( J ) Transwell migration assay for NMuMG cells treated as in G . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(J) Transwell migration assay for NMuMG cells treated as in G. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


. ( E ) ChIP assay using H3K27me3 - specific antibody in epithelial cells ( day 0 ) ) or NMuMG cells treated for seven days with TGF - β ( mesenchymal ) and DMSO or JNKi . qRT - PCRs were performed for indicated gene promoters and enrichments are plotted on the right y - axis as ratio of precipitated DNA ( bound ) to total input DNA . On the left y - axis RNA levels of analyzed genes derived from RNA - seq data ( fold change to untreated normalized tag counts ) are plotted . Mean and SEM is plotted from two independent biological replicates .

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. (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates.


( F ) FAIRE assay was performed in cells treated as in ( D ) and qRT - PCRs were performed for the same gene promoters and plotted as in ( E ) .

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(F) FAIRE assay was performed in cells treated as in (D) and qRT-PCRs were performed for the same gene promoters and plotted as in (E).


JNK - induced transcription factors are critically required for EMT . ( A ) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors . Right panel shows RNA levels ( normalized tag counts ) for these factors .

JNK-induced transcription factors are critically required for EMT. (A) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors. Right panel shows RNA levels (normalized tag counts) for these factors.


( B ) Same overlap as in ( A ) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT . Right panel displays RNA levels as in ( A ) for 10 transcription factors identified in this overlap .

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(B) Same overlap as in (A) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT. Right panel displays RNA levels as in (A) for 10 transcription factors identified in this overlap.


( C ) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days ( d4 ) of siRNA - mediated depletion of factors compared to non - targeting control ( NTC ) . Staining was performed with antibodies against epithelial markers E - cadherin and ZO1 , mesenchymal markers Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . Scale bar , 100 μm , 40X magnification . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(C) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days (d4) of siRNA-mediated depletion of factors compared to non-targeting control (NTC). Staining was performed with antibodies against epithelial markers E-cadherin and ZO1, mesenchymal markers Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. Scale bar, 100 μm, 40X magnification. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Depletion of Prkrir , Pcbp4 and Tsc22d1 rescues TGF - β - induced reduction in the rate of cell division . ( A ) Lineage trees tracked by live imaging under control , Non treated control ( NTC ) and siRNA - mediated depletion of factors ( X = cell death / cells could not be tracked till the end ) .

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Depletion of Prkrir, Pcbp4 and Tsc22d1 rescues TGF-β-induced reduction in the rate of cell division. (A) Lineage trees tracked by live imaging under control, Non treated control (NTC) and siRNA-mediated depletion of factors (X= cell death/cells could not be tracked till the end).


( B ) Average number of rounds of division per clone in NTC compared with control and siRNA - mediated depletion of factors ( ANOVA , Tukeys post - test , * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(B) Average number of rounds of division per clone in NTC compared with control and siRNA-mediated depletion of factors (ANOVA, Tukeys post-test, *=p<0.05,**=p<0.01).


PRKRIR , PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate . ( A ) Scratch assay in MDA - MB - 231 cells treated for four days with DMSO or JNKi . Scale bar , 200 μm ; 20 X magnification . Mean and SEM is plotted from three independent biological replicates .

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PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates.


( B ) mRNA levels for key EMT marker genes in MDA - MB - 231 treated with DMSO or JNKi for 4 days were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological

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(B) mRNA levels for key EMT marker genes in MDA-MB-231 treated with DMSO or JNKi for 4 days were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates


( C ) mRNA levels for transcription factors in isogenic epithelial ( MDA - MB - 361 ) and mesenchymal ( MDA - MB - 231 ) breast cancer cells were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

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(C) mRNA levels for transcription factors in isogenic epithelial (MDA-MB-361) and mesenchymal (MDA-MB-231) breast cancer cells were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


( D ) Scratch assay in MDA - MB - 231 cells transfected with NTC or siRNA against PRKRIR , NFIL3 , PCBP4 and TSC22D1 for 4 days as in A . Mean and SEM is plotted from three independent biological replicates as bar plot on right .

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(D) Scratch assay in MDA-MB-231 cells transfected with NTC or siRNA against PRKRIR, NFIL3, PCBP4 and TSC22D1 for 4 days as in A. Mean and SEM is plotted from three independent biological replicates as bar plot on right .


( E ) Invasion assay in MDA - MB - 231 cells treated as in ( D ) . Mean and SEM is plotted from three independent biological replicates .

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(E) Invasion assay in MDA-MB-231 cells treated as in (D). Mean and SEM is plotted from three independent biological replicates.


( F ) mRNA levels of key EMT marker genes in MDA - MB - 231 and MDA - MB - 231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor ( SP600125 ) for 24 hours were measured by qRT - PCRs relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(F) mRNA levels of key EMT marker genes in MDA-MB-231 and MDA-MB-231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor (SP600125) for 24 hours were measured by qRT-PCRs relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


( C ) mRNA levels for representative genes highlighted in ( A ) for cells treated like in ( Fig 5D ) were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates .

(C) mRNA levels for representative genes highlighted in (A) for cells treated like in (Fig 5D) were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.


F ) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag - HA tagged Tsc22d1 and detection using an anti - Flag antibody , Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus . Y - axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions . SEM is derived from independent biological replicates .

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F) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag-HA tagged Tsc22d1 and detection using an anti-Flag antibody, Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus. Y-axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions. SEM is derived from independent biological replicates.


( G ) ChIP assay using anti - Flag antibody following expression of Flag - HA tagged TSC22D1 in MDA - MB - 231 cells . Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y - axis as ratio of precipitated DNA ( bound ) to total input DNA and then further divided by the same obtained in the empty vector transfected cells . SEM is derived from independent biological replicates .

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(G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates.


( H ) mRNA levels for PRKRIR , PCBP4 , TSC22D1 in non - invasive ( DCIS ) ( n = 20 ) and invasive tumor ( n = 20 ) samples were measured by qRT PCRs relative to TBP and plotted on the y - axis . ZEB1 was used as an established positive control for invasive tumors . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(H) mRNA levels for PRKRIR, PCBP4, TSC22D1 in non-invasive (DCIS) (n=20) and invasive tumor (n=20) samples were measured by qRT PCRs relative to TBP and plotted on the y-axis. ZEB1 was used as an established positive control for invasive tumors. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration . ( A ) RT - qPCR analysis for key EMT markers in embryonic stem cells ( ES ) , neuronal progenitors ( NP ) and terminally differentiated neurons ( TN ) . mRNA levels were measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration. (A) RT-qPCR analysis for key EMT markers in embryonic stem cells (ES), neuronal progenitors (NP) and terminally differentiated neurons (TN). mRNA levels were measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( B ) Western blot analyzing total JNK and phospho - JNK ( p - JNK ) during neuronal differentiation of stem cells . β - actin serve as a loading control .

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(B) Western blot analyzing total JNK and phospho-JNK (p-JNK) during neuronal differentiation of stem cells. β-actin serve as a loading control.


( C ) RNA levels of Pcbp4 , Prkrir and Tsc22d1 during neuronal differentiation as shown in A , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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(C) RNA levels of Pcbp4, Prkrir and Tsc22d1 during neuronal differentiation as shown in A, measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( D ) RNA levels of Pcbp4 , Prkrir , Tsc22d1 , represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 ( JNKi ) for 6 hours .

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(D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours.


( E ) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in ( A ) , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates .

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(E) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in (A), measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.


( F ) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA - seq data from Ventricular Zone ( VZ ) , Sub - Ventricular Zone ( SVZ ) and Cortical plate ( CP ) of E14 . 5 mouse Cortex .

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(F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex.


( G ) RNA levels of genes shown in Figure ( E ) , represented by average normalized tag counts derived from RNA - seq data of TGF - β - induced EMT in NMuMG cells and plotted on the y - axis . Error bars represent SEM from three biological replicates .

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(G) RNA levels of genes shown in Figure (E), represented by average normalized tag counts derived from RNA-seq data of TGF-β-induced EMT in NMuMG cells and plotted on the y-axis. Error bars represent SEM from three biological replicates.


( I ) A representative image from immunofluorescence analysis performed with anti - GFP and anti - Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain . The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining . y axis represent percentage of cells above or below the Tbr1 stain region . Error bars represent SEM from 3 independent biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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(I) A representative image from immunofluorescence analysis performed with anti-GFP and anti-Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain. The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining. y axis represent percentage of cells above or below the Tbr1 stain region. Error bars represent SEM from 3 independent biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


( A - B ) qRT - PCR analysis of TGF ligands , TGF receptors , and TGF target expression in control and FRS2 knockdown HASMCs . ( NS : not significant compared to control , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) . - actin was used for sample loading normalization . Histogram of qRT - PCR results are representative of three independent experiments .

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(A-B) qRT-PCR analysis of TGF ligands, TGF receptors, and TGF target expression in control and FRS2 knockdown HASMCs. (NS: not significant compared to control, *p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test). -actin was used for sample loading normalization. Histogram of qRT-PCR results are representative of three independent experiments.


( 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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

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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 .

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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 .

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

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

(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 B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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 .

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

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 ) .

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

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 ) .

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

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

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

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

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

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

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

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 ) .

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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 .

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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 .

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

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 .

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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 .

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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 .

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

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 .

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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 .

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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 .

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

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 .

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

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 .

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

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 .

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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 ) .

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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.