( B ) Phase contrast images displaying morphology of untreated and treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs . Morphology was quantified as circularity ratio , where 1 = perfect circle and 0 = line ( experimental replicates = 3 ) . Statistical analysis ( paired t test ) was performed on the first quartile to enhance detection of morphological changes ( error bars : SD ) .

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(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm. (C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).


( E ) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours . Scale bar : 75 μm . ( F ) Bar graphs quantifying EdU experiment shown in ( E ) ( experimental replicates = 3 ; error bars : SEM ) . Statistical analysis based on an unpaired t - test .

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(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.


( H ) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in the presence or absence of γ - secretase inhibitor L685458 and immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : SEM ) . Statistical significance based on one - way ANOVA with Tukey ' s multiple comparison .

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(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.


( J ) Immunofluorescence panels showing human specific LAMIN A / C + ( nuclei ) and SPECTRIN ( sarcolemma ) staining in tibialis anterior muscles of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 muscles ) and untreated ( n = 3 muscles ) N5 hiMPs . ( K ) Quantification of LAMIN A / C + grafted human cells across each muscle . Data information : full gene list for heatmaps in ( A ) and ( D ) available in Appendix Table S4 .

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(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs. (K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.


( F ) Representative fluorescence images of CMFDA - stained untreated and DLL4 & PDGF - BB - treated hiMPs within the top perfusion channel , 15 minutes after delivery and kept on the OrganoFlow ® . ( G ) Bar graph quantifying adhesion images in ( E ) . Statistical significance was calculated based on a paired t - test ( experimental replicates = 3 ) . Each point on the plot represents the number of adhered cells after 15 minutes within a single chip .

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(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.


( H ) Assessment of DLL4 & PDGF - BB - treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells . Representative images showing the lower side of the trans - well membrane on which treated and untreated hiMPs ( stained with the transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying the average number of CFDA - positive cells / mm2 , that have migrated through the endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : SEM ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : SEM ) . Statistical significance based on one - way ANOVA with Bonferroni ' s multiple comparison .

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(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.


( C ) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels , α - Tubulin ( Tubulin ) as loading control .

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(C) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels, α-Tubulin (Tubulin) as loading control.


( B ) Top : Experimental outline of CRISPRa targeting of GBP1 and ASCL1 ( control ) by transient transfection with plasmids containing dCAS9 - SAM . Bottom : HeLa cells were transfected with dCas9 - SAM technology with gRNA for indicated genes , either alone or in combination with a 24h exposure to IFNγ . RNA was isolated and GBP1 mRNA expression was measured by RT - qPCR after 48h of transfection and normalized to ACTB expression . Statistical significance was determined using two - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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(B) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM. Bottom: HeLa cells were transfected with dCas9-SAM technology with gRNA for indicated genes, either alone or in combination with a 24h exposure to IFNγ. RNA was isolated and GBP1 mRNA expression was measured by RT-qPCR after 48h of transfection and normalized to ACTB expression. Statistical significance was determined using two-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


( C ) Top : Experimental outline of CRISPRa targeting of GBP1 and ASCL1 ( control ) by transient transfection with plasmids containing dCAS9 - SAM , followed by induction with IFNγ . Bottom : RNA was isolated , GBP1 mRNA level was determined as indicted in experimental outline in top , by RT - qPCR and normalized to ACTB mRNA level . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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(C) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM, followed by induction with IFNγ. Bottom: RNA was isolated, GBP1 mRNA level was determined as indicted in experimental outline in top, by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


( D ) Experiment as in C but with addition of IFNγ during priming . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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(D) Experiment as in C but with addition of IFNγ during priming. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


( B ) Stable CRISPR knockouts were generated for indicated genes in HeLa cells . Knockout ( KO ) cells or their parental controls ( WT ) were induced with IFNγ for 24 hours or left untreated and prepared for SDS - PAGE and immunoblotting . Blots incubated with GBP5 antibody assess gene expression . α - Tubulin ( Tubulin ) was used as a loading control . Note that GBP5 and Tubulin blot of parental cells is as in 1C .

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(B) Stable CRISPR knockouts were generated for indicated genes in HeLa cells. Knockout (KO) cells or their parental controls (WT) were induced with IFNγ for 24 hours or left untreated and prepared for SDS-PAGE and immunoblotting. Blots incubated with GBP5 antibody assess gene expression. α-Tubulin (Tubulin) was used as a loading control. Note that GBP5 and Tubulin blot of parental cells is as in 1C.


HeLa cells were primed with IFNγ for 24 hours , followed with IFNγ washout . After 48h , naïve and primed cells were induced by IFNγ for 1h and 3h . Cells were harvested at indicated time points and processed for CUT & RUN . Representation of processed data of CUT & RUN for STAT1 ( D ) and IRF1 ( E ) occupancy at GBP5 and GBP4 genes . Sequenced reads were mapped to the human genome ( hg38 ) , coverage data are displayed as reads per million ( RPM ) at equal scaling .

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HeLa cells were primed with IFNγ for 24 hours, followed with IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for CUT&RUN. Representation of processed data of CUT&RUN for STAT1 (D) and IRF1 (E) occupancy at GBP5 and GBP4 genes. Sequenced reads were mapped to the human genome (hg38), coverage data are displayed as reads per million (RPM) at equal scaling.


( A ) Single - cell RNA - seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve ( n = 91 ) , induction ( n = 90 ) and reinduction ( n = 92 ) . Error bars , SEM .

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(A) Single-cell RNA-seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve (n=91), induction (n=90) and reinduction (n=92). Error bars, SEM.


( C ) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout ( STAT1KO ) , STAT1 rescued ( STAT1 , constitutive ) and parental control ( WT ) , to confirm knockout and rescue status . α - Tubulin ( Tubulin ) was used as a loading control .

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(C) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout (STAT1KO), STAT1 rescued (STAT1, constitutive) and parental control (WT), to confirm knockout and rescue status. α-Tubulin (Tubulin) was used as a loading control.


( D ) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A . Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A . α - Tubulin ( Tubulin ) was used as a loading control .

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(D) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A. Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A. α-Tubulin (Tubulin) was used as a loading control.


( G ) Cells constitutively expressing STAT1 ( as in B ) were primed with IFNγ , followed by IFNγ washout . After 48h , naïve and primed cells were induced by IFNγ for different time points ( 5 , 15 , 30 , 60 , 180 min ) . Cell extracts were prepared at indicated timepoints and processed for western blotting . Immunoblot of pSTAT1 - Y701 , and α - Tubulin ( Tubulin ) as a loading control .

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(G) Cells constitutively expressing STAT1 (as in B) were primed with IFNγ, followed by IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for different time points (5, 15, 30, 60, 180 min). Cell extracts were prepared at indicated timepoints and processed for western blotting. Immunoblot of pSTAT1-Y701, and α-Tubulin (Tubulin) as a loading control.


( A ) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time ( primed state ) after IFNγ washout . Cell extracts were prepared at indicated timepoints , processed for western blotting and probed for STAT1 , pSTAT1 - Y701 and pSTAT1 - S727 . α - Tubulin ( Tubulin ) as a loading control .

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(A) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time (primed state) after IFNγ washout. Cell extracts were prepared at indicated timepoints, processed for western blotting and probed for STAT1, pSTAT1-Y701 and pSTAT1-S727. α-Tubulin (Tubulin) as a loading control.


( C ) ( H ) STAT1KO : : STAT1 - S727E and STAT1KO : : STAT1 - S727D expressing cells or their parental controls ( WT ) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A , RNA was isolated and GBP5 mRNA level was determined by RT - qPCR and normalized to ACTB mRNA level . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean ( error bars , SD ; n = 3 biological replicates ) .

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(C) (H) STAT1KO::STAT1-S727E and STAT1KO::STAT1-S727D expressing cells or their parental controls (WT) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A, RNA was isolated and GBP5 mRNA level was determined by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates).


( E ) Characterization of dTAG - induced destruction and recovery . STAT1 - EGFP - dTAG expressing cells were induced with IFNγ for 24h , then treated with dTAG13 ( 100 nM ) for DMSO vehicle control ( I ) or STAT1 depletion ( II ) for 24h . To determine recovery of STAT1 expression , cells were washed three times with medium and harvested 48h after IFNγ washout ( 120 hour timepoint , lane 4 ) . In Parallel , ( III ) cells were treated with dTAG13 ( 100 nM ) for 48h during 24h induction with IFNγ . Immunoblot of STAT1 - GFP - dTAG at indicated treatments confirm STAT1 depletion and recovery , respectively . α - Tubulin ( Tubulin ) was used as a loading control .

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(E) Characterization of dTAG-induced destruction and recovery. STAT1-EGFP-dTAG expressing cells were induced with IFNγ for 24h, then treated with dTAG13 (100 nM) for DMSO vehicle control (I) or STAT1 depletion (II) for 24h. To determine recovery of STAT1 expression, cells were washed three times with medium and harvested 48h after IFNγ washout (120 hour timepoint, lane 4). In Parallel, (III) cells were treated with dTAG13 (100 nM) for 48h during 24h induction with IFNγ. Immunoblot of STAT1-GFP-dTAG at indicated treatments confirm STAT1 depletion and recovery, respectively. α-Tubulin (Tubulin) was used as a loading control.


( G ) RNA was isolated and GBP5 mRNA level as indicated in ( F ) was determined by RT - qPCR , normalized to ACTB mRNA level . Statistical significance was determined using two - way ANOVA . Data are shown as mean ( error bars , SD ; n = 3 biological replicates )

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(G) RNA was isolated and GBP5 mRNA level as indicated in (F) was determined by RT-qPCR, normalized to ACTB mRNA level. Statistical significance was determined using two-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates)


A Western blot analysis STAT expression and phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ for 0 . 5 , 6 , 12 and 24 h . Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 ( Y701 ) and STAT2 ( Y689 ) and total level of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments . C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments .

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B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.


A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs . Spleens , livers or bone marrow derived macrophages ( BMDMs ) were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice . Whole cell extracts were collected and tested for levels of total Stat1 in western blot . The blots are representative of more than 3 independent experiments .

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A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers or bone marrow derived macrophages (BMDMs) were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice. Whole cell extracts were collected and tested for levels of total Stat1 in western blot. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701 . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice and stimulated for 30 min with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ . Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot . The blots are representative of more than 3 independent experiments .

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B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice and stimulated for 30 min with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ. Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot. The blots are representative of more than 3 independent experiments.


C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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 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

C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ-induced genes. BMDMs of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05); the p-values were calculated using paired ratio t-test.


D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs were isolated from wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice treated with 250 IU / mL of IFNβ for 4 , 8 , 12 , 24 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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

D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN-induced genes (ISG). BMDMs were isolated from wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice treated with 250 IU/mL of IFNβ for 4, 8, 12, 24 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01); the p-values were calculated using paired ratio t-test.


E STAT1 and STAT2 phosphorylation in Stat1 - / - , Stat1Y701F , Stat2 - / - and Irf9 - / - macrophages . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice and treated with 250 IU / mL of IFNβ for 30 min or 6 , 12 or 24 h . The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689 . The same cell extracts were tested for total levels of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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

E STAT1 and STAT2 phosphorylation in Stat1-/-, Stat1Y701F, Stat2-/- and Irf9-/- macrophages. BMDMs were isolated from wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice and treated with 250 IU/mL of IFNβ for 30 min or 6, 12 or 24 h. The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


A IFNβ - stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT2 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

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

A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test.


B Impact of STAT2 deficiency on IFNβ - stimulated STAT1 association with the Mx2 ISRE . BMDMs of wild type ( WT ) , Stat1 - / - ( S1 ) and Stat2 - / - ( S2 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT1 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard error of mean ( SEM ) .

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

B Impact of STAT2 deficiency on IFNβ -stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM).


C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP - reChIP . BMDMs of wild type ( WT ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with either STAT1 - specific antibody and re - immunoprecipitated with STAT2 - specific antibody or vice versa . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard deviation ( SD ) .

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 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 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

C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).


D Impact of STAT1Y701F on delayed , STAT2 - mediated expression of IFN - induced genes . Stat1 - / - fibroblasts were transfected with plasmids driving expression of the indicated proteins . 24 h after transfection , 250 IU / ml of IFNβ was added to the transfected cells and ISG expression was determined by Q - PCR after 48h of cytokine treatment . Gene expression was measured by Q - PCR and normalized to Gapdh . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) and asterisks denote the level of statistical significance ( * , p ≤ 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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 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

D Impact of STAT1Y701F on delayed, STAT2-mediated expression of IFN-induced genes. Stat1-/- fibroblasts were transfected with plasmids driving expression of the indicated proteins. 24 h after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by Q-PCR after 48h of cytokine treatment. Gene expression was measured by Q-PCR and normalized to Gapdh. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*, p≤ 0.05); the p-values were calculated using paired ratio t-test.


A Analysis of STAT2 nuclear translocation by immunofluorescence . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were seeded on cover slips and stimulated with 250 IU / mL of IFNβ for 30 min or 24 h . The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor ® 488 conjugated secondary antibody ( green ) . Nuclei were stained with DAPI ( blue ) . Studies are representative of more than three independent experiments . The scale bars represent 10 µm . B Quantitative evaluation of STAT2nuclear translocation . The intensity of STAT2 - dependent immunofluorescence over DNA staining ( DAPI ) was quantified using ImageJ software in 20 cells from two independent experiments . Bars represent a mean with standard deviation ( SD ) and asterisks denote the level of statistical significance ( * * * , p ≤ 0 . 001 ) ; p - value was calculated using unpaired t - test .

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 B-CONTROLLED_VAR 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 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 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 B-MEASURED_VAR 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

A Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were seeded on cover slips and stimulated with 250 IU/mL of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.B Quantitative evaluation of STAT2nuclear translocation. The intensity of STAT2-dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***, p≤ 0.001); p-value was calculated using unpaired t-test.


A Legionella pneumophila growth in unstimulated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded on in the 24 - well plates and infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection . B Legionella pneumophila growth in IFNβ - treated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded in 24 - well plates , treated with 500 U / mL of IFNβ and then infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection .

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 O O 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 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 O O O O O O O O O O O O O O O O O O O O O O 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 Legionella pneumophila growth in unstimulated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded on in the 24-well plates and infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.B Legionella pneumophila growth in IFNβ-treated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded in 24-well plates, treated with 500 U/mL of IFNβ and then infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.


A Western blot analysis of STAT1 tyrosine phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 5 or 6 h . Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine ( Y701 ) . The blots are representative of more than 3 independent experiments .

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 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 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

A Western blot analysis of STAT1 tyrosine phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected with L. monocytogenes (LO28, MOI 10) for 5 or 6 h. Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine (Y701). The blots are representative of more than 3 independent experiments.


B Impact of Stat1Y701F mutation , or of deletion of ISGF3 subunits on the expression of the IFNβ gene . BMDMs of wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 4 , 8 , 12 , 24 or 48 h . Levels of Ifnβ gene expression were determined by Q - PCR . Bars represent mean values of three independent experiments . Error bars represent standard error of mean ( SEM ) .

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 B-CONTROLLED_VAR 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 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

B Impact of Stat1Y701F mutation, or of deletion of ISGF3 subunits on the expression of the IFNβ gene. BMDMs of wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice were infected with L. monocytogenes (LO28, MOI 10) for 4, 8, 12, 24 or 48 h. Levels of Ifnβ gene expression were determined by Q-PCR. Bars represent mean values of three independent experiments. Error bars represent standard error of mean (SEM).


A Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F and Stat1 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) . Colony forming unit ( CFU ) numbers were determined 1 , 2 , 4 , 6 or 8 h after infection by plating on brain - heart - infusion ( BHI ) agar plates . The graph represents biological triplicates and the data are represented as mean values . Error bars represent standard deviation ( SD ) and asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - test .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F and Stat1-/- mice were infected with L. monocytogenes (LO28, MOI 10). Colony forming unit (CFU) numbers were determined 1, 2, 4, 6 or 8 h after infection by plating on brain-heart-infusion (BHI) agar plates. The graph represents biological triplicates and the data are represented as mean values. Error bars represent standard deviation (SD) and asterisks denote statistically significant differences (ns, p>0.05; **, p≤0.01; ***, p≤ 0.001); p-values were calculated using unpaired t-test.


B Survival of mice infected with L . monocytogenes . 10 wild type ( WT ) , Stat1Y701F and Stat1 - / - mice per group were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Survival was monitored over 10 days . The study is representative of more than 3 independent experiments .

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

B Survival of mice infected with L. monocytogenes. 10 wild type (WT), Stat1Y701F and Stat1-/- mice per group were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Survival was monitored over 10 days. The study is representative of more than 3 independent experiments.


C Organ pathogen burdens of mice infected with L . monocytogenes . Wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Number of colony forming units ( CFU ) in organs was determined at 48 h , 72 h or at the terminal stage of infection by plating homogenates on brain - heart - infusion ( BHI ) agar plates or Oxford agar plates ( for lungs ) . Dots represent pooled data of 3 independent experiments . Lines represent the median and asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 ) ; p - values were calculated using Mann - Whitney test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Organ pathogen burdens of mice infected with L. monocytogenes. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Number of colony forming units (CFU) in organs was determined at 48 h, 72 h or at the terminal stage of infection by plating homogenates on brain-heart-infusion (BHI) agar plates or Oxford agar plates (for lungs). Dots represent pooled data of 3 independent experiments. Lines represent the median and asterisks denote statistically significant differences (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001; ****, p≤ 0.0001); p-values were calculated using Mann-Whitney test.


A Immunohistochemical analysis of infection and inflammatory infiltrates . Wild type ( WT ) , Stat1Y701F and Stat1 - / - mice were infected by intraperitoneal injection of 1x102 viable L . monocytogenes for 48 h . Liver sections were examined by immunohistochemistry with L . monocytogenes or Ly6C / Ly6G specific antibody . The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images . B Quantitative evaluation of inflammatory infiltrates . Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size .

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 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

A Immunohistochemical analysis of infection and inflammatory infiltrates. Wild type (WT), Stat1Y701F and Stat1-/- mice were infected by intraperitoneal injection of 1x102 viable L. monocytogenes for 48 h. Liver sections were examined by immunohistochemistry with L. monocytogenes or Ly6C/Ly6G specific antibody. The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images.B Quantitative evaluation of inflammatory infiltrates. Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size.


C Liver pathology in infected mice . Wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected by intraperitoneal injection of 1x105 viable L . monocytogenes for 72 h . Serum was collected and tested for ALT activity . Lines represent the median and asterisks denote statistically significant differences ( * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - test .

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 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

C Liver pathology in infected mice. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x105 viable L. monocytogenes for 72 h. Serum was collected and tested for ALT activity. Lines represent the median and asterisks denote statistically significant differences (*, p≤ 0.05; ***, p≤ 0.001); p-values were calculated using unpaired t-test.


Western blot analysis ( B ) of DCLK1 in aortas of LFD and HFD - fed ApoE - / - mice . β - actin was used as the loading control ( n = 10 biological replicates ) .

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

Western blot analysis (B) of DCLK1 in aortas of LFD and HFD-fed ApoE-/- mice. β-actin was used as the loading control (n=10 biological replicates).


( E ) Representative immunofluorescence staining of CD31 ( green ) and DCLK1 ( red ) in aortic roots ( scale bar = 25 μm ) . ( F ) Representative immunofluorescence staining of α - SMA ( green ) and DCLK1 ( red ) in aortic roots ( scale bar = 25 μm ) . ( G ) Representative immunofluorescence staining of F4 / 80 ( green ) and DCLK1 ( red ) in aortic roots ( scale bar = 25 μm ) . White arrows indicate co - location of F4 / 80 and DCLK1 .

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(E) Representative immunofluorescence staining of CD31 (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). (G) Representative immunofluorescence staining of F4/80 (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). White arrows indicate co-location of F4/80 and DCLK1.


( H - I ) Time - course of DCLK1 induction in response to oxLDL in mouse primary peritoneal macrophages ( MPMs ) . MPMs were exposed to oxLDL ( 50 μg / mL ) for indicated time . Western blot analysis ( H ) and densitometric quantification ( I ) of DCLK1 were shown . GAPDH was used as the loading control ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(H-I) Time-course of DCLK1 induction in response to oxLDL in mouse primary peritoneal macrophages (MPMs). MPMs were exposed to oxLDL (50 μg/mL) for indicated time. Western blot analysis (H) and densitometric quantification (I) of DCLK1 were shown. GAPDH was used as the loading control (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


Representative en face Oil Red O staining ( A ) of Oil Red O - positive plaque lesion area in aortas . Plaque area was defined as percentage of total surface area of the aorta ( n = 10 biological replicates ) .

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Representative en face Oil Red O staining (A) of Oil Red O-positive plaque lesion area in aortas. Plaque area was defined as percentage of total surface area of the aorta (n=10 biological replicates).


Representative images ( C ) of plaque lesion area in aortic arches ( n = 10 biological replicates ) .

Representative images (C) of plaque lesion area in aortic arches (n=10 biological replicates).


( E - F ) Representative images of Oil Red O staining ( E ) and quantification ( F ) of atherosclerotic lesion in aortic roots ( scale bar = 250 μm , n = 10 biological replicates ) . Plaque area was quantified by the proportion of plaque area to aortic root area . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(E-F) Representative images of Oil Red O staining (E) and quantification (F) of atherosclerotic lesion in aortic roots (scale bar=250 μm, n=10 biological replicates). Plaque area was quantified by the proportion of plaque area to aortic root area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( G - H ) Representative images of Masson ' s Trichrome staining ( G ) and quantification ( H ) for collagen deposition in aortic roots ( scale bar = 25 μm , n = 10 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(G-H) Representative images of Masson's Trichrome staining (G) and quantification (H) for collagen deposition in aortic roots (scale bar=25 μm, n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


Representative immunofluorescence staining images ( A ) of F4 / 80 ( green ) in aortic roots . Tissues were counterstained with DAPI ( blue ) . Scale bar = 250 μm , n = 10 biological replicates .

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Representative immunofluorescence staining images (A) of F4/80 (green) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar=250 μm, n=10 biological replicates.


Representative immunohistochemistry staining images of Ly6G ( C in aortic roots ( scale bar = 25 μm , n = 10 biological replicates ) .

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Representative immunohistochemistry staining images of Ly6G (C in aortic roots (scale bar=25 μm, n=10 biological replicates).


Representative immunohistochemistry staining images of Ly6C ( E in aortic roots ( scale bar = 25 μm , n = 10 biological replicates ) .

Representative immunohistochemistry staining images of Ly6C (E in aortic roots (scale bar=25 μm, n=10 biological replicates).


Scatter diagram ( G ) and quantification of monocyte in plasma measured by an automated blood cell analyzer ( n = 10 biological replicates ) . SSC , side scatter ; SFL , side fluorescence . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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Scatter diagram (G) and quantification of monocyte in plasma measured by an automated blood cell analyzer (n=10 biological replicates). SSC, side scatter; SFL, side fluorescence. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( J - K ) Protein ( J ) and mRNA ( K ) levels of inflammatory cytokines TNF - α and IL - 6 in serum and aortas . The values of mRNA levels were normalized to Rn18s ( n = 10 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(J-K) Protein (J) and mRNA (K) levels of inflammatory cytokines TNF-α and IL-6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( L ) mRNA levels of proinflammatory chemokines and adhesion molecules in aortas ( n = 10 biological replicates ) . The values were normalized to Rn18s . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(L) mRNA levels of proinflammatory chemokines and adhesion molecules in aortas (n=10 biological replicates). The values were normalized to Rn18s. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( A - B ) Mouse primary peritoneal macrophages ( MPMs ) isolated from DCLK1f / f and DCLK1MCKO mice were challenged with oxLDL ( 50 μg / mL ) for 24 h . Protein levels of TNF - α ( A ) and IL - 6 ( B ) were analyzed using ELISA ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(A-B) Mouse primary peritoneal macrophages (MPMs) isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α (A) and IL-6 (B) were analyzed using ELISA (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( C - D ) MPMs isolated from DCLK1f / f and DCLK1MCKO mice were challenged with oxLDL ( 50 μg / mL ) for 6 h . mRNA levels of Tnf - α ( C ) and Il - 6 ( D ) were determined via RT - qPCR ( n = 3 biological replicates ) . The values were normalized to β - actin . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(C-D) MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/mL) for 6 h. mRNA levels of Tnf-α (C) and Il-6 (D) were determined via RT-qPCR (n=3 biological replicates). The values were normalized to β-actin. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( J , K ) Western blot analysis ( J ) and densitometric quantification ( K ) of p - p65 and IκBα . GAPDH and p65 were used as loading controls ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(J, K) Western blot analysis (J) and densitometric quantification (K) of p-p65 and IκBα. GAPDH and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( L - M ) MPMs were treated as described in panel ( J ) . Western blot analysis ( L ) and densitometric quantification ( M ) of p65 in nucleus and cytoplasm . GAPDH was used as the loading control for cytosolic fractions . Lamin B1 was used as the loading control for nuclear fractions ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(L-M) MPMs were treated as described in panel (J). Western blot analysis (L) and densitometric quantification (M) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( N - O ) MPMs were treated as described in panel ( J ) . Representative immunofluorescence staining images ( N ) and quantification ( O ) of NF - κB p65 ( red ) translocating into nucleus in MPMs . Cells were counterstained with DAPI ( blue ) . Scale bar = 25 μm , n = 3 biological replicates . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(N-O) MPMs were treated as described in panel (J). Representative immunofluorescence staining images (N) and quantification (O) of NF-κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( P - Q ) Representative immunofluorescence staining images ( P ) and quantification ( Q ) of p - p65 ( red ) in aortic roots . Tissues were counterstained with DAPI ( blue ) . Scale bar = 100 μm , n = 10 biological replicates . p - p65 area was quantified by the proportion of p - p65 positive area to plaque area . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(P-Q) Representative immunofluorescence staining images (P) and quantification (Q) of p-p65 (red) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar=100 μm, n=10 biological replicates. p-p65 area was quantified by the proportion of p-p65 positive area to plaque area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( B ) 293T cells were transfected with Flag - DCLK1 plasmid for 24 h . Control cells were transfected with empty vector ( EV ) . Levels of Flag were measured by Western blot ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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(B) 293T cells were transfected with Flag-DCLK1 plasmid for 24 h. Control cells were transfected with empty vector (EV). Levels of Flag were measured by Western blot (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


( D ) Co - immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag - DCLK1 . Flag - DCLK1 was immunoprecipitated by anti - Flag antibody . IgG , immunoglobulin G . ( E ) Co - immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL ( 50 μg / mL ) for 1 h . DCLK1 was immunoprecipitated by anti - DCLK1 antibody .

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(D) Co-immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag-DCLK1. Flag-DCLK1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G. (E) Co-immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL (50 μg/mL) for 1 h. DCLK1 was immunoprecipitated by anti-DCLK1 antibody.


293T cells were co - transfected with Flag - DCLK1 and si - IKKβ for 24 h . Western blot analysis ( H ) of IκBα , p - IKKβ and p - p65 . GAPDH , IKKβ and p65 were used as loading controls ( n = 3 biological replicates ) .

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293T cells were co-transfected with Flag-DCLK1 and si-IKKβ for 24 h. Western blot analysis (H) of IκBα, p-IKKβ and p-p65. GAPDH, IKKβ and p65 were used as loading controls (n=3 biological replicates).


( B ) MPMs were pretreated with DCLK1 - IN - 1 ( 5 and 10 μM ) or vehicle ( DMSO , 1 ‰ ) for 1 h , followed by exposure of oxLDL ( 50 μg / mL ) for 24 h . Protein levels of TNF - α and IL - 6 were analyzed using ELISA ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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(B) MPMs were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α and IL-6 were analyzed using ELISA (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


( D - E ) MPMs were pretreated with DCLK1 - IN - 1 ( 5 and 10 μM ) or vehicle ( DMSO , 1 ‰ ) for 1 h , followed by exposure of oxLDL ( 50 μg / mL ) for 1 h . Western blot analysis ( D ) and densitometric quantification ( E ) of IκBα , p - IKKβ and p - p65 . GAPDH , IKKβ and p65 were used as loading controls ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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(D-E) MPMs were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 1 h. Western blot analysis (D) and densitometric quantification (E) of IκBα, p-IKKβ and p-p65. GAPDH, IKKβ and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


( F - G ) MPMs were treated as described in panel ( E ) . Western blot analysis ( F ) and densitometric quantification ( G ) of p65 in nucleus and cytoplasm . GAPDH was used as the loading control for cytosolic fractions . Lamin B1 was used as the loading control for nuclear fractions ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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(F-G) MPMs were treated as described in panel (E). Western blot analysis (F) and densitometric quantification (G) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


( H - I ) MPMs were treated as described in panel ( D ) . Representative immunofluorescence staining images ( H ) and quantification ( I ) of NF - κB p65 ( red ) translocating into nucleus in MPMs . Cells were counterstained with DAPI ( blue ) . Scale bar = 25 μm , n = 3 biological replicates . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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(H-I) MPMs were treated as described in panel (D). Representative immunofluorescence staining images (H) and quantification (I) of NF-κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


Representative images ( C ) of plaque lesion area in aortic arches ( n = 10 biological replicates ) . The plaque area was quantified by the proportion of plaque area to aortic arches area .

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Representative images (C) of plaque lesion area in aortic arches (n=10 biological replicates). The plaque area was quantified by the proportion of plaque area to aortic arches area.


( K - N ) Protein ( K - L ) and mRNA ( M - N ) levels of inflammatory cytokines TNF - α and IL - 6 in serum and aortas . The values of mRNA levels were normalized to Rn18s ( n = 10 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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(K-N) Protein (K-L) and mRNA (M-N) levels of inflammatory cytokines TNF-α and IL-6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


B Flow cytometry based on CD49f and EpCAM staining separates lineage - negative breast tissue cells into stromal ( CD49f - EpCAM - ) and epithelial cells , which includes basal ( CD49f + EpCAMlo / - ) , luminal progenitor ( LP ) ( CD49f + EpCAM + ) and mature luminal ( ML ) ( CD49f - EpCAM + ) cells .

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B Flow cytometry based on CD49f and EpCAM staining separates lineage-negative breast tissue cells into stromal (CD49f-EpCAM-) and epithelial cells, which includes basal (CD49f+EpCAMlo/-), luminal progenitor (LP) (CD49f+EpCAM+) and mature luminal (ML) (CD49f-EpCAM+) cells.


C t - SNE plot of the integrated scRNA - seq profiles of epithelial cells from 11 reduction mammoplasties . Cell colors correspond to tissue specimens . D Same t - SNE plot as ( C ) but separated by hormonal status of the donor ( 8 premenopausal and 3 postmenopausal ) .

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C t-SNE plot of the integrated scRNA-seq profiles of epithelial cells from 11 reduction mammoplasties. Cell colors correspond to tissue specimens. D Same t-SNE plot as (C) but separated by hormonal status of the donor (8 premenopausal and 3 postmenopausal).


A t - SNE map of combined scRNA - seq transcriptomes of total tissue cells from 13 reduction mammoplasties . Cell colors correspond to tissue specimens . B Same t - SNE map as ( A ) but colored by cell cluster ( with cluster resolution 0 . 05 ) . C Ternary plot positioning each cell according the proportion of basal , LP or ML signature genes expressed by that cell . The plot shows the same cells and cell colors as for ( B ) .

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A t-SNE map of combined scRNA-seq transcriptomes of total tissue cells from 13 reduction mammoplasties. Cell colors correspond to tissue specimens. B Same t-SNE map as (A) but colored by cell cluster (with cluster resolution 0.05). C Ternary plot positioning each cell according the proportion of basal, LP or ML signature genes expressed by that cell. The plot shows the same cells and cell colors as for (B).


D Re - clustered EPCAM - negative non - epithelial cells revealed seven clusters ( resolution 0 . 05 ) representing immune and stromal cell lineages . E Multidimensional scaling plot showing expression profile distances between the pseudo - bulk samples . Each dot corresponds to aggregated expression for a cell cluster for one patient . Cluster colors are overlaid from ( D , right panel ) . Distances on the plot correspond to leading log2 - fold - change , defined as the average log2 - fold - change for the 500 most differential genes between each pair of profiles . F Heat map of pseudo - bulk samples showing marker genes for each non - epithelial cluster . The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo - bulk read counts . Color bars at the top of the plot indicate the cluster and menopausal status ( blue / yellow for pre / post menopause ) of each sample . G Same t - SNE map of non - epithelial cells as in ( D ) but colored by menopausal status . Barplot shows relative cluster sizes ( percentage of total cells ) for each status condition . Clusters 1 and 2 have significantly different sizes in post - menopause samples after allowing for inter - patient variability ( P = 0 . 040 and P = 0 . 032 by quasi - binomial F - test ) . Sizes are not significantly different for other clusters ( P > 0 . 15 ) . H Relative cluster sizes as for ( G ) but by individual patients . Cluster colors correspond to ( D ) . by cluster . A quasi - multinomial F - test was used to test for differences in cluster frequencies between pre - and post - menopausal samples ( P = 0 . 007 ) . I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre - and post - menopausal samples and the cell clusters identified in Fig 2D .

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D Re-clustered EPCAM-negative non-epithelial cells revealed seven clusters (resolution 0.05) representing immune and stromal cell lineages. E Multidimensional scaling plot showing expression profile distances between the pseudo-bulk samples. Each dot corresponds to aggregated expression for a cell cluster for one patient. Cluster colors are overlaid from (D, right panel). Distances on the plot correspond to leading log2-fold-change, defined as the average log2-fold-change for the 500 most differential genes between each pair of profiles. F Heat map of pseudo-bulk samples showing marker genes for each non-epithelial cluster. The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Color bars at the top of the plot indicate the cluster and menopausal status (blue/yellow for pre/post menopause) of each sample. G Same t-SNE map of non-epithelial cells as in (D) but colored by menopausal status. Barplot shows relative cluster sizes (percentage of total cells) for each status condition. Clusters 1 and 2 have significantly different sizes in post-menopause samples after allowing for inter-patient variability (P = 0.040 and P=0.032 by quasi-binomial F-test). Sizes are not significantly different for other clusters (P > 0.15). H Relative cluster sizes as for (G) but by individual patients. Cluster colors correspond to (D). by cluster. A quasi-multinomial F-test was used to test for differences in cluster frequencies between pre- and post-menopausal samples (P = 0.007). I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre- and post-menopausal samples and the cell clusters identified in Fig 2D.


A Microenvironment t - SNE map separated by menopausal status and colored by expression ( red = high expression , grey = undetectable ) of selected fibroblast markers ( upper panel ) . The dotted lines indicate the pericyte subsets . Bottom panel shows percentage of cells expressing the markers for the cell

A Microenvironment t-SNE map separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clusters


B Co - immunofluorescence staining of pre - versus post - menopausal tissue for E - cadherin ( cyan ) , PDGFRβ ( yellow ) and F - actin ( pink ) . DAPI is shown in grey . The arrowheads in ( v ) depict fibroblasts in direct contact with the myoepithelial layer . For 2D and 3D confocal imaging : n = 13 premenopausal and n = 10 postmenopausal specimens . Scale bars : wholemount and optical sections : 100 μm ( panels i - iv ) ; enlargements , 30 μm ( panel v ) .

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B Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).


C t - SNE plot of the integrated scRNA - seq profiles of fibroblasts from pre - and post - menopausal tissue Cell colors correspond to tissue specimens . D Same t - SNE plot as in ( C ) showing 5 distinct clusters ( clusters 3 and 5 were specific to one patient ) .

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C t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue Cell colors correspond to tissue specimens. D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient).


F Heat map of same cells as ( D ) showing expression of the top 20 marker genes in each cluster . Color bars at the top of the plot show cluster membership ( colors as in ( D ) ) and pre - or post - menopausal status ( blue and yellow , respectively ) .

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F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).


A t - SNE map of combined scRNA - seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers ( BRCA1 ; n = 4 ) and non - BRCA premenopausal women ( n = 8 ) with no family history of breast cancer . Cell colors represent individual samples . B Same t - SNE map as in ( A ) but colored by cluster ( cluster resolution 0 . 12 ) . C Epithelial clusters were identified by EPCAM expression and the non - epithelial cells were re - clustered to reveal immune and stromal cell populations ( cluster resolution 0 . 08 ) . Lineage identity was determined by hierarchical clustering according to top marker

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A t-SNE map of combined scRNA-seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers (BRCA1; n=4) and non-BRCA premenopausal women (n=8) with no family history of breast cancer. Cell colors represent individual samples. B Same t-SNE map as in (A) but colored by cluster (cluster resolution 0.12). C Epithelial clusters were identified by EPCAM expression and the non-epithelial cells were re-clustered to reveal immune and stromal cell populations (cluster resolution 0.08). Lineage identity was determined by hierarchical clustering according to top marker genes


D t - SNE plot showing the combined single - cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs ( n = 4 for each ) , colored according to individual patients ( B1 = preneoplastic BRCA1 + / - tissue ; TN - B1 = BRCA1 - associated TNBCs ) . E Same t - SNE map as ( D ) but colored by cluster ( cluster resolution 0 . 15 ) . F Expression of epithelial markers indicated on the combined t - SNE plot as in ( D , E ) for BRCA1 preneoplastic and BRCA1 - associated tumor cells . G Epithelial clusters were identified by EPCAM expression and non - epithelial cells are indicated by the dotted line . H Same t - SNE map as in ( D , E ) but colored according to cancerous state : preneoplastic tissue ( blue ) and BRCA1 - associated TNBCs ( yellow ) . I Box plots of signature expression scores for the 13 cell clusters in ( D , E ) according to human breast epithelial and stromal gene signatures . Cluster 1 corresponds to tumor cells , while clusters 6 , 9 and 10 represent adjacent normal LP , basal and ML cells , respectively . Box plots show quartiles , minimum and maximum .

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D t-SNE plot showing the combined single-cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs (n=4 for each), colored according to individual patients (B1 = preneoplastic BRCA1+/- tissue; TN-B1= BRCA1-associated TNBCs). E Same t-SNE map as (D) but colored by cluster (cluster resolution 0.15). F Expression of epithelial markers indicated on the combined t-SNE plot as in (D, E) for BRCA1 preneoplastic and BRCA1-associated tumor cells. G Epithelial clusters were identified by EPCAM expression and non-epithelial cells are indicated by the dotted line. H Same t-SNE map as in (D, E) but colored according to cancerous state: preneoplastic tissue (blue) and BRCA1-associated TNBCs (yellow). I Box plots of signature expression scores for the 13 cell clusters in (D, E) according to human breast epithelial and stromal gene signatures. Cluster 1 corresponds to tumor cells, while clusters 6, 9 and 10 represent adjacent normal LP, basal and ML cells, respectively. Box plots show quartiles, minimum and maximum.


A Reclustered EPCAM - negative cells ( excluding clusters 1 , 6 , 9 and 10 revealed immune / stromal cells in the microenvironment , identified using lineage markers . B Combined t - SNE plot as in ( A ) of the single - cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers ( blue ; preneo ) and BRCA1 - associated TNBCs ( yellow ; tumor ) . C t - SNE plots showing expression profiles of cardinal markers of immune and stromal cells . D Bar plots showing the percentage of cells expressing typical immune cell ( including Treg ) genes for clusters in ( A ) by preneoplastic vs tumor . E Left panel , same t - SNE map as in ( A ) but separated into cells from preneoplastic ( blue ) and tumor specimens ( yellow ) . Endothelial cell ( endo ) , fibroblast cell ( fibro ) and pericytes ( peri ) clusters are marked . Right panel , proportion of clusters as in ( A ) according to tissue type . F Relative cluster sizes as in ( E ) but by individual patient .

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A Reclustered EPCAM-negative cells (excluding clusters 1, 6, 9 and 10 revealed immune/stromal cells in the microenvironment, identified using lineage markers. B Combined t-SNE plot as in (A) of the single-cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers (blue; preneo) and BRCA1-associated TNBCs (yellow; tumor). C t-SNE plots showing expression profiles of cardinal markers of immune and stromal cells. D Bar plots showing the percentage of cells expressing typical immune cell (including Treg) genes for clusters in (A) by preneoplastic vs tumor. E Left panel, same t-SNE map as in (A) but separated into cells from preneoplastic (blue) and tumor specimens (yellow). Endothelial cell (endo), fibroblast cell (fibro) and pericytes (peri) clusters are marked. Right panel, proportion of clusters as in (A) according to tissue type. F Relative cluster sizes as in (E) but by individual patient.


G Co - immunofluorescence of tissue stained for the epithelial marker ( cytokeratin 19 ; yellow ) and the tumor - associated macrophage marker CX3CR1 ( magenta ) . DAPI is shown in white ( n = 2 preneoplastic samples ; n = 2 tumors ) . Scale bar , 100 μm .

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G Co-immunofluorescence of tissue stained for the epithelial marker (cytokeratin 19; yellow) and the tumor-associated macrophage marker CX3CR1 (magenta). DAPI is shown in white (n=2 preneoplastic samples; n=2 tumors). Scale bar, 100 μm.


H Heat map of top differentially expressed genes in the major immune / stromal cell clusters , identified in ( A ) . BRCA1 + / - preneoplastic cells , blue ; BRCA1 - associated tumor cells , yellow .

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H Heat map of top differentially expressed genes in the major immune/stromal cell clusters, identified in (A). BRCA1+/- preneoplastic cells, blue; BRCA1-associated tumor cells, yellow.


A - C t - SNE plots of combined scRNA - seq profiles of total cells from 8 TNBC tumors , 6 HER2 + tumors and 13 ER + tumors respectively . Integration and cluster sizes for the same cells Cells colored by cluster ( top left panels ) , EPCAM expression ( top right ) , cancer subtype marker ( bottom left ) or MKI67 expression ( bottom right ) . Dotted lines delineate epithelial cells ( top panels ) and cycling epithelial cells ( bottom panels ) . Normal epithelial subsets ( normal ) are also demarcated by dotted lines in the upper - right panels of B and C . D InferCNV plots to map inferred copy number variation ( CNV ) for the combined tumor scRNA - seq expression data for the epithelial clusters indicated in panels A - C . scRNA - seq data from normal breast epithelial cells ( N - 1105 - epi ) served as a reference for normalization . Each row represents a gene and each column cells from a cluster in a single tumor . The tumor clusters are color - coded as in A - C . Amplifications ( red ) and deletions ( blue ) are inferred from the gene expression . Tumor cells were distinguished from normal ( N ) cells by abundance of CNV .

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A-C t-SNE plots of combined scRNA-seq profiles of total cells from 8 TNBC tumors, 6 HER2+ tumors and 13 ER+ tumors respectively. Integration and cluster sizes for the same cells Cells colored by cluster (top left panels), EPCAM expression (top right), cancer subtype marker (bottom left) or MKI67 expression (bottom right). Dotted lines delineate epithelial cells (top panels) and cycling epithelial cells (bottom panels). Normal epithelial subsets (normal) are also demarcated by dotted lines in the upper-right panels of B and C. D InferCNV plots to map inferred copy number variation (CNV) for the combined tumor scRNA-seq expression data for the epithelial clusters indicated in panels A-C. scRNA-seq data from normal breast epithelial cells (N-1105-epi) served as a reference for normalization. Each row represents a gene and each column cells from a cluster in a single tumor. The tumor clusters are color-coded as in A-C. Amplifications (red) and deletions (blue) are inferred from the gene expression. Tumor cells were distinguished from normal (N) cells by abundance of CNV.


H Combined t - SNE transcriptome profiles of two distinct ER + tumors isolated from the same breast of a patient : ER - 0029 - 7C ( blue ) and ER - 0029 - 9C ( yellow ) . The corresponding t - SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right - hand panel .

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H Combined t-SNE transcriptome profiles of two distinct ER+ tumors isolated from the same breast of a patient: ER-0029-7C (blue) and ER-0029-9C (yellow). The corresponding t-SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right-hand panel.


A - C t - SNE maps of reclustered EPCAM - negative non - epithelial cells Cluster resolutions 0 . 136 , 0 . 1 and 0 . 1 respectively . The major cell clusters within the microenvironment were identified based on expression of lineage - specific genes . Heat maps of pseudo - bulk samples show marker genes for each cluster . The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo - bulk read counts . Cluster 9 in HER2 + tumors ( B ) expressed myeloid and luminal epithelial markers , suggesting phagocytosis of the latter by macrophages . Color bars at the top of the heat map indicate the cluster of each sample ; top genes that mark each cluster are indicated .

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A-C t-SNE maps of reclustered EPCAM-negative non-epithelial cells Cluster resolutions 0.136, 0.1 and 0.1 respectively. The major cell clusters within the microenvironment were identified based on expression of lineage-specific genes. Heat maps of pseudo-bulk samples show marker genes for each cluster. The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Cluster 9 in HER2+ tumors (B) expressed myeloid and luminal epithelial markers, suggesting phagocytosis of the latter by macrophages. Color bars at the top of the heat map indicate the cluster of each sample; top genes that mark each cluster are indicated.


A , B Image quantification showing number of CD8 + Ki67 + cells ( A ) or CD68 + Ki67 + cells ( B ) per mm2 of tissue from TNBC , HER2 + and ER + tumors , either within the tumor region ( K8 / 18 + ) or the stroma ( defined as > 5 μm from tumor border ) . Error bars represent s . e . m . CD8 / Ki67 : n = 11 for TNBC , n = 12 for ER + , n = 5 for HER2 + . CD68 / Ki67 : n = 6 for TNBC , n = 8 for ER + , n = 5 for HER2 + .

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A, B Image quantification showing number of CD8+Ki67+ cells (A) or CD68+Ki67+ cells (B) per mm2 of tissue from TNBC, HER2+ and ER+ tumors, either within the tumor region (K8/18+) or the stroma (defined as >5 μm from tumor border). Error bars represent s.e.m. CD8/Ki67: n=11 for TNBC, n=12 for ER+, n=5 for HER2+. CD68/Ki67: n=6 for TNBC, n=8 for ER+, n=5 for HER2+.


C - F Representative confocal images of ER + ( ER - 0032 ) and triple negative tumors ( TN - 0066 ) immunolabeled for K8 / 18 ( yellow ) , CD68 ( green ) and Ki67 ( red ) ( C , E ) or K8 / 18 ( yellow ) , CD8 ( green ) and Ki67 ( red ) ( D , F ) . DAPI is shown in blue . Arrows depict proliferative T cells ( CD8 + Ki67 + ) or macrophages ( CD68 + Ki67 + ) . Enlargements on shown in the right - hand panels . Scale bars , 200 µm for large tilescans ; 50 µm for enlargements and smaller tilescans .

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C-F Representative confocal images of ER+ (ER-0032) and triple negative tumors ( TN-0066) immunolabeled for K8/18 (yellow), CD68 (green) and Ki67 (red) (C, E) or K8/18 (yellow), CD8 (green) and Ki67 (red) (D, F). DAPI is shown in blue. Arrows depict proliferative T cells (CD8+Ki67+) or macrophages (CD68+Ki67+). Enlargements on shown in the right-hand panels. Scale bars, 200 µm for large tilescans; 50 µm for enlargements and smaller tilescans.


C Immunostaining of tumor and LN from patient ER - 0064 for expression of ER , PR and pan - cytokeratin . Insets show sections stained with control isotype antibodies . PR , progesterone receptor . Scale bar , 100 μm .

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C Immunostaining of tumor and LN from patient ER-0064 for expression of ER, PR and pan-cytokeratin. Insets show sections stained with control isotype antibodies. PR, progesterone receptor. Scale bar, 100 μm.