( C ) Left : Immunoblot analysis of TGFRs , phosphorylated Smad2 ( p - Smad2 ) , and phosphorylated Smad3 ( p - Smad3 ) in control and FRS2 knockdown HASMCs . Blots are representative of four independent experiments . Right : Band intensities of p - Smad2 , p - Smad3 , TGFR1 , and TGFR2 were normalized to Smad2 / 3 or GAPDH and expressed as a fraction of a control value . Results are expressed as means ± SD ( * p < 0 . 05 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) .

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(C) Left: Immunoblot analysis of TGFRs, phosphorylated Smad2 (p-Smad2), and phosphorylated Smad3 (p-Smad3) in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of p-Smad2, p-Smad3, TGFR1, and TGFR2 were normalized to Smad2/3 or GAPDH and expressed as a fraction of a control value. Results are expressed as means ± SD (*p<0.05; ***p<0.001 compared to control; unpaired two-tailed Student's t test).


( A ) Left : Immunoblot analysis of smooth muscle marker gene expression in control and FRS2 knockdown HASMCs . Blots are representative of four independent experiments . Right : Band intensities of SM - actin , SM22 , and SM - calponin were normalized to - tubulin and expressed as a fraction of a control value . Results are expressed as means ± SD ( * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) .

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(A) Left: Immunoblot analysis of smooth muscle marker gene expression in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of SM -actin, SM22, and SM-calponin were normalized to -tubulin and expressed as a fraction of a control value. Results are expressed as means ± SD (**p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test).


( B ) qRT - PCR analysis of SMC transcription factor gene expression in control and FRS2 knockdown HASMCs . ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test . N = 3 ) . - actin was used for sample loading normalization .

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(B) qRT-PCR analysis of SMC transcription factor gene expression in control and FRS2 knockdown HASMCs. (*p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test. N=3). -actin was used for sample loading normalization.


( C ) Collagen gel contraction assays were used to determine the contractile ability of control or FRS2 knockdown HASMCs ( * p < 0 . 05 compared to control ; unpaired two - tailed Student ' s t test . N = 3 ) .

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(C) Collagen gel contraction assays were used to determine the contractile ability of control or FRS2 knockdown HASMCs (*p<0.05 compared to control; unpaired two-tailed Student's t test. N=3).


( D - F ) Upper panels : Immunoblots of smooth muscle markers , phosphorylated Smad2 ( p - Smad2 ) , and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 ( 10 m ) , TGFR2 or Smad2 shRNA lentiviruses . Blots are representative of three independent experiments . Bottom panels : Band intensities of SM - calponin and p - Smad2 were normalized to - tubulin , HSP90 , or Smad2 and expressed as a fraction of a control value .

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(D-F) Upper panels: Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 (10 m), TGFR2 or Smad2 shRNA lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to -tubulin, HSP90, or Smad2 and expressed as a fraction of a control value.


( A ) Quantitative real - time PCR analysis of mature let - 7 family in control and FRS2 knockdown HASMCs . SNORD47 was used to normalize the variability in template loading . Histogram of qRT - PCR results are three independent experiments .

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(A) Quantitative real-time PCR analysis of mature let-7 family in control and FRS2 knockdown HASMCs. SNORD47 was used to normalize the variability in template loading. Histogram of qRT-PCR results are three independent experiments.


( B ) Upper panel : Immunoblots of SM - calponin , phosphorylated Smad2 ( p - Smad2 ) , and TGFR1 expression in control and FRS2 knockdown HASMCs transduced with or without let - 7b lentiviruses . Blots are representative of three independent experiments . Bottom panels : Band intensities of SM - calponin and p - Smad2 were normalized to GAPDH or Smad2 and expressed as a fraction of a control value .

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(B) Upper panel: Immunoblots of SM-calponin, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs transduced with or without let-7b lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to GAPDH or Smad2 and expressed as a fraction of a control value.


( C ) Phase - contrast and immunofluorescence staining of smooth muscle markers ( red ) in HASMCs . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 12 m . Images are representative of three independent experiments .

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(C) Phase-contrast and immunofluorescence staining of smooth muscle markers (red) in HASMCs. Nuclei were counterstained with DAPI (blue). Scale bar: 12 m. Images are representative of three independent experiments.


( D ) HASMCs were cultured in the growth medium ( M231 + SMGS ) at day 0 then switched from growth conditions to differentiation medium ( M231 + SMDS ) for 8 days . Quantitative real - time PCR analysis of mature let - 7 family in HASMCs . SNORD47 was used to normalize the variability in template loading . Histogram of qRT - PCR results are three independent experiments .

(D) HASMCs were cultured in the growth medium (M231 + SMGS) at day 0 then switched from growth conditions to differentiation medium (M231 + SMDS) for 8 days. Quantitative real-time PCR analysis of mature let-7 family in HASMCs. SNORD47 was used to normalize the variability in template loading. Histogram of qRT-PCR results are three independent experiments.


( E ) Control and FRS2 knockdown HASMCs were cultured in the growth medium ( M231 + SMGS ) at day 0 then switched from growth conditions to differentiation medium ( M231 + SMDS ) for 6 days with or without let - 7b lentiviruses . Immunoblots of smooth muscle markers , phosphorylated Smad2 ( p - Smad2 ) , and TGFR1 expression in control and FRS2 knockdown HASMCs with or without let - 7b lentiviruses . Blots are representative of three independent experiments .

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(E) Control and FRS2 knockdown HASMCs were cultured in the growth medium (M231 + SMGS) at day 0 then switched from growth conditions to differentiation medium (M231 + SMDS) for 6 days with or without let-7b lentiviruses. Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs with or without let-7b lentiviruses. Blots are representative of three independent experiments.


( A ) Coronary arteries dissected from the human heart . Left main ( LM ) , left anterior descending ( LAD ) , and left circumflex ( LCX ) branches . Scale bar : 1 cm .

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(A) Coronary arteries dissected from the human heart. Left main (LM), left anterior descending (LAD), and left circumflex (LCX) branches. Scale bar: 1 cm.


( B ) Elastic - Van Gieson ( EVG ) staining of human coronary arteries demonstrating various degrees of atherosclerosis .

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(B) Elastic-Van Gieson (EVG) staining of human coronary arteries demonstrating various degrees of atherosclerosis.


( C - D ) Representative images of immunofluorescence staining for CD31 ( green ) and SM - actin ( red ) or SM - MHC ( red ) in No / mild , moderate , and severe disease human left main coronary arteries . No : no - disease . Nuclei were stained with DAPI ( blue ) . ( b ) & amp ; ( d ) & amp ; ( f ) are high - magnification view from ( a ) & amp ; ( c ) & amp ; ( e ) . Images are representative of ten No / mild , nine moderate and ten severe disease human left main coronary artery samples . L : lumen . Scale bar : 16 m .

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(C-D) Representative images of immunofluorescence staining for CD31 (green) and SM -actin (red) or SM-MHC (red) in No/mild, moderate, and severe disease human left main coronary arteries. No: no-disease. Nuclei were stained with DAPI (blue). (b)&(d)&(f) are high-magnification view from (a)&(c)&(e). Images are representative of ten No/mild, nine moderate and ten severe disease human left main coronary artery samples. L: lumen. Scale bar: 16 m.


( E & amp ; G ) Representative images of immunofluorescence staining for p - FGFR1 ( red ) or FGFR1 ( red ) in the same patient cohort . Nuclei were counter - stained with DAPI ( blue ) . Scale bar : 16 m .

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(E&G) Representative images of immunofluorescence staining for p-FGFR1 (red) or FGFR1 (red) in the same patient cohort. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m.


( F & amp ; H ) Percentage of medial p - FGFR1 + SMC and FGFR1 + SMC ( * * * p < 0 . 001 compared to No / mild disease , NS : not significant compared to No / mild disease ; one - way ANOVA with Newman - Keuls post hoc test for multiple comparison correction ) .

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(F&H) Percentage of medial p-FGFR1+ SMC and FGFR1+ SMC (***p<0.001 compared to No/mild disease, NS: not significant compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction).


Figure 5 . TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis . ( A & C & E ) Representative images of immunofluorescence staining for TGF ( red ) , p - Smad2 ( red ) , or p - Smad3 ( red ) from patients with No / mild , moderate , or severe disease . Nuclei were counter - stained with DAPI ( blue ) . Scale bar : 16 m . ( B & D & F ) Medial TGF area and percentage of medial p - Smad2 , and p - Smad3 positive SMCs ( * * * p < 0 . 001 compared to No / mild disease ; one - way ANOVA with Newman - Keuls post hoc test for multiple comparison correction ) . A full table of p - values for this figure is shown in Appendix Table S1 .

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Figure 5. TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis. (A&C&E) Representative images of immunofluorescence staining for TGF (red), p-Smad2 (red), or p-Smad3 (red) from patients with No/mild, moderate, or severe disease. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m. (B&D&F) Medial TGF area and percentage of medial p-Smad2, and p-Smad3 positive SMCs (***p<0.001 compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction). A full table of p-values for this figure is shown in Appendix Table S1.


( A ) Dissected mouse aorta demonstrating lipid - rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe - / - mice . ( b ) & ( d ) Cross - section of brachiocephalic artery from ( a ) & ( c ) stained with Oil Red O . L : lumen . Scale bar : 4 mm . 3 mice per group .

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(A) Dissected mouse aorta demonstrating lipid-rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe-/- mice. (b) & (d) Cross-section of brachiocephalic artery from (a) & (c) stained with Oil Red O. L: lumen. Scale bar: 4 mm. 3 mice per group.


( B ) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti - SM - actin , anti - Notch3 , and anti - SM - MHC antibodies . Nuclei were counterstained with DAPI ( blue ) . L : lumen . Scale bar : 62 m . 3 mice per group .

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(B) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti-SM -actin, anti-Notch3, and anti-SM-MHC antibodies. Nuclei were counterstained with DAPI (blue). L: lumen. Scale bar: 62 m. 3 mice per group.


( C - F ) Analysis of brachiocephalic artery of Apoe - / - mice maintained for 4 months on either normal or high fat diet using anti - CD31 ( green ) , anti - p - FGFR1 ( red ) , anti - FGFR1 ( red ) , anti - p - Smad2 ( red ) , and anti - p - Smad3 ( red ) antibodies . Nuclei counterstained with DAPI ( blue ) . Scale bar : 62 m . L : lumen . M : Media . 6 mice per group .

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(C-F) Analysis of brachiocephalic artery of Apoe-/- mice maintained for 4 months on either normal or high fat diet using anti-CD31 (green), anti-p-FGFR1 (red), anti-FGFR1 (red), anti-p-Smad2 (red), and anti-p-Smad3 (red) antibodies. Nuclei counterstained with DAPI (blue). Scale bar: 62 m. L: lumen. M: Media. 6 mice per group.


( G - J ) Quantification of the number of medial smooth muscle cells expressing p - FGFR1 , FGFR1 , p - Smad2 , and p - Smad3 ( * * * p < 0 . 001 compared to ND , NS : not significant compared to ND ; unpaired two - tailed Student ' s t test . ) . ND : Normal diet . HFD : High fat diet . A full table of p - values for this figure is shown in Appendix Table S1 .

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(G-J) Quantification of the number of medial smooth muscle cells expressing p-FGFR1, FGFR1, p-Smad2, and p-Smad3 (***p<0.001 compared to ND, NS: not significant compared to ND; unpaired two-tailed Student's t test.). ND: Normal diet. HFD: High fat diet. A full table of p-values for this figure is shown in Appendix Table S1.


( A ) Representative photomicrographs of Oil Red O - stained atherosclerotic lesions in the aortic arch , of Apoe - / - or Frs2SMCKO / Apoe - / - mice after 16 weeks of high fat diet . 3 mice per group . Scale bar : 5 mm .

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(A) Representative photomicrographs of Oil Red O-stained atherosclerotic lesions in the aortic arch, of Apoe-/- or Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet. 3 mice per group. Scale bar: 5 mm.


( B ) ( Left ) Microphotographs of aortas ( en face ) from Apoe - / - and Frs2SMCKO / Apoe - / - mice after 16 weeks of high fat diet after staining with Oil Red O . ( Right ) Lesion area quantification . All data shown as mean ± SD . ( * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) . 12 mice per group .

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(B) (Left) Microphotographs of aortas (en face) from Apoe-/- and Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet after staining with Oil Red O. (Right) Lesion area quantification. All data shown as mean ± SD. (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test). 12 mice per group.


( C - D ) Representative cross - sections of brachiocephalic arteries Apoe - / - and Frs2SMCKO / Apoe - / - mice stained with hematoxylin and eosin ( H & amp ; E ) ( C ) and Movat ( D ) . ( b ) & amp ; ( d ) are high - magnification view from ( a ) & amp ; ( c ) . NC : necrotic core . Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 .

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(C-D) Representative cross-sections of brachiocephalic arteries Apoe-/- and Frs2SMCKO/Apoe-/- mice stained with hematoxylin and eosin (H&E) (C) and Movat (D). (b)&(d) are high-magnification view from (a)&(c). NC: necrotic core. Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.


( E ) Histological analysis of atheosclerotic plaque with anti - Ki67 antibody . Nuclei were counterstained with DAPI ( blue ) . ( b ) & ( d ) are high - magnification images from ( a ) & ( c ) . Scale bar : 62 m ( low - magnification images ) ; 10 m ( high - magnification images ) . Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 .

(E) Histological analysis of atheosclerotic plaque with anti-Ki67 antibody. Nuclei were counterstained with DAPI (blue). (b)&(d) are high-magnification images from (a)&(c). Scale bar: 62 m (low-magnification images); 10 m (high-magnification images). Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.


( F ) Quantification of plaque cellularity ; Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 ( * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

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(F) Quantification of plaque cellularity; Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12 (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test).


( G ) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe - / - and Frs2SMCKO / Apoe - / - mice . ( * p < 0 . 05 , * * p < 0 . 01 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

(G) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe-/- and Frs2SMCKO/Apoe-/- mice. (*p<0.05, **p<0.01 compared to Apoe-/-; unpaired two-tailed Student's t test).


( H ) Measurement of Ki67 + cells ( * p < 0 . 05 , * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

(H) Measurement of Ki67+ cells (*p<0.05, ***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test).


( A ) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC - labeled zymosan ( 3 mg / ml , 10 µl ) in one hind paw . White dotted lines show the position of zymosan .

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(A) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC-labeled zymosan (3 mg/ml, 10 µl) in one hind paw. White dotted lines show the position of zymosan.


Quantification of the number of macrophages and their subtypes 8 , 24 , 48 and 72 hours after zymosan injection based on the MELC images . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * * p < 0 . 001 . # p < 0 . 05 , # # # p < 0 . 001 as compared to naïve mice .

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Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.


FACS analysis of macrophages and their subtypes 8 , 24 and 48 hours after zymosan injection . Data are shown as mean ( n = 8 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 .

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FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.


( A ) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM ( n . d . , not determinable ) .

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(A) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM (n.d., not determinable).


Representative MELC images showing the IL - 4 and Siglec F expression 4 hours ( panel C ) after zymosan injection . The white dotted lines depict the area where the transition between the neighboring regions occurs .

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Representative MELC images showing the IL-4 and Siglec F expression 4 hours (panel C) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.


Representative MELC images showing the IL - 4 and Siglec F expression 24 hours ( panel D ) after zymosan injection . The white dotted lines depict the area where the transition between the neighboring regions occurs .

Representative MELC images showing the IL-4 and Siglec F expression 24 hours (panel D) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.


( E ) Number of IL - 4 - expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM , two - tailed Students t - test * * * p < 0 . 001 .

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(E) Number of IL-4-expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, two-tailed Students t-test ***p<0.001.


( F ) Number of IL - 4 - expressing eosinophils in the three regions 24 hours after zymosan injection . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) Number of IL-4-expressing eosinophils in the three regions 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.


( G ) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw . Data are shown as mean ( n = 5 mice ) ± SEM , One Way ANOVA / Bonferroni * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(G) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, **p<0.01, ***p<0.001.


FACS analysis of eosinophils in the blood B ; n = 6 mice ) at the indicated time points after zymosan injection . Anti - Siglec F antibody or the IgG - control ( control ) were administered with 0 , 883mg / kg 24 hours prior zymosan injection . Data are shown as mean ± SEM , student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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 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

FACS analysis of eosinophils in the blood B; n= 6 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.


FACS analysis of eosinophils in the paws ( panel C n = 8 mice ) at the indicated time points after zymosan injection . Anti - Siglec F antibody or the IgG - control ( control ) were administered with 0 , 883mg / kg 24 hours prior zymosan injection . Data are shown as mean ± SEM , student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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 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

FACS analysis of eosinophils in the paws (panel C n= 8 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.


Zymosan - induced mechanical ( panel E ) hypersensitivity of control or anti - Siglec F antibody - treated mice . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

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

Zymosan-induced mechanical (panel E) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.


Zymosan - induced thermal ( panel F ) hypersensitivity of control or anti - Siglec F antibody - treated mice . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 vs . control mice .

Zymosan-induced thermal (panel F) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.


Quantification of eosinophils , the sum of all macrophages , M2 - like , M1 - like and M0 - macrophages in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


Quantification of eosinophils , the sum of all macrophages , as well as efferocytosis ( panel H ) in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Quantification of eosinophils, the sum of all macrophages, as well as efferocytosis (panel H) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


Quantification of eosinophils , the sum of all macrophages , neutrophils ( panel I ) in the MELC images of paws from control or eosinophil - depleted mice 24 hours after zymosan injection . Data are presented as mean ± SEM ( n = 5 ) . Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Quantification of eosinophils, the sum of all macrophages, neutrophils (panel I) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.


( A ) IL - 4 levels in paws from naive and zymosan - injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil - depleted mice . Data are shown as mean ± SEM ( n = 6 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) IL-4 levels in paws from naive and zymosan-injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil-depleted mice. Data are shown as mean ± SEM (n=6 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: **p<0.01, ***p< 0.001.


( B ) FACS analysis of the eosinophil number in paws from control and eosinophil - depleted mice with or without IL - 4c treatment 24h prior zymosan injection . Data are shown as the mean ± SEM ( n = 8 mice ) . Two - way ANOVA / Bonferroni * * * p < 0 . 001 .

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

(B) FACS analysis of the eosinophil number in paws from control and eosinophil-depleted mice with or without IL-4c treatment 24h prior zymosan injection. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni ***p< 0.001.


( C ) Same as panel B except that resident ( Ly6C - ) and monocyte - derived macrophages ( Ly6C + ) were determined .

O O 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) Same as panel B except that resident (Ly6C-) and monocyte-derived macrophages (Ly6C+) were determined.


Same as panel B except that M2 - like ( panel D ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

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

Same as panel B except that M2-like (panel D) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


Same as panel B except that M1 - like ( panel E ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

Same as panel B except that M1-like (panel E) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


Same as panel B except that M0 macrophages ( panel F ) were determined . Data are shown as mean ± SEM ( n = 8 mice ) . Student ' s t - test / Two - way ANOVA / Bonferroni , control vs anti - Siglec F antibody treatment : * p < 0 . 05 , * * * p < 0 . 001 .

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

Same as panel B except that M0 macrophages (panel F) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.


( A ) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil - depleted mice . Data are mean ± SEM ( n = 6 mice ) . Student ' s One - way ANOVA , significance between control and anti - Siglec F antibody treatment * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O B-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

(A) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil-depleted mice. Data are mean ± SEM (n=6 mice). Student's One-way ANOVA, significance between control and anti-Siglec F antibody treatment *p<0.05; **p<0.01; ***p<0.001.


FACS analysis of the neutrophil count in paws ( panel B ) from control or eosinophil - depleted mice with or without IL - 4c treatment . Data are shown as the mean ± SEM ( n = 6 - 8 ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 .

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

FACS analysis of the neutrophil count in paws (panel B) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.


FACS analysis of the neutrophil count in peripheral blood ( panel C ) from control or eosinophil - depleted mice with or without IL - 4c treatment . Data are shown as the mean ± SEM ( n = 6 - 8 ) . Two - way ANOVA / Bonferroni , * * p < 0 . 01 , * * * p < 0 . 001 .

FACS analysis of the neutrophil count in peripheral blood (panel C) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.


( D ) Same as panel B except that efferocytosis was determined . * p < 0 . 05 ; * * * p < 0 .

O O O 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) Same as panel B except that efferocytosis was determined. *p<0.05; ***p<0.001


( E ) FACS analysis of alive and dying neutrophils in paws from control or eosinophil - depleted mice . Data are shown as the mean ± SEM ( n = 5 mice ) .

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

(E) FACS analysis of alive and dying neutrophils in paws from control or eosinophil-depleted mice. Data are shown as the mean ± SEM (n=5 mice).


( C ) Zymosan - induced thermal hypersensitivity in control or eosinophil - depleted mice with or without IL - 4c treatment 24h hours prior zymosan injection . The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points . Data are shown as the mean ± SEM ( n = 6 mice ) . Two - way ANOVA / Bonferroni , * p < 0 . 05 , * * p < 0 . 01 ; ns , not significant .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Zymosan-induced thermal hypersensitivity in control or eosinophil-depleted mice with or without IL-4c treatment 24h hours prior zymosan injection. The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points. Data are shown as the mean ± SEM (n=6 mice). Two-way ANOVA/Bonferroni, *p< 0.05, **p< 0.01; ns, not significant.


Northern blotting shows that Gasz depletion ( Gasz KD ) led to the accumulation of flam - piRNA precursors but reduced the level of mature idefix - piRNA arising from the precursors , as in the case of Zuc depletion ( Zuc KD ) . U6 snRNA was detected as a 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

Northern blotting shows that Gasz depletion (Gasz KD) led to the accumulation of flam-piRNA precursors but reduced the level of mature idefix-piRNA arising from the precursors, as in the case of Zuc depletion (Zuc KD). U6 snRNA was detected as a loading control.


Western blotting showing the protein levels of endogenous Armi , Piwi , SoYb , Yb , Vret , and Gasz in total OSC lysate ( total ) , the mitochondrial fraction ( mito ) , and the cytoplasmic fraction after mitochondrial isolation ( cyto ) . β - Tubulin ( β - Tub ) and HSP60 were detected as markers for the " cyto " and " mito " fractions , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 blotting showing the protein levels of endogenous Armi, Piwi, SoYb, Yb, Vret, and Gasz in total OSC lysate (total), the mitochondrial fraction (mito), and the cytoplasmic fraction after mitochondrial isolation (cyto). β-Tubulin (β-Tub) and HSP60 were detected as markers for the "cyto" and "mito" fractions, respectively.


Left : Western blotting comparing the abundances of Armi and Piwi in total OSC lysate ( total ) and the mitochondrial fraction ( mito ) before ( control ) and after ( Zuc KD ) Zuc knockdown in OSCs . Relative mito - localization intensity ( * ) shows the amount of proteins in " mito " normalized by the amount of proteins in " total " and HSP60 . Right : Immunofluorescence shows that the Armi signal ( green ) is strongly overlapped with the mitochondrial signal ( red ) in Zuc - depleted OSCs ( Zuc KD ) . Armi is localized to Yb bodies in control OSCs ( control ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Left : Western blotting comparing the abundances of Armi and Piwi in total OSC lysate ( total ) and the mitochondrial fraction ( mito ) before ( control ) and after ( Gasz KD ) Gasz depletion in Zuc - depleted OSCs ( Zuc KD ) . Relative mito - localization intensity ( * ) shows the amount of proteins in " mito " normalized by the amount of proteins in " total " and HSP60 . Right : Immunofluorescence shows that the Armi mitochondrial signal ( green ) found in Zuc - depleted cells ( Zuc KD / control ) mostly disappeared after additional Gasz depletion in the cells ( Zuc KD / Gasz KD ) , but was found at Yb bodies . Mitochondrial signal is shown in red . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Armi and Piwi co - immunoprecipitated with Flag - Gasz ( F - Gasz ) from the mitochondrial fraction ( mito fraction ) of OSCs ( input ) . F - Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation . * : Heavy chains of antibodies used for immunoprecipitation . n . i . : Non - immune IgG used as a negative control .

Armi and Piwi co-immunoprecipitated with Flag-Gasz (F-Gasz) from the mitochondrial fraction (mito fraction) of OSCs (input). F-Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation. *: Heavy chains of antibodies used for immunoprecipitation. n.i.: Non-immune IgG used as a negative control.


Co - immunoprecipitation from whole - cell lysate of OSCs before ( control ) and after ( Zuc KD ) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag - Gasz ( F - Gasz ) complex increased upon Zuc depletion ( Zuc KD ) .

Co-immunoprecipitation from whole-cell lysate of OSCs before (control) and after (Zuc KD) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag-Gasz (F-Gasz) complex increased upon Zuc depletion (Zuc KD).


Northern blotting shows that the flam - piRNA precursors in the Flag - Gasz complex ( Flag ) accumulated upon Zuc depletion ( Zuc KD ) .

Northern blotting shows that the flam-piRNA precursors in the Flag-Gasz complex (Flag) accumulated upon Zuc depletion (Zuc KD).


Upper : The flam - piRNA precursors associated with Piwi disappeared upon Armi depletion ( Armi KD ) in Zuc - lacking OSCs ( Zuc KD ) . Lower : The abundance of immunopurified Piwi is shown by western blotting .

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

Upper: The flam-piRNA precursors associated with Piwi disappeared upon Armi depletion (Armi KD) in Zuc-lacking OSCs (Zuc KD). Lower: The abundance of immunopurified Piwi is shown by western blotting.


Depletion of endogenous Piwi ( Piwi KD ) in Zuc - lacking OSCs ( Zuc KD ) caused Armi ( green ) to relocate to Yb bodies . Localization of Armi at Yb bodies is shown in B . Mitochondrial ( upper panel ) and Yb ( lower panel ) signals are shown in red . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


The Armi complex was immunopurified from Zuc - lacking OSCs ( Zuc KD ) before ( control ) and after ( Piwi KD ) Piwi depletion and probed for detecting Armi and Gasz . Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc - lacking OSCs . Co - IP index ( * ) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc - depleted cells ( Zuc KD / control ) and Zuc - and Piwi - depleted cells ( Zuc KD / Piwi KD ) is 1 : 0 . 05 .

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

The Armi complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and Gasz. Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc-lacking OSCs. Co-IP index (*) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.05.


The Flag - Gasz ( F - Gasz ) complex was immunopurified from Zuc - lacking OSCs ( Zuc KD ) before ( control ) and after ( Piwi KD ) Piwi depletion and probed for detecting Armi and F - Gasz . F - Gasz was ectopically expressed prior to immunoprecipitation . Co - IP index ( * ) shows that the ratio of Armi amount in the F - Gasz complex isolated from Zuc - depleted cells ( Zuc KD / control ) and Zuc - and Piwi - depleted cells ( Zuc KD / Piwi KD ) is 1 : 0 . 31 .

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

The Flag-Gasz (F-Gasz) complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and F-Gasz. F-Gasz was ectopically expressed prior to immunoprecipitation. Co-IP index (*) shows that the ratio of Armi amount in the F-Gasz complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.31.


In vitro pull - down assays show that Armi - Flag ( Armi - F ) directly binds with GST - Gasz ∆ C113 but hardly with GST . Armi - F was immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F was detected by western blotting using anti - Flag antibodies .

In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.


Upper : Northern blotting shows that Flag - Piwi WT ( WT ) and PAZ mutant ( PAZmt ) but not MID mutant ( MIDmt ) bound with flam - piRNA precursors in Zuc - depleted OSCs . Endogenous Piwi was depleted simultaneously with Zuc ( Zuc KD + Piwi KD ) . Zuc was depleted by RNAi and so residual Zuc may have remained in cells . Therefore , a tiny amount of piRNAs was loaded onto Piwi . F - Piwi WT , PAZmt , and MIDmt were made to be RNAi - resistant by mutagenesis ( see Materials and Methods ) . Lower : The abundance of immunopurified Flag - Piwi ( F - Piwi ) is shown by western blotting .

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

Upper: Northern blotting shows that Flag-Piwi WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with flam-piRNA precursors in Zuc-depleted OSCs. Endogenous Piwi was depleted simultaneously with Zuc (Zuc KD + Piwi KD). Zuc was depleted by RNAi and so residual Zuc may have remained in cells. Therefore, a tiny amount of piRNAs was loaded onto Piwi. F-Piwi WT, PAZmt, and MIDmt were made to be RNAi-resistant by mutagenesis (see Materials and Methods). Lower: The abundance of immunopurified Flag-Piwi (F-Piwi) is shown by western blotting.


Armi localization to Yb bodies observed in Zuc - and Piwi - depleted OSCs ( Zuc KD + Piwi KD ) ( Figs 3A and B ) was restored by ectopic expression of Flag - Piwi WT and PAZmt but not of MIDmt ( see also Fig EV4B ) . Armi , mitochondria , and F - Piwi are shown in green , red , and yellow , respectively . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Western blotting shows that Flag - Piwi ( F - Piwi ) WT ( WT ) and PAZ mutant ( PAZmt ) but not MID mutant ( MIDmt ) bound with Armi in Zuc / Piwi - depleted OSCs ( Zuc KD + Piwi KD ) .

O O O O O O O O O O O O O O O O O O O O O O O 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 blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).


Armi - Flag ( Armi - F ) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Immunoprecipitation and subsequent western blotting show that Armi - F WT bound with Piwi and Gasz . The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz . Luciferase - Flag ( Luc - F ) was used as a negative 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 O O O O O

Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT bound with Piwi and Gasz. The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz. Luciferase-Flag (Luc-F) was used as a negative control.


Armi - Flag ( Armi - F ) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Armi - F WT ( green ) localized onto mitochondria ( red ) , whereas Armi - F N756A mutant ( green ) localized to Yb bodies ( see also Fig EV5D ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F N756A mutant (green) localized to Yb bodies (see also Fig EV5D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


In vitro pull - down assays show that both Armi - Flag ( Armi - F ) WT and N756A mutant directly bind with GST - Gasz ∆ C113 but not with GST . Armi - F WT and mutant were immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F WT and N756A mutant were detected by western blotting using anti - Flag antibodies .

In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and N756A mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and N756A mutant were detected by western blotting using anti-Flag antibodies.


Armi - Flag ( Armi - F ) WT and the ∆ N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Immunoprecipitation and subsequent western blotting show that Armi - F WT but not Armi ∆ N34 mutant bound with Gasz in the cells . Both Armi WT and ∆ N34 mutant interacted with Piwi to similar extents . Luciferase - Flag ( Luc - F ) was used as a negative 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 O O O O O O O O O O O O O

Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT but not Armi ∆N34 mutant bound with Gasz in the cells. Both Armi WT and ∆N34 mutant interacted with Piwi to similar extents. Luciferase-Flag (Luc-F) was used as a negative control.


Armi - Flag ( Armi - F ) WT and the ∆ N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Armi - F WT ( green ) localized onto mitochondria ( red ) , whereas Armi - F ∆ N34 mutant ( green ) localized to Yb bodies ( see also Fig EV6D ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


In vitro pull - down assays show that both Armi - Flag ( Armi - F ) WT and ∆ N34 mutant directly bind with GST - Gasz ∆ C113 but not with GST . Armi - F WT and mutant were immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F WT and mutant were detected by western blotting using anti - Flag antibodies .

In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies.


Immunoprecipitation and subsequent western blotting shows that Armi - Flag ( Armi - F ) but not Flag - Armi ( F - Armi ) strongly bound with Gasz in Zuc / Armi - depleted OSCs ( Zuc KD + Armi KD ) . Piwi bound with both Armi ( F - Armi and Armi - F ) . Luciferase - Flag ( Luc - F ) was used as a negative 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

Immunoprecipitation and subsequent western blotting shows that Armi-Flag (Armi-F) but not Flag-Armi (F-Armi) strongly bound with Gasz in Zuc/Armi-depleted OSCs (Zuc KD + Armi KD). Piwi bound with both Armi (F-Armi and Armi-F). Luciferase-Flag (Luc-F) was used as a negative control.


G IP of UbcH7 by PknG or PknGΔUbl in U937 cells . Cells were infected with WT , ΔpknG , or ΔpknG : pknGΔUbl Mtb strains as in B . H IP of UbcH7 by PknG or PknG E190A in U937 cells . Cells were infected with WT , ΔpknG , or ΔpknG : pknG E190A Mtb strains as in B .

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G IP of UbcH7 by PknG or PknGΔUbl in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknGΔUbl Mtb strains as in B. H IP of UbcH7 by PknG or PknG E190A in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknG E190A Mtb strains as in B.


E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His - UBA1 at 37 ° C for 30 min . The reaction products were analyzed by immunoblotting with antibodies against Ub ( top panel ) , the His - tag on UBA1 ( middle panel ) , and PknG ( bottom panel ) , or by staining with Coomassie Brilliant Blue ( bottom ) . For reducing conditions , samples were treated with 500 mM β - mercaptoethanol ( βME ) prior to SDS - PAGE gel analysis .

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E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol (βME) prior to SDS-PAGE gel analysis.


A MS / MS analysis of UbcH7 in the absence ( top ) or presence ( bottom ) of PknG to reveal ubiquitination sites . Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high - confidence peptides possessing di - Gly modifications . The ubiquitinated residue ( Lys82 ) in the peptide sequence is highlighted in red . The peak heights are the relative abundances of the corresponding fragmentation ions . Matched amino terminus - containing ions ( b ions ) are indicated in red , and matched carboxyl terminus - containing ions ( y ions ) are indicated in blue . Only the major peaks that were identified are labeled .

O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled.


A Luciferase assay of NF - κB activation in HEK293T cells transfected with WT or mutant Mtb PknG . The graph shows the mean ± SEM of three independent biological replicates , n = 3 . * P < 0 . 05 , * * P < 0 . 01 . Data were analyzed according to one - way analysis of variance ( ANOVA ) using GraphPad Prism 7 .

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A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7.


D Survival of Mtb strains in primary human monocyte - derived macrophages treated as in B and C . The graph shows the mean ± SEM of three independent biological replicates , n = 3 . * P < 0 . 05 , * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

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D Survival of Mtb strains in primary human monocyte-derived macrophages treated as in B and C. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.


B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells . Cells were infected with the indicated Mtb strains at an MOI of 1 . Non - infected cells were used as controls . After 4 hours , the cells were lysed and immunoprecipitated with the antibody against PknG . The immunoprecipitated proteins were immunoblotted with TRAF2 , TAK1 , TAB2 ( negative control ) , or TAB3 ( negative control ) antibodies .

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B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies.


E Immunoblotting of TRAF2 , TAK1 , and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours . Cells were treated with CHX ( 50 μg / mL ) with or without MG132 ( 5 μM ) . Cell lysates were analyzed by immunoblotting for TRAF2 ( anti - TRAF2 ) , TAK1 ( anti - TAK1 ) , and PknG ( anti - PknG ) . GAPDH was used as the loading control . Densitometric quantification of immunoblots is indicated below the immunoblots .

O O O O O O O O O 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 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

E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.


G , H In vivo ubiquitination assay of TRAF2 ( G ) and TAK1 ( H ) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours . Cells were lysed and immunoprecipitated using the antibody against Flag . Immunoprecipitated proteins were immunoblotted with the antibody against HA .

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G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.


A , B qPCR analysis of Tnf mRNA ( A ) and Il6 mRNA ( B ) in splenic cells from C57BL / 6 mice infected with WT or mutant Mtb stains for 0 - 20 days . Data are representative of one experiment with two independent biological replicates ( mean ± SEM of n = 6 mice per group ) . * P < 0 . 05 and * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

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A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.


D H & E staining of lungs and livers of C57BL / 6 mice treated as in A . Arrows indicate puncta of cellular infiltration . Scale bars , 200 μm .

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D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm.


A . BAZ2A expression is higher in ESC + 2i than in differentiated cells ( neural progenitors , NPC ) . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and NPC . Tubulin is shown as a protein loading control .

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A. BAZ2A expression is higher in ESC+2i than in differentiated cells (neural progenitors, NPC). Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Right panel. Western blot showing BAZ2A protein levels in ESC+2i and NPC. Tubulin is shown as a protein loading control.


B . BAZ2A associates with chromatin of ESC and NPC . Chromatin - bound ( Chrom . ) and soluble ( Sol . ) fractions of equivalent cell number of ESC + 2i and NPCs were analyzed by western blot for BAZ2A levels . Total , total protein . Tubulin and histones are shown as loading and fractionation control .

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B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.


C . siRNA - knockdown efficiency of BAZ2A shown by qRT - PCR and western blot . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to each ESC line . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * * < 0 . 0001 ) . Right panel . Western blot showing BAZ2A protein levels . Tubulin is shown as a protein 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 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. siRNA-knockdown efficiency of BAZ2A shown by qRT-PCR and western blot. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to each ESC line. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (**** < 0.0001). Right panel. Western blot showing BAZ2A protein levels. Tubulin is shown as a protein loading control.


D . BAZ2A knockdown affects proliferation of ESC + 2i but not of ESC + serum . Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 ; ns , non - significant ) .

D. BAZ2A knockdown affects proliferation of ESC+2i but not of ESC+serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01; ns, non-significant).


E . BAZ2A is expressed at similar levels in both ESC + 2i and ESC + serum . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and ESC + serum . Tubulin and histone H3 are shown as a protein loading controls .

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E. BAZ2A is expressed at similar levels in both ESC+2i and ESC+serum. Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. Right panel. Western blot showing BAZ2A protein levels in ESC+2i and ESC+serum. Tubulin and histone H3 are shown as a protein loading controls.


F . BAZ2A is required for the differentiation of ESC + 2i but not of ESC + serum . Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10 % serum and in the absence of LIF .

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F. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF.


G . Quantification of differentiated cells . Values represent relative number of differentiated cells from three independent experiments relative to control cells . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ; ns , non - significant ) .

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G. Quantification of differentiated cells. Values represent relative number of differentiated cells from three independent experiments relative to control cells. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001; ns, non-significant).


H . Western blot showing BAZ2A protein levels in two BAZ2A - KO ESC lines obtained via CRISPr / Cas9 directly in ESC + serum . PARP1 is shown as a protein loading control .

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H. Western blot showing BAZ2A protein levels in two BAZ2A-KO ESC lines obtained via CRISPr/Cas9 directly in ESC+serum. PARP1 is shown as a protein loading control.


I . Cell proliferation is not affected in BAZ2A - KO ESC + serum . Data represent relative cell numbers 2 days culture starting with the same number of cells . Average values of four independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns , non - significant ) .

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I. Cell proliferation is not affected in BAZ2A-KO ESC+serum. Data represent relative cell numbers 2 days culture starting with the same number of cells. Average values of four independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns, non-significant).


J . Representative images of wt and Baz2a - KO ESC + serum before and after transition in 2i conditions .

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J. Representative images of wt and Baz2a-KO ESC+serum before and after transition in 2i conditions.


A . Volcano plot showing fold change ( log2 values ) in transcript level of ESC + 2i and ESC + serum with two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Gene expression values of three replicates were averaged and selected for 1 . 5 fold changes and P < 0 . 05 . Statistical significance ( P - values ) was calculated using R package DEseq2 .

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A. Volcano plot showing fold change (log2 values) in transcript level of ESC+2i and ESC+serum with two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Gene expression values of three replicates were averaged and selected for 1.5 fold changes and P < 0.05. Statistical significance (P-values) was calculated using R package DEseq2.


B . Validation by qRT - PCR of genes regulated by BAZ2A in ESC + 2i but not in ESC + serum ( upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red ) . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) . Data without P values are statistically non significant .

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B. Validation by qRT-PCR of genes regulated by BAZ2A in ESC+2i but not in ESC+serum (upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red). Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001). Data without P values are statistically non significant.


A . Western blot showing equal BAZ2A levels in wt - ESC and F / H - BAZ2A ESC line containing FLAG - HA sequences at the N - terminus of both Baz2a alleles . Whole cell lysates from equivalent amounts of cells were analysed . SNF2H , and Tubulin are shown as protein loading controls .

A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls.


B . Anti - FLAG ChIP of wt - ESCs and F / H - BAZ2A - ESCs cultured in 2i or serum . Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 ) .

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B. Anti-FLAG ChIP of wt-ESCs and F/H-BAZ2A-ESCs cultured in 2i or serum. Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01).


C . Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3 . The grey rectangles highlighted some of BAZ2A associated regions .

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C. Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3. The grey rectangles highlighted some of BAZ2A associated regions.


D . Pearson correlation heat map for the indicated ChIPseqs in ESC + 2i . BAZ2A ChIPseqs were performed with FLAG antibodies in F / H - BAZ2A - ESCs cultured in 2i or serum and with HA antibodies in F / H - BAZ2A - ESC + 2i . Data of H3K4me3 , H3K27ac and H3K27me3 are from this work . H3K36me3 , H3K9me3 and H3K4me1 values in ESC + 2i were taken from published data

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D. Pearson correlation heat map for the indicated ChIPseqs in ESC+2i. BAZ2A ChIPseqs were performed with FLAG antibodies in F/H-BAZ2A-ESCs cultured in 2i or serum and with HA antibodies in F/H-BAZ2A-ESC+2i. Data of H3K4me3, H3K27ac and H3K27me3 are from this work. H3K36me3, H3K9me3 and H3K4me1 values in ESC+2i were taken from published data sets


E . Boxplot showing the mean of normalized BAZ2A occupancies in ESC + 2i and ESC + serum at BAZ2A - bound regions . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

E. Boxplot showing the mean of normalized BAZ2A occupancies in ESC+2i and ESC+serum at BAZ2A-bound regions. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


F . Differential BAZ2A - binding in 2i and serum correlates with H3K27ac occupancies . Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC + 2i and ESC + serum at all BAZ2A - bound regions and BAZ2A - bound regions only in 2i were calculated and their log2 - fold changes are plotted . H3K27ac in ESC + serum Statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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F. Differential BAZ2A-binding in 2i and serum correlates with H3K27ac occupancies. Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC+2i and ESC+serum at all BAZ2A-bound regions and BAZ2A-bound regions only in 2i were calculated and their log2-fold changes are plotted. H3K27ac in ESC+serum Statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . Representative images showing 2i - specific BAZ2A - bound regions and H3K27ac and H3K27me3 in ESC + 2i and ESC + serum . The grey box highlights strong TIP5 - bound regions and corresponding histone modifications .

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G. Representative images showing 2i-specific BAZ2A-bound regions and H3K27ac and H3K27me3 in ESC+2i and ESC+serum. The grey box highlights strong TIP5-bound regions and corresponding histone modifications.


H . Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3 , depleted of H3K27ac and not bound by BAZ2A .

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H. Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3, depleted of H3K27ac and not bound by BAZ2A.


I . Heat map profiles of H3K27me3 and H3K27ac at ± 5 kb from the TSS of genes differentially regulated by BAZ2A in ESC + 2i and random genes . Differentially regulated genes are shown as upregulated or downregulated in ESC + 2i upon BAZ2A knockdown . Data were ranked by H3K27me3 levels . Transcription start site ( TSS ) .

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I. Heat map profiles of H3K27me3 and H3K27ac at ±5 kb from the TSS of genes differentially regulated by BAZ2A in ESC+2i and random genes. Differentially regulated genes are shown as upregulated or downregulated in ESC+2i upon BAZ2A knockdown. Data were ranked by H3K27me3 levels. Transcription start site (TSS).


J . Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over + / - 1kb of TSSs from upregulated , downregulated and random genes . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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J. Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over +/- 1kb of TSSs from upregulated, downregulated and random genes. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Number of BAZ2A - bound regions in A and B compartments of ESC + serum . B . Hi - C contact matrices for a zoomed in region on chromosome 3 at 100 - kb resolution showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Low panels . HiC contacts of a zoomed region at 50 - kb resolution in ESC + serum , NPCs and CNs are shown . Data are from ( Bonev et al . , 2017 ) . HiC contacts were visualized using Juicebox C . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + serum . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . D . Long - range contacts marked by BAZ2A binding are specific for ESCs . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in all genome or in A compartments of ESC + serum , NPC and CN . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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A. Number of BAZ2A-bound regions in A and B compartments of ESC+serum. B. Hi-C contact matrices for a zoomed in region on chromosome 3 at 100-kb resolution showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Low panels. HiC contacts of a zoomed region at 50-kb resolution in ESC+serum, NPCs and CNs are shown. Data are from (Bonev et al., 2017). HiC contacts were visualized using Juicebox C. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+serum. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (****< 0.0001). D. Long-range contacts marked by BAZ2A binding are specific for ESCs. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in all genome or in A compartments of ESC+serum, NPC and CN. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


E . Number of BAZ2A - bound regions in A and B compartments in ESC + 2i . F . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + 2i . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments of ESC + 2i . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * < 0 . 001 , * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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E. Number of BAZ2A-bound regions in A and B compartments in ESC+2i. F. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+2i. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments of ESC+2i. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (** < 0.001, ****< 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


G . BAZ2A binding correlates the best with eigenvector values . Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC + serum and ESC + 2i , respectively .

G. BAZ2A binding correlates the best with eigenvector values. Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC+serum and ESC+2i, respectively.


H . Boxplots showing H3K4me1 and H3K27ac content of BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in ESC + 2i . Data from regions with lowest 10 % and top 10 % contact strength are shown . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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H. Boxplots showing H3K4me1 and H3K27ac content of BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in ESC+2i. Data from regions with lowest 10% and top 10% contact strength are shown. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Numbers of ATAC peaks called separately in ESC + 2i and ESC + serum including their localization according to compartmentalization . B . Numbers of significant changed ATAC peaks in ESC + 2i vs . ESC + serum and their localization according to compartmentalization . Increased chromatin accessibility sites ( IAS ) , decreased accessibility sites ( DAS ) . C . Average profile of normalized ATAC read counts over differential peaks in ESC + 2i and ESC + serum changed ATAC peaks . The signal is plotted over + / - 1kb on the center of the ATAC peak .

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A. Numbers of ATAC peaks called separately in ESC+2i and ESC+serum including their localization according to compartmentalization. B. Numbers of significant changed ATAC peaks in ESC+2i vs. ESC+serum and their localization according to compartmentalization. Increased chromatin accessibility sites (IAS), decreased accessibility sites (DAS). C. Average profile of normalized ATAC read counts over differential peaks in ESC+2i and ESC+serum changed ATAC peaks. The signal is plotted over +/- 1kb on the center of the ATAC peak.


D . H3K27ac correlates with differential accessible sites between ESC + 2i and ESC + serum . Boxplot showing the mean values of normalized H3K27ac at ATAC peaks ( + / - 500bp ) that are increased and decreased in ESC + 2i compared to ESC + serum , respectively . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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D. H3K27ac correlates with differential accessible sites between ESC+2i and ESC+serum. Boxplot showing the mean values of normalized H3K27ac at ATAC peaks (+/- 500bp) that are increased and decreased in ESC+2i compared to ESC+serum, respectively. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


E . BAZ2A depletion increases chromatin accessibility only in ESC + 2i . Numbers of significant changed ATAC peaks in ESC + 2i and ESC + serum upon BAZ2A knockdown and their localization according to compartmentalization . F . Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC + 2i and ESC + serum treated with siRNA - Baz2a and siRNA - control over changed ATAC peaks . The signal is plotted over + / - 1kb centered on the ATAC peak .

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E. BAZ2A depletion increases chromatin accessibility only in ESC+2i. Numbers of significant changed ATAC peaks in ESC+2i and ESC+serum upon BAZ2A knockdown and their localization according to compartmentalization. F. Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC+2i and ESC+serum treated with siRNA-Baz2a and siRNA-control over changed ATAC peaks. The signal is plotted over +/- 1kb centered on the ATAC peak.


G . The distances of increased ( IAS ) and decreased ( DAS ) chromatin accessibility sites in BAZ2A - depleted ESC + 2i to the nearest gene promoter ( + / - 5kb of TSS ) were calculated . Distances in bp to the nearest promoter of genes upregulated ( UP ) , downregulated ( DOWN ) or not affected ( NOT ) upon BAZ2A knockdown in ESC + 2i are plotted . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * < 0 . 01 ) . Error bars represent s . d . The mean is represented by a horizontal line .

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G. The distances of increased (IAS) and decreased (DAS) chromatin accessibility sites in BAZ2A-depleted ESC+2i to the nearest gene promoter (+/- 5kb of TSS) were calculated. Distances in bp to the nearest promoter of genes upregulated (UP), downregulated (DOWN) or not affected (NOT) upon BAZ2A knockdown in ESC+2i are plotted. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (** < 0.01). Error bars represent s.d. The mean is represented by a horizontal line.


H . Genomic annotation of IAS and DAS in ESC + 2i depleted of BAZ2A perfomed with

H. Genomic annotation of IAS and DAS in ESC+2i depleted of BAZ2A perfomed with ChIPseeker


I . Fold - enrichment relative to expected of regions with increased chromatin accessibility ( IAS ) and upregulated genes upon BAZ2A KD at sub - regions within compartment A and B ( center , inside and boundaries ) . Compartments smaller than 500 kb were excluded from the analysis . Statistical significance ( P - values ) was calculated with permutation test ( ns : non significant , * < 0 . 05 , * * * < 0 . 001 ) .

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I. Fold-enrichment relative to expected of regions with increased chromatin accessibility (IAS) and upregulated genes upon BAZ2A KD at sub-regions within compartment A and B (center, inside and boundaries). Compartments smaller than 500 kb were excluded from the analysis. Statistical significance (P-values) was calculated with permutation test (ns: non significant, * < 0.05, *** < 0.001).


J . BAZ2A - upregulated genes are enriched in the B compartment . Numbers of BAZ2A - regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC + 2i including their localization according to compartmentalization .

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J. BAZ2A-upregulated genes are enriched in the B compartment. Numbers of BAZ2A-regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC+2i including their localization according to compartmentalization.


K . Active histone marks increase in the B compartment upon BAZ2A knockdown . Boxplots indicating the log2 fold - changes of normalized H3K27ac and H3K4me3 read counts in ESC + 2i treated with siRNA - Baz2a and siRNA - control at boundaries and centers of A and B compartments . ) . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , * < 0 . 05 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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K. Active histone marks increase in the B compartment upon BAZ2A knockdown. Boxplots indicating the log2 fold-changes of normalized H3K27ac and H3K4me3 read counts in ESC+2i treated with siRNA-Baz2a and siRNA-control at boundaries and centers of A and B compartments. ). Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, * < 0.05). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


A . Hi - C contact matrices for chromosome 1 of ESC + serum , ESC + 2i / siRNA - control and ESC + 2i / siRNA - Baz2a showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Cells were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Eigenvector values are shown . Increase in far - cis contacts in ESC + 2i upon BAZ2A - KD are highlighted with a rectangle . B . Relative contact frequencies over genomic distances in ESCs treated with siRNA - control and siRNA - BAZ2A . Statistical significance ( P - values ) was calculated using the Kolmogorov - Smirnov test . C . Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments . Differences in eigenvector values were calculated from HiC data sets from ESC + 2i treated with siRNA - Baz2a and siRNA - control . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , ns : non significant ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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A. Hi-C contact matrices for chromosome 1 of ESC+serum, ESC+2i/siRNA-control and ESC+2i/siRNA-Baz2a showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Cells were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Eigenvector values are shown. Increase in far-cis contacts in ESC+2i upon BAZ2A-KD are highlighted with a rectangle. B. Relative contact frequencies over genomic distances in ESCs treated with siRNA-control and siRNA-BAZ2A. Statistical significance (P-values) was calculated using the Kolmogorov-Smirnov test. C. Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments. Differences in eigenvector values were calculated from HiC data sets from ESC+2i treated with siRNA-Baz2a and siRNA-control. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, ns: non significant). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.