell survival assay of HGSOC cell lines upon stable RAD51 overexpression. Mean with standard deviation is shown of at least three biological replicates per point. Extra-sum-of-squares F test.


B In - vitro colony forming assays comparing RAD51 - overexpressing and control HGSOC cell line , Caov - 3 , after treatment with increasing doses of carboplatin . Mean and SD of four biological replicates ( left ) a representative experiment ( right ) . P - value for a comparison between cell lines for each drug treatment condition is indicated above the bars . T - test .

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B In-vitro colony forming assays comparing RAD51-overexpressing and control HGSOC cell line, Caov-3, after treatment with increasing doses of carboplatin. Mean and SD of four biological replicates (left) a representative experiment (right). P-value for a comparison between cell lines for each drug treatment condition is indicated above the bars. T-test.


E Volcano plot for fold changes of immune genes enriched or depleted in RAD51 - High tumours vs RAD51 - Low tumours from TCGA . T - test ; dashed line denoted threshold of significance , Bonferroni corrected for multiple testing .

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E Volcano plot for fold changes of immune genes enriched or depleted in RAD51-High tumours vs RAD51-Low tumours from TCGA. T-test; dashed line denoted threshold of significance, Bonferroni corrected for multiple testing.


F Immune genes enriched in RAD51 - High and RAD51 - Low tumours across four EOC mRNA cohorts ( TCGA , AOCS , MGH , Duke ) .

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F Immune genes enriched in RAD51- High and RAD51-Low tumours across four EOC mRNA cohorts (TCGA, AOCS, MGH, Duke).


G Example unmixed fluorescent IHC images demonstrating the presence of CD8 + T cells in the Tumour ( T ) and Stroma ( S ) in a RAD51 - High and a RAD51 - Low tumour from the BCC cohort . Scale bar is 50μm .

G Example unmixed fluorescent IHC images demonstrating the presence of CD8+ T cells in the Tumour (T) and Stroma (S) in a RAD51-High and a RAD51-Low tumour from the BCC cohort. Scale bar is 50μm.


H CD8 + cytotoxic T - cell ( Tcyt ) infiltration analysis in the BCC cohort . Absolute tumour cytotoxic T - cell infiltration numbers and tumour / stroma ( T / S ) cytotoxic T - cell number ratio in RAD51 - High and - Low cases ( left ) . Bar is median . Mann - Whitney test .

H CD8+ cytotoxic T-cell (Tcyt) infiltration analysis in the BCC cohort. Absolute tumour cytotoxic T-cell infiltration numbers and tumour/stroma (T/S) cytotoxic T-cell number ratio in RAD51-High and -Low cases (left). Bar is median. Mann-Whitney test.


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( A ) Supernatants were harvested after 18 h , and concentrations of IL - 6 and IL - 12 were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Mean ± SD of triplicate determinations from > 3 independent experiments are shown .

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A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( B ) mRNA was harvested after 2 h , and expression of IL - 6 and IL - 12 was measured by real - time PCR and normalized to G6pdx . Unpaired two - tailed t - test . Data ( mean ± SD ) are representative of three independent experiments .

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A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.


C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC . Periods of oxidation were 24 h for CuSO4 ( 10 μM ) , 48 h for FeSO4 ( 10 μM ) , and 72 h for air . BMDCs were treated for 60 min with lipids prior stimulation with R837 ( 5 μg / ml ) for 18 h . Cytokine concentrations in supernatants were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Data represent mean ± SEM of triplicate determinations .

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C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.


D PAPC ( 40 μg / ml ) oxidized with CuSO4 ( 10 μM ) for the indicated times was used for treatment of BMDCs prior to R837 ( 5 μg / ml ) stimulation and measurement of IL - 12 secretion . Mean ± SEM of triplicate determinations from three different oxidation series are shown .

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D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.


E , F Treatment of BMDCs with the indicated concentrations of CuSO4 - oxidized PAPC suppressed IL - 6 ( E ) and IL - 12 ( F ) secretion triggered by a variety of different TLR agonists . After OxPAPC treatment for 60 min , BMDCs were stimulated for 18 h with LTA ( 500 ng / ml ) , Poly I : C ( 50 μg / ml ) , LPS ( 10 ng / ml ) , R837 ( 5 μg / ml ) , and CpG ( 100 nM ) . Cytokine concentrations in the supernatant were quantified by ELISA . Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.


G - I Splenicdendritic cells were treated with OxPAPC or DPPC ( 40 μg / ml ) before co - culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61 . ( G ) After 4 days of cell culture , T - cell polarization was assessed by intracellular staining for the cytokines IL - 4 ( Th2 ) and IFN - γ ( Th1 ) . ( H ) Bar graphs represent the frequencies of IL - 4 - and IFN - γ - producing T cells after 4 days of co - culture with OxPAPC - treated and DPPC - treated splenicdendritic cells . Bars represent mean ± SD of duplicate experiments . * P ≤ 0 . 05 by unpaired two - tailed t - test . ( I ) IFN - γ production in supernatants of SMARTA CD4T cells ( stimulated with 1 , 000 nM gp61 ) and co - cultured with OxPAPC - and DPPC - treated splenicdendritic cells for 4 days . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Bars represent mean ± SD .

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G-I Splenicdendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenicdendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenicdendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.


Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4 - catalyzed oxidation of PAPC for 2 and 24 h . Mean ± SEM of duplicate determinations are shown .

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Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.


Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL - 12 secretion of thioglycollate - elicited macrophages . Dots represent data of individual OxPAPC preparations .

Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.


Selected candidate lipids were tested for their inhibitory activity on R837 - induced ( 5 μg / ml ; 18 h ) cytokine secretion in BMDCs . Concentrations of indicated lipids : PECPC ( 10 μM ) , PEIPC ( 10 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , and KOdiAPC ( 40 μM ) . Representative data ( mean ± SD of triplicate determinations ) from one of three independent experiments are shown . * * * * P < 0 . 0001 ; ns , not significant ; as determined by one - way ANOVA adjusted by Sidak ' s multiple comparisons test .

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Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.


Dose - response curves of IL - 12 secretion in BMDCs pulsed for 60 min with the indicated lipids , followed by stimulation with R837 ( 5 μg / ml ) for 18 h . Toxic concentrations of lipids were excluded from analysis . Mean ± SEM of triplicate determinations are shown .

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Dose-response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.


EC50 values of anti - inflammatory lipid products normalized to the capacity of 15d - PGJ2 to suppress IL - 12 production in BMDCs ( left panel ) and their respective bioactivities ( right panel ) depicted as the fold increase relative to 15d - PGJ2 . Mean ± SEM of triplicate determinations are shown .

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EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.


IL - 12 production of BMDC stimulated via TLR 4 ( LPS ; 100 ng / ml ) , TLR 9 ( CpG ; 100 nM ) , and TLR 7 ( R837 ; 5 μg / ml ) after pretreatment with the indicated free and esterified versions of EC and 15d - PGJ2 . Lipids were used at 1 μM ( EC ) , 10 μM ( PECPC ) , or 20 μM ( 15d - PGJ2 and 15d - PGJ2PC ) . Data ( mean ± SEM ) are representative of 3 independent experiments . * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; determined by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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IL-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.


Analysis of the capacity of various OxPL - derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset . Concentrations of indicated lipids : EC ( 1 μM ) , PECPC ( 10 μM ) , 15d - PGJ2 ( 20 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , KOdiAPC ( 40 μM ) .

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Analysis of the capacity of various OxPL-derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).


A IL - 12 production of BMDC from wild - type , Nrf2 − / − , Pparg − / − , and Pparg litter mate control mice normalized to medium control ( open bars ) . Cells were treated with the indicated lipids ( filled bars ) for 60 min prior TLR 7 ligation with R837 ( 5 μg / ml ) for 18 h . Lipids were used at starting concentrations of 40 μM ( POVPC , PGPC , and KOdiAPC ) , 40 μg / ml ( OxPAPC ) , 20 μM ( 15d - PGJ2 ) , and 1 . 25 μM ( EC ) , depicted as black bars , and 2 - fold serial dilutions thereof ( gray bars ) . Data represent mean ± SEM of triplicates from one of three independent experiments .

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A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.


B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild - type and Nrf2 − / − BMDM stimulated with EC ( 2 μM ) for 60 min . Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx . Data ( mean ± SEM ) are representative of two independent experiments .

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B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/−BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.


C , D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 ( C ) and of the pro - inflammatory cytokines IL - 6 and IL - 12 ( D ) in wild - type , PPAR - γ - deficient , and Nrf2 - deficient BMDM after treatment with EC or 15d - PGJ2 for 60 min followed by LPS treatment for 3 h . Expression levels are normalized to G6pdx . Data represent mean ± SEM of triplicate cultures from one of two independent experiments .

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C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.


E mRNA expression of the indicated chemokines as determined by qPCR . Wild - type BMDCs were treated with EC ( 1 μM ) or 15d - PGJ2 ( 20 μM ) for 60 min followed by R837 stimulation ( 5 μg / ml ) for 3 h . Expression levels are shown normalized to G6pdx . Data ( mean ± SEM , n = 2 ) are representative of three independent experiments .

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E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.


A C57BL / 6mice were treated ( i . v . ) with 500 μg EC or DPPC control 2 h prior to i . p injection of 150 ng / g LPS together with 800 μg / g D - galactosamine . 4 h after LPS application , lungs were perfused with PBS and embedded in paraffin . Tissue sections were hematoxylin - stained to visualize adherent cells . Bars represent 100 µm . B , C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in ( B ) and as averages of single mice in ( C ) . Pooled data of two independent experiments are shown ( n = 10 for EC , n = 14 for DPPC ) . Unpaired two - tailed t - test .

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A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.


D , E C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) and 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS and 800 μg / g D - galactosamine . Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils ( D ) . Unpaired two - tailed t - test . Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL cells of EC - / LPS - treated and DPPC - / LPS - treated mice .

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D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


F , G The concentrations of IL - 6 ( F ) and IL - 12 ( G ) in the BAL of mice treated as in ( D ) were quantified by ELISA . Data ( mean ± SEM ) are representative of two independent experiments with 3 mice per group . * * * * P < 0 . 0001 ; unpaired two - tailed t - test .

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F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test.


C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) and 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS and 800 μg / g D - galactosamine . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL cells of EC - / LPS - treated and DPPC - / LPS - treated mice .

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C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


Dose - response curves showing the modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h . Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in ( A ) , presented relative to EC .

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Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.


Quantification of IL - 6 and IL - 12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 ( 5 μg / ml ; 18 h ) . Bars represent mean ± SEM from one of three independent experiments . * * P < 0 . 01 ; * * * P < 0 . 001 ; one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.


mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression . BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed t - test .

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mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.


ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in ( E ) . Dose - response curves showing the modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h .

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ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.


mRNA quantification of the indicated chemokines relative to G6pdx expression . BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed t - test .

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mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.


Quantification and characterization of cellular infiltrates in BAL . Groups of six C57BL / 6mice were pretreated i . t . with 50 μg cEC , EC , or BisRed 24 and 2 h before challenge with 150 ng / g LPS in the presence of 800 μg / g D - galactosamine . BAL was harvested 4 h after LPS injection , and inflammatory cells were characterized by FACS analysis . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; one - way ANOVA with Sidak ' s multiple comparisons test .

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Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.


Comparison of the capacity of cEC , EC , and PECPC ( 1 μM ) to license splenicdendritic cells to polarize naïve CD4T cells into IFN - γ - producing ( Th1 ) and IL - 4 - producing ( Th2 ) effector cells . Data ( mean ± SD ) are representative of two independent experiments . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenicdendritic cells to polarize naïve CD4T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


A . Immunostaining of TUJ1 , DESMIN and GATA6 after six days of mESC differentiation towards ectoderm , mesoderm and endoderm . Bar : 50 μm .

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A. Immunostaining of TUJ1, DESMIN and GATA6 after six days of mESC differentiation towards ectoderm, mesoderm and endoderm. Bar: 50 μm.


C . Venn diagram of genes with differential expression during mESC differentiation towards the three germ layers .

C. Venn diagram of genes with differential expression during mESC differentiation towards the three germ layers.


D - F . Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm ( E ) , mesoderm ( F ) and endoderm ( G ) .

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D-F. Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm (E), mesoderm (F) and endoderm (G).


A . Hierarchical clustering of gene expression profiles of mESCs differentiated towards the three germ layers and differentiated mouse tissues .

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A. Hierarchical clustering of gene expression profiles of mESCs differentiated towards the three germ layers and differentiated mouse tissues.


C . Hierarchical clustering of mRNA expression during differentiation of mESCs towards endoderm ( yellow ) or EpiSCs ( green ) .

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C. Hierarchical clustering of mRNA expression during differentiation of mESCs towards endoderm (yellow) or EpiSCs (green).


B . Ectoderm differentiation of the 3KI mESC line ( marked by the expression of Sox1 : : GFP ) . C . Mesendoderm differentiation of the 3KI mESC line ( marked by the expression of T : : 3xTagBFP ) .

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B. Ectoderm differentiation of the 3KI mESC line (marked by the expression of Sox1::GFP). C. Mesendoderm differentiation of the 3KI mESC line (marked by the expression of T::3xTagBFP).


D . Endoderm differentiation of the 3KI mESC line ( marked by the expression of Eomes : : mCherry ) .

D. Endoderm differentiation of the 3KI mESC line (marked by the expression of Eomes::mCherry).


E , F . Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers . Bar : 100 μm .

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E, F. Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers. Bar: 100 μm.


B . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - and Zfp354c - / - mESCs in the 3KI background to ectoderm . C . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - and Zfp354c - / - mESCs in the 3KI background to mesendoderm . D . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - and Zff354c - / - mESCs in the 3KI background to definitive endoderm .

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B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm. C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm. D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm.


F . Differentiation of wild type ( WT ) , Sp1 + / - , Nr5a2 - / - and Fos - / - Jun - / - mESCs in the 3KI background to definitive endoderm .

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F. Differentiation of wild type (WT), Sp1+/-, Nr5a2-/- and Fos-/-Jun-/- mESCs in the 3KI background to definitive endoderm.


( B , C ) HCT116 , PKcs - / - , and Lig4 - / - cells were subjected to Agilent Seahorse XFe24 analyzer to measure oxidative phosphorylation ( B ) via the oxygen consumption rate ( OCR ) and glycolysis ( C ) via the extracellular acidification rate ( ECAR ) .

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(B, C) HCT116, PKcs-/-, and Lig4-/- cells were subjected to Agilent Seahorse XFe24 analyzer to measure oxidative phosphorylation (B) via the oxygen consumption rate (OCR) and glycolysis (C) via the extracellular acidification rate (ECAR).


( J , K ) M059K / J cells were also subjected to the OCR and ECAR analyses .

(J, K) M059K/J cells were also subjected to the OCR and ECAR analyses.


( B ) HeLa S3 total cell lysate , nuclear and cytosolic and mitochondrial fractions were analyzed by western blot to show sub - cellular localization of DNA - PKcs and ATM .

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(B) HeLa S3 total cell lysate, nuclear and cytosolic and mitochondrial fractions were analyzed by western blot to show sub-cellular localization of DNA-PKcs and ATM.


( C , D ) 100 μg of purified HeLa S3 mitochondria were subjected to limited protease K ( at the indicated amount ) digestion in a final 50 μl reaction volume at 37C for 30 minutes ( C ) or sodium carbonate ( 100 mM ) verse sodium chloride ( 133 mM ) extraction followed by western blot ( D ) .

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(C, D) 100 μg of purified HeLa S3 mitochondria were subjected to limited protease K (at the indicated amount) digestion in a final 50 μl reaction volume at 37C for 30 minutes (C) or sodium carbonate (100 mM) verse sodium chloride (133 mM) extraction followed by western blot (D).


( A ) HeLa cells expressing Flag - tagged ANT1 - 3 were subjected to Co - IP using anti - Flag antibody , then analyzed by western blot as indicated .

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(A) HeLa cells expressing Flag-tagged ANT1-3 were subjected to Co-IP using anti-Flag antibody, then analyzed by western blot as indicated.


( E ) HeLa cells expressing Flag - tagged VDAC1 - 3 were subjected to Co - IP using anti - Flag antibody and analyzed by western blot as indicated .

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(E) HeLa cells expressing Flag-tagged VDAC1-3 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated.


( F ) PLA analysis show that the interaction between DNA - PKcs and Flag - ANT2 was disrupted by knockdown of VDAC2 , but not VDAC1 or VDAC3 . Quantification shown on the right . * * * , P < 0 . 001 . Data information : The bar graphs were generated from three independent biological replicates analyses ; data are represented as mean ± SD . Statistical significance was established using a two - way analysis of variance ( ANOVA , * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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(F) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by knockdown of VDAC2, but not VDAC1 or VDAC3. Quantification shown on the right. ***, P < 0.001. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).


( A , B ) HCT116 , DNA - PKcs - / - , and Ligase 4 - / - cells were subjected to ADP - ATP exchange assay using MgGreen 5K + fluorescent dye . The exchange was stimulated by 5mM ADP and halted by ANTs inhibitor carboxyatractyloside ( cATR ) . Normalized MgGreen 5K + fluorescence was first measured to indicate changes in free magnesium concentration ( A ) . The decreased free magnesium concentration was converted into efflux of ATP ( [ ATP ] e , mM ) after the addition of ADP ( B ) . Bar graph on the right shows the normalized MgGreen 5K + fluorescence and efflux of ATP concentration after ADP until the reaction was stopped by cATR treatment .

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(A, B) HCT116, DNA-PKcs-/-, and Ligase 4-/- cells were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The exchange was stimulated by 5mM ADP and halted by ANTs inhibitor carboxyatractyloside (cATR). Normalized MgGreen 5K+ fluorescence was first measured to indicate changes in free magnesium concentration (A). The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP (B). Bar graph on the right shows the normalized MgGreen 5K+ fluorescence and efflux of ATP concentration after ADP until the reaction was stopped by cATR treatment.


( F ) shGFP and shATM pair of HeLa ( Right ) or MCF7 ( Left ) cells were subjected to an ADP - ATP exchange assay using MgGreen 5K + fluorescent dye .

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(F) shGFP and shATM pair of HeLa (Right) or MCF7 (Left) cells were subjected to an ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye.


( A ) Western blot results show attenuation of ANT2 protein level in DNA - PKcs - deficient and depleted cells .

(A) Western blot results show attenuation of ANT2 protein level in DNA-PKcs-deficient and depleted cells.


( L ) Treatment with VDAC2 siRNA in Hela cells attenuated ADP - ATP exchange . Bar graph on the right shows the efflux of ATP concentration after adding ADP .

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(L) Treatment with VDAC2 siRNA in Hela cells attenuated ADP-ATP exchange. Bar graph on the right shows the efflux of ATP concentration after adding ADP.